The theme of the previous post concerned how human diseases could be triggered by environmental compounds with slow and subtle effects, with an emphasis on those which occur naturally. (The interest in natural exemplars of such effects arises from earlier posts on ‘Natural Molecular Space’). A principal theme in this follow-up post will be comparing cancer and cellular degeneration induced by environmental agents.
Subtle Carcinogens and Other Problems
With the exceptions of Polonium-210 and asbestos, the ‘subtle poisons’ considered previously were neurotoxic organic molecules. But organic cancer-causing compounds have been described for a long time. The first description of an association between a specific cancer and an industrial (work-related) activity dates back to the 18th century, when a rare form of scrotal cancer was linked to chimney sweeping. From the time of publication of this finding, it took almost 160 years for science to advance enough such that the active component in soot and coal tars was identified as benzo[a]pyrene, a polycyclic aromatic hydrocarbon. Certain other such polycyclic aromatics are also carcinogenic, and they are thus known collectively as PAHs.
Of course, we now know that a whole zoo of both natural and artificial compounds can induce cancer, with varying degrees of potency. It isn’t the intended scope of this post to review a great number of specific cases here, but among the natural set of known carcinogens, an important group are derived as secondary metabolites of various fungal organisms (metabolic products which are not essential components of fundamental life-support processes). While some secondary metabolites (such as antibiotics) have been extremely beneficial to humans, a ‘dark side’ of such secondary metabolism also exists. Not all toxic fungal products (or mycotoxins) are proven carcinogens, but some most certainly are. Probably the most significant in economic and human disease impact are a group of closely related compounds called aflatoxins, produced by various species of the fungal Aspergillus genus (most notably A. flavus). Aflatoxin B1 is the most potent known natural liver carcinogen, and a major problem as a side-effect of fungal contamination of foodstuffs, such as peanuts.
Sometimes it is the case that carcinogens are not directly found in certain natural food materials, but are actually formed during cooking processes. There is a certain irony here, because on the whole, cooking of many foods is beneficial through the killing of potentially dangerous parasites, especially those harbored in raw meat. And apart from the generally detrimental effects of parasites on health, a number of such organisms are themselves directly linked to the generation of specific cancers. Yet during ordinary cooking of meats, carcinogenic heterocyclic amines can form, and if charring is involved (as with barbecuing), polycyclic aromatic hydrocarbons can be created. Among the latter is found benzo[a]pyrene, the same compound of chimney sweep fame as noted above. Strictly speaking, carcinogens formed by cooking are not ‘natural’, since they require human intervention for their formation. Indeed, cooking itself has been considered a useful marker for distinguishing humans from all other organisms, including our primate relatives, and may have even shaped evolutionary pathways leading to modern humans. Still, while carcinogenic compounds resulting from cooking clearly arise from human agency, their formation has always been completely inadvertent, and occurred long before the faintest glimmerings of modern chemistry.
Subtlety of effect, at least as measured by the time between exposures and onset of disease, is practically a by-word for carcinogens, as well as the ‘subtle’ neurotoxic agents considered in the previous post. This is not to say that these two broad areas of pathology cover everything where subtlety rears its head, but they may safely be grouped as the major concerns. Beyond this, one needs to consider other physiological systems which may be damaged or negatively affected slowly and subtly by non-biological environmental agents, but not with tumorigenic outcomes. One case in point is the immune system, and there are precedents for natural compounds with immunosuppressant qualities. In this respective, it should be noted that toxic compounds can have multiple effects, and aflatoxins (for example) have immunosuppressive activity as well as their other noxious manifestations. Reproductive systems can be adversely affected by natural phytoestrogens, as considered in more detail in a previous post .
These other issues aside, cancer and toxic neurological disease can be seen as book-ends in terms of the gross effects leading to divergent pathological results. Let’s consider this statement a little further.
Growth or Degeneration, and a Problem Either Way
A toxic challenge will by definition perturb normal cellular functions. Following such an event, broadly speaking three things can happen. Firstly, an affected cell may, through its endogenous repair system, correct the damage and resume its normal functions. Failing this (in the second alternative) it can die, through a number of alternative mechanisms noted in the legend to the figure below. The best-defined form of directed or ‘programmed’ cell death is the process termed apoptosis. But if death itself should fail and replication continue, chromosomal changes induced in the cell may eventually lead to ‘transformation’, where the normal controls on growth are circumvented and a tumor phenotype acquired, the third possible outcome. Successive genetic changes can accumulate, and transformed cells with invasive properties become amplified through their enhanced growth and survival properties. It is no accident that important genes regulating apoptosis are frequently mutated in cancer cells. If checkpoints on cell growth are removed through blockade of cell death, barriers to transformation may be greatly reduced. Indeed, while most carcinogens are also potent mutagens (inducing genetic mutations in genomic DNA), some are not. The latter have been a long-standing puzzle, but it has been shown that non-mutagenic chemical carcinogens are direct blockers of apoptosis, thereby allowing cells with mutations (normally removed by apoptosis) to persist and proceed down transformation pathways.
As noted in the previous post, recovery from a toxic insult might not necessarily be complete, in the sense that the post-toxic state may be sub-optimal relative to the norm, predisposing the cellular victims to future risk. But leaving such complications and the general area of damage repair aside, the major enduring pathological consequences of a low-level toxic assault revolve around cancer vs. degeneration. These outcomes might seem like diametrically opposed processes, since in one case cells grow wildly without normal constraints, and in the other, they die. While the final end-points are clearly quite divergent, it is interesting that the factors which push cells along these pathways have many regions of overlap. Genetic analyses have shown that many mutations which predispose towards Parkinson’s disease are also associated with certain cancers.
But toxic chemicals can also have dramatically different effects depending on the cellular context in which they act. A specific genotoxic (DNA-damaging) compound found in cycad plants (methylazomethanol) can induce neurological damage and degeneration in mice without tumor formation, whereas a high frequency of tumors are induced in the colon. Major determinants of the outcome of such toxic challenge are the levels of appropriate DNA repair enzymes (the effectiveness of the DNA damage response), and differential effects on cellular signaling pathways. Up- or down-regulation of specific pathways operating in diverse cell lineages can evidently result in outcomes as distinct as death or degeneration. A clear distinction between neurons and most other differentiated cells is their cell division status, where non-dividing and long-lived neurons can be contrasted with lineages with active turnover through cell division. Neurons thus permanently exit from the cell cycle into a ‘post-mitotic’ state for the lifetime of the organism.
Indeed, trying to force a mature neuron towards re-entering the cell cycle (by artificially expressing viral gene products which ‘kick-start’ cycling in other quiescent cells) has been observed to promote cell death. Given this piece of information, a differential response to at least some toxic agents can conceptualized in fairly simple terms: Forcing a quiescent cell which is nonetheless ‘primed’ for mitotic cycling (active division) may lead to carcinogenic transformation; doing the same thing to a mature neuron will kill it. This dichotomy is portrayed in the figure below:
Outcomes of mutational damage through low-level genotoxic exposure for neurons vs. non-neuronal cell lineages. In both cases, repair mechanisms exist, which may be insufficient to deal with the problem. Dividing cells may then be diverted into a programmed cell death pathway (usually apoptosis) and thus removed. In a population of renewable replicating cells, this is unlikely to be a direct problem, and of course eliminates a potentially dangerous altered cell. Yet if the shunt towards apoptosis fails for any reason, the altered cell may continue to proliferate and acquire further mutations, with the ultimate consequence of malignant transformation into a fully cancerous phenotype. For neural cells, beyond a certain damage threshold, death is inevitable, even for stimuli that normally promote mitosis in other cell types. Note here that cell death in general can occur by at least three mechanisms, shown specifically for neurons in this schematic. The process of autophagy (a kind of self-recycling of cellular components) is associated with repair processes, but can also constitute a specific cell death mechanism in some circumstances. Apoptosis is a programmed form of death operating through specific cellular signaling interactions, and autophagy can interface with some of these apoptotic pathways (as shown by arrows). Necrosis was originally categorized as non-specific and passive cell death brought on by severe physical or chemical insults. While cell death in such a disordered manner is presumably still a possibility, recent evidence has shown that at least one form of non-apoptotic ‘necrotic’ cell death is also a regulated process, which has been duly termed ‘necroptosis’. In any event, unlike loss of cells with a high mitotic turnover, death of non-dividing neurons will ultimately have significant functional consequences.
And yet the above figure might, upon further reflection, appear overly simplistic. Two pieces of extra widely-known information are relevant here: neurological tumors, and neurological plasticity. If neurons die upon transformation, what is the source of brain tumors? And what about the considerable publicity which has been given to the previously unsuspected potential for recovery from substantial brain damage, indicative of ‘neuroplasticity’? In both cases, a simple answer is that neither the ‘dark side’ of neural tumors nor much brighter prospects for neural regeneration derive from irrevocably post-mitotic cells. In both cases, neural stem cells, identified only relatively recently, may be the central players. With respect to tumors, the “neural stem cell hypothesis” proposes their origin as the source of primary brain tumors, as opposed to metastatic tumors which originate elsewhere but migrate to the brain and grow there.
The above figure addresses genotoxic substances, but this does not include other neurotoxic agents such as the compound MPTP, discussed in the previous post owing to its specific role in inducing cell death in the substantia nigral brain region, and thereby leading to induced Parkinson’s disease. As noted previously, MPTP has a distinct toxic mechanism via the enzymatic formation of a specific metabolic product, which is taken up by dopamine-producing neurons. This metabolic derivative then inhibits mitochondrial respiration, leading to cell death. Studies have found, however, that MPTP also has mutagenic properties – or at least, once again, one of its metabolic products is the active compound in such assays. Yet even if MPTP indirectly caused unrepairable genomic lesions in target neurons, the above observations suggest that cell death would still be the case anyway, rather than prolonged growth and transformation.
To conclude then with a summary of sorts upon this theme:
It seems cancer and neural decay
Are opposed, in a particular way
For to die or to grow
Is the question, you know
And the source of young Hamlet’s dismay
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘ ….an association between a specific cancer ….. chimney sweeping‘ This resulted from observations by Dr. Percival Pott (1714-1788) for scrotal cancer in young chimney sweeps, first published in 1775. See Brown & Thornton 1957 for relevant historical information.
‘….the active component in soots and coal tars was identified as benzo[a]pyrene….’ See Ling et al. 2004 for more information, especially including the structure of benzo[a]pyrene-DNA adducts, by which its carcinogenicity is manifested.
‘…..a whole zoo of both natural and artificial compounds can induce cancer…..’ For a review on the diversity of carcinogens (including not limited to organic compounds), see Yang 2011.
‘……toxic fungal products (or mycotoxins)…..’ For a useful review, See Pitt 2000.
‘Aflatoxin B1 is the most potent known natural liver carcinogen….’ See a review by Hedayati et al. 2007.
‘…..a number of different parasites are themselves directly linked to the generation of specific cancers….’ If we consider ‘parasites’ in the broadest sense, then there are numerous precedents of viral and bacterially-generated cancers. But in food-related circumstances, ‘parasite’ will most often refer to various worms, some of which are indeed associated with cancer. For example, see Vennervald & Polman 2009 for a review of the status of helminth worms as carcinogenic agents.
‘…..if charring is involved……polycyclic aromatic hydrocarbons can be created….’ See Daniel et al. 2011.
‘……cooking ….. may have even shaped evolutionary pathways leading to modern humans…’ The ‘cooking hypothesis’ has lead to a very interesting book by Richard Wrangham, Catching Fire: How Cooking Made Us Human; Basic Books 2009.
‘……there are precedents for natural compounds with immunosuppressant qualities….’ Although in normal circumstances immunosuppression is obviously undesirable, for some medical applications certain natural immunosuupressants have proved a great boon. Such compounds have proven highly useful for suppressing unwanted immune rejections of transplanted organs, and thereby greatly facilitated the efficacy of transplant surgery in general. These include cyclosporin A and FK506, which form ternary complexes between cellular proteins (cyclophilin and FKBP respectively) and the protein phosphatase calcineurin. See Fox & Heitman 2002 for a review.
‘……while most carcinogens are also potent mutagens…….some are not….’ See Kokel et al. 2006, for presentation of evidence that specific non-genotoxic carcinogens act by suppressing apoptotic cell death.
‘…..many mutations which predispose towards Parkinson’s disease are also associated with certain cancers….’ See Devine et al. 2011.
‘……found in cycad plants…..’ See the previous post for more detail regarding the neurotoxic effects of cycads.
‘A specific genotoxic (DNA-damaging) compound found in cycad plants (methylazomethanol) induces neurological damage ….’ This refers to an interesting publication of Kisby et al. 2011.
‘…..cell death…..’ For further information regarding autophagy, see Kaushik & Cuervo 2006. For a perspective on apoptosis in the light of the recently described necroptosis, see Christofferson & Yuan 2010.
‘……publicity which has been given to the previously unsuspected potential of recovery from substantial brain damage….’ Neuroplasticity has received much popular notice largely owing to the book The Brain That Changes Itself, by Norman Doidge. Viking, 2007. Note that one aspect of neuroplasticity, the ability of neurons to exhibit ‘regenerative sprouting’ from axons, is not the same as acquiring the ability to undergo full cell division. See Weiloch & Nikolich 2006.
‘…..the “neural stem cell hypothesis” proposes their origin…..’ For more information, see Germano et al. 2010.
Next Post: Late March.
The past series of posts have largely been preoccupied with the benefits to be had from ‘natural molecular space’, whether the molecules in question are large, small, or functionally linked together in complex (but useful) entire biosystems.
Obviously, some biomolecules are not merely useless, but may be actively harmful. There are a great many bioproducts which are of both high toxicity and obvious impact, at least to the unfortunate victims of serious or even life-threatening natural poisonings or envenomations. But toxic effects can be much more subtle, and therefore much less easily noticed. In fact, the insidious slowness of some toxic effects can render the actual molecular culprits very hard to pin down, and inevitably controversy is thus generated. These ‘subtle negative’ environmental influences are the principle theme for this discussion, which will include natural products, but will also heavily feature both artificial compounds and non-biological but ‘natural’ substances. (The quotation marks are used here since it is very often only through human activities that natural materials with potentially harmful effects are processed and brought into contact with sizable numbers of people).
What Does Subtlety Mean in a Toxic Context?
When we speak of a subtle toxic effect, what is actually meant? It might result from several factors, or any combination of them, including potency, exposure dose, frequency of exposure over time, and the in vivo persistence of the toxic substance. Any ingested toxic compound must by definition interfere with an important biochemical process, with ensuing negative consequences for the functioning of the organism. A poisonous substance might interact with many different biological molecules, but some of these will be of greater import than others in terms of how the resulting deleterious effects are produced. And the affinity of the poison for such biological targets is a determinant of potency.
Potency and dosage over time are inter-related. To qualify as ‘subtle’, intake of a highly potent compound (one whose toxic threshold is reached with very small amounts) would need to be in exceedingly low quantities, where no immediate effects are apparent. If that was the end to it, then obviously such a low-level exposure to the toxic agent has no further consequences. But a subtle deleterious effect might exist if the compound had produced some kind of persistent tissue or cellular damage, of a type that was very hard to detect without sophisticated intervention, and that was not at all appreciable by the individual concerned. Then, several possibilities could exist which in the end would result in a manifested disease state. Firstly, if the individual is re-exposed to the same source of the toxin on more than one occasion, the damage might be cumulative and accrete until it becomes of such significance that an overt illness is produced. If the body’s repair systems cannot comprehensively deal with the low-level induced damage, in some cases even long intervals between exposures might still result in noticeable pathology. But even if the repair is effective, regular intake of similar low doses of the toxic material over time might eventually overwhelm the host defenses, again leading to disease.
These scenarios assume repeated exposures, but even a single exposure could potentially have significant consequences. It might be supposed that a single bout of damage, if not fully repaired, might be another negative event in an individual’s ‘wear and tear’ list that increases with ageing. In other words, any such a low-grade but persistent toxic ‘insult’ might become more significant over time, in combination with other problems inevitably occurring through life. But a much more serious possibility also has been proposed, where short-lived exposure to certain chemical agents might actually set up an on-going pathological inflammatory process, even long after the original poison has been removed from the host system. This theme will be looked at in a little more detail in a later post in this series.
At this point, it’s very relevant to consider that there is an important issue relating to the physiological removal of toxic agents, or (in other words) how long it may be that noxious substances of any description can persist once taken into a host organism. Persistence has clear-cut implications for the ability of a substance to contribute to long-term and subtle deleterious effects. While water-soluble (hydrophilic) compounds are generally metabolized and excreted reasonably quickly, lipid-soluble (hydrophobic) compounds can be taken up by fat reserves and remain there for years, with only a slow diminution with time. A classic example in this regard is the insecticide DDT, whose tendency to persist in adipose (fat) tissue is well-described. Poisons which are themselves toxic elements obviously cannot be further ‘broken down’ chemically, and can persist through their interactions with normal biomolecules. For example, heavy metals such as lead and mercury can bind and inhibit numerous enzymes. Although the resulting complexes between metals and protein molecules may be physiologically degraded, release of the metal component may simply liberate it for another cycle of inhibition. In some cases, a noxious element may be physically or chemically similar to a normal biologically-used element, and replace it in certain biomolecules, with disastrous effects on metabolic activities. This is case for the toxic elements arsenic (capable of competing with phosphorus) and thallium (capable of competing with potassium).
Another major class of persistent and dangerous substances are certain mineral fibers, most notably asbestos. Poorly biodegraded long fibers (such as some mineral silicates, of which asbestos is a case in point) can persist indefinitely in specific anatomical sites. Although the mechanism is still incompletely understood, this can be associated with the generation of a chronic inflammatory process and ultimately carcinogenesis. The link between asbestos and mesothelioma is well recorded.
If we cast a wide enough net, another class of non-biological poisons must certainly be included: radionuclides, or radioactive isotopic versions of the elements. These can be either radioactive isotopic versions of normal elements of biological significance, or radioisotopes of non-biological elements. All such cases can be of either natural or artificial origins. Many examples of the former group can be cited, but potassium-40 (40K) is a natural radioisotope of interest, since it is contributes the largest portion of the radioactive background in living organisms. As such, it has been proposed as a major source of natural mutation, although experimental results have suggested that its contribution to mutation must indeed be (if anything) a subtle influence. Cases of relevant non-biological radioisotopes are likewise exceedingly numerous. Briefly, consider the example of polonium-210 (210Po), which can occur naturally, or can be generated by artificial nuclear reactions. This radioisotope is present in tobacco smoke, and it has been implicated as a major factor in the generation of smoking-induced cancer. Polonium-210 has also been in the news in recent years, through its use an exceedingly potent poison in the murder of the ex-Russian agent Alexander Litvinenko in London in 2006. There’s obviously nothing subtle about that, but as with any toxic agent, even polonium-210 can exert low-level effects if ingested in small enough doses. At that lower end of the exposure scale, the effects will vary among different individuals, but may contribute to cancers or other conditions, with an overall shortening of life expectancy.
Individual variation in responses to low-level toxic exposure reflect genetic variation in the metabolic processing of foreign compounds, or how the body reacts to the presence of noxious materials. There is much more to be said on this topic, which will be picked up at a later time within this series of posts. But for the time being, we can note this as one of a number of influences bearing on whether a low-level toxic exposure will have longer-term ‘subtle’ effects, depicted in the figure below:
A depiction of the range of various influences which can determine whether a substance could manifest a slow or insidious ‘subtle’ toxicity. Note that an implicit issue within ‘Generation of Ongoing Pathology’ is the ability of host systems to repair and contain toxic insults, as opposed to the generation of responses which are ultimately self-damaging.
The influence termed ‘cofactors’ in the above diagram simply refers to any other non-host factor which can interact with a proposed environmental toxic substance to exacerbate its action, or even be essential for the insidious toxic effect to be manifested in the first place. An interesting example is a putative requirement for the presence of simian virus 40 (SV40) for the generation of mesothelioma by asbestos.
For the rest of this post, I’ll move on to some specific examples of effects which have revealed subtlety in several senses of the word. The first case involves an artificial compound which is not strictly speaking an ‘environmental’ effect, since it required self-administration, if inadvertently. However, the experience with this compound has had many ramifications which do impinge on environmental influences, both man-made and natural.
(1) Parkinson’s Disease & Toxic Agents
In the early 1980s a remarkable series of events occurred which had implications across several fields of science and medicine. Although terrible and tragic in many ways, it provided a dramatic example of how a toxin can produce quite specific neurological effects, and had direct implications for the origins of Parkinson’s disease (PD). At that time in California, clinicians were confronted with a series of drug addicts in a state of ‘frozen’ mobility, which had many similarities to severe PD. Subsequent scientific detective work showed that this apparent similarity was more than just superficial. The sporadic condition of human PD is characterized by ongoing degeneration in a region of the brain called the substantia nigra, where destruction of neurons normally producing the crucial neurotransmitter dopamine leads to loss of muscular motor functions, eventually immobilizing the patient. These neurons are also pigmented, through the production of a type of melanin (‘neuromelanin’), an early observation which provided the name of this brain area (‘substantia nigra’ = Latin for ‘black substance’). A compound, L-dihydroxyphenylalanine (L-DOPA, which can access the brain and becomes metabolized to dopamine itself) can greatly alleviate symptoms, especially when first applied. The ‘frozen’ addicts likewise generally showed responsiveness to L-DOPA. By analyzing their common activities, the source of the problem was tracked down to their injection of a street drug preparation, a ‘synthetic heroin’, which in actuality was a botched attempt to make the drug meperidine (pethidine). The preparation that the clandestine chemists had produced contained sizable amounts of a different compound, N-methyl-4-phenyl-1,2,5,6-tetrahydopyridine (MPTP), eventually identified as the toxic culprit by means of animal testing. These studies also showed that MPTP ingestion resulted in specific damage to the substantia nigra, with associated loss of dopamine-producing neurons and the onset of parkinsonian symptoms.
Structures of some relevant molecules for the Parkinson’s / MPTP story. The amino acid phenylalanine is included as the precursor to dopamine, and to show its chemical similarity to L-DOPA. Meperidine is the drug towards which abortive synthetic attempts led to the formation of MPTP. MPP+ is the actively toxic metabolic product derived from MPTP itself.
The striking features of this story were widely reported in the scientific literature, and even found their way into popular fiction quite quickly. Those unmistakably victimized by MPTP had varying fates, ranging from death within a relatively short time, to survival for over a decade. But behind the initial cadre of severely affected patients, the prospect still remains of many more people developing PD from short-term exposure to MPTP (and initially subclinical damage) even decades ago. And this naturally raises one of the major implications of the whole MPTP saga: if a defined toxin can have such amazingly specific effects, could there not be other toxins in the environment with similar properties, which induce the neurodegeneration seen in ‘sporadic’ parkinsonian patients? In the course of these kinds of speculations, it was noted that the very description of this disease was a relative latecomer in 1817. Could the apparent lack of reporting of this disease in earlier times mean that ‘natural’ PD is actually a toxic condition, associated with the beginnings of the industrial revolution and newly introduced environmental pollutants?
Many studies have been conducted in order to evaluate this and related questions. In particular, exposure to certain insecticides has been a long-standing suspect as a potential agent of PD, but despite ‘probable cause’, this has not been firmly nailed down. These kinds of analyses must distinguish between genetic influences and environmental factors. (Many distinct genes are known to affect an individual’s susceptibility to PD, and this will be further considered in a subsequent post in this ‘subtle’ series). Studies with monozygotic (identical) twins illustrate this. In one detailed 1999 investigation, sets of monozygotic twins showed no significant differences in the concordance (common incidence in both twin pairs) of PD compared to non-identical twin pairs, but only (and this a crucial point) if the age of onset for either twin was after 51 years of age. Non-concordance of a disease in twin pairs in a controlled study is highly suggestive of environmental causes at least being contributing factors. Consider that if a disease does have a simple genetic origin, significant concordance would be expected in the (essentially) genetically identical pairs. Most cases of sporadic PD occur later in life, also consistent with (but far from proof of) a slow induction from environmental sources. But where PD does occur at younger ages, genetic influences (rare mutations, possibly in combination with environmental factors) might be postulated, and this is consistent with the higher concordance observed with identical twins with relatively young ages at the onset of PD. But the only general conclusion typically made at present is that the origin of sporadic PD is complex, with multiple genetic and environmental influences implicated directly or as suspects. And yet there is no question that, at least in certain genetic backgrounds, MPTP alone can induce a pathology with the key characteristics of PD. How does it do this?
A Stealth Poison At Work
Intensive studies on the mechanism of MPTP toxicity revealed that it was not the direct perpetrator of the neuronal damage. MPTP itself is acted upon by a specific enzyme within the brain, monoamine oxidase (MAO) B, which converts this compound into a positively charged species, the N-methyl-4-phenyl-pyridinium ion (MPP+, as shown in the above chemical structure figure). Consistent with this observation, inhibitors of MAO enzymes are protective against the effects of MPTP in animal models. MPP+ itself is capable of using the machinery for dopamine transport into neurons (using specific dopamine receptors), and this promotes its accumulation in very specific neuronal sites. It is important to note that this particular uptake mechanism also explains the high selectivity of MPTP (the precursor to MPP+) in its toxic action. Once taken up by dopamine neurons, MPP+ itself acts as a primary toxic agent towards mitochondria, through its inhibition of Complex I of the mitochondrial respiratory electron transport chain.
With the MPTP story, a series of processes are thus required for the ultimate toxic effect to be manifested: conversion to MPP+, uptake by dopamine neurons, and inhibition of mitochondrial activities. (These are primary factors; other issues such as specific genetic backgrounds certainly contribute to individual susceptibility, as will be discussed further in a subsequent post). So, it has been noted that this conjunction of requirements would (hopefully) render the occurrence of compounds with analogous properties to MPTP quite rare. With this in mind, are there natural precedents for this kind of noxious chemical agent? This raises the second case set to be considered (as noted above): natural toxic substances with ‘subtle’ actions. In many such circumstances, the subtlety is bound up with the difficulty of pinning down the true identity of the pathogenic culprit.
(2) Cycads, Soursops, and other ‘Environmental’ Neurological Diseases
In certain Western Pacific islands, epidemiologists have noted for decades an unusual incidence of a degenerative neurological condition called Amyotrophic Lateral Sclerosis / Parkinsonism-Dementia complex (ALS-PDC). In the language of the Chamorros of Guam, a people living on one of the afflicted island groups, the disease is known as ‘lytico-bodig’. A strong role for genetic influences in the origin of ALS-PDC seemed unlikely, given that it was recorded in diverse ethnic groups in varied Western Pacific locations. For a considerable time, though, a dietary item has been implicated: the consumption of a flour made from the seeds of cycad plants available in the affected locales. This remains unproven and controversial, particularly since a specific compound has not been conclusively identified. Yet the general ‘cycad hypothesis’ has support from a number of linked observations. Cycad flour fed to experimental animals over time induces a neurological condition with features of progressive parkinsonism, with associated damage to the substantia nigra. Also, the incidence of ALS-PDC has been in decline in recent years, and this correlates with changes in diet where the amounts of cycad-derived material have markedly declined. A specific amino acid, β-methylamino-L-alanine (BMAA; not found in normal proteins) has been repeatedly linked with cycad-induced disease, but proof of its role has consistently fallen short of the mark. Another contender is methylazoxymethanol (MAM, a metabolite derivative of the cycad compound cycasin), which has been shown to produce neurological genotoxicity.
Whatever the outcome of these studies, there is no question that raw cycad seeds (from which flour is derived) are quite poisonous, and this has long been known to Western Pacific peoples. But by using extensive washing and soaking procedures, they have ingeniously found a way to exploit this otherwise-useless material as a valuable foodstuff. The great irony implicit in the ‘cycad hypothesis’ is that although they succeeded in eliminating the acute toxicity of the cycad seeds, they could not remove traces of toxic substances which may have been the agents of subtle and insidious neurological damage.
Another potential natural molecular assailant of neurons is also found in an island setting, but in the West Indies. A high incidence of an ‘atypical’ parkinsonism has been identified on the island of Guadeloupe. (One example of the atypical nature of this condition is its failure to respond to L-DOPA.) This has been linked by epidemiological studies with the consumption of the tropical fruit called soursop, and a specific compound from this fruit (annonacin) has implicated as the probable underlying source of the pathology. Annonacin is an inhibitor of mitochondrial Complex I, and can also induce loss of dopamine neurons in the substantia nigra of experimental animals – findings which cannot help but stimulate recollection of the MPTP story, even if there are many points of divergence.
Finally, it’s interesting to note that both ALS-PDC of the Pacific and the Guadeloupe disease also have pathological features of ‘tauopathies’, or diseases associated with abnormal intercellular distribution of a protein called tau, which is normally found in conjunction with neuronal microtubules (a part of the cytoskeleton). In addition, one aspect of the neuropathy induced by annonacin is abnormal neuronal tau behavior. But a massively more frequent and consequential tauopathy is Alzheimer’s disease, so these findings raise the fascinating question as to whether environmental toxic agents might contribute to the burgeoning world-wide caseload of Alzheimer’s – and if so, how much, and under what genetic circumstances? The significance of such questions for public health in countries with increasingly ageing populations is obvious.
One point already alluded to above is the notion that a transient ‘hit and run’ exposure to a toxic substance might set up a continuous and actually self-perpetuating cycle of damage. Such a possibility could considerably complicate attempts to identify causative toxic agents. If a single short-live exposure (or transient set of exposures) to an agent can result in disease many years later, it is clear that fingering the original culprit becomes correspondingly more difficult. It remains a possibility that such effects are relevant to the cycad saga at least, but a more detailed consideration of this notion is a topic for a later post in this series.
In the meantime, a biopoly-verse rumination:
Bring genetics and host factors to view
Where some insidious poisons can brew
To stay and remain?
Or start off a chain
Of damage in an unfortunate few.
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘ A classic example in this regard is the insecticide DDT……’ (With respect to persistence in fat). See Turusov et al. 2002.
‘….arsenic (capable of replacing phosphorus) and thallium (capable of replacing potassium).’ With respect to arsenic, it is interesting to recall the recent controversy regarding ‘arsenical life’, where arsenic in a specific bacterium was reputedly replacing phosphorus (see a previous post for brief detail on this). Arsenic can compete with phosphorus when it is in the form of arsenate (See Kaur et al. 2011; and also Dani 2011 for a discussion of the biological significance of this). For more details regarding thallium and its competition with potassium, see Hoffman 2003.
‘….release of the metal component may simply liberate it for another cycle of inhibition. This can be overcome if a chemical agent (a chelator) is administered which is capable of tightly binding the metal, solubilizing it, and allowing it to be excreted. See Flora & Pachauri 2010; Jang & Hoffman 2011.
‘….potassium-40 (40K) …. has been proposed as a major source of natural mutation, although experimental results suggest that its contribution to mutation must indeed be subtle influence.’ See Gevertz et al. 1985 for more detail and a refutation of the importance of this radioisotope for mutation, at least in bacteria.
‘…..polonium-210 (210Po), …is present in tobacco smoke, and it has been attributed a major role in the generation of smoking-induced cancer….’ See Zagà et al. 2010.
‘ Polonium-210 has been in the news in recent years, through its use an exceedingly potent poison in the murder of the Russian Alexander Litvinenko…..’ Polonium-210 is an α-emitter (Helium-4 nuclei). While these emitted particles are relatively massive and poorly penetrating, they are very dangerous if an α-source has been ingested. Doses as little as 1 μg may be lethal in susceptible individuals, and doses of several hundred μg will be universally fatal. See Scott 2007. For more details on the Litvinenko case, see a BBC timeline article.
‘….polonium-210 can exert low-level effects if ingested in small enough doses.’ See also Scott 2007.
‘ The influence termed ‘cofactors’ ….. example is a putative requirement for the presence of simian virus 40 (SV40) for the generation of mesothelioma by asbestos….’ See Rivera et al. 2008; Qi et al. 2011. Note that SV40 was a contaminant of early Salk polio vaccine preparations (see Vilchez & Butel 2004).
‘….origins of Parkinson’s disease…..’ This disease (the ‘shaking palsy’) was first described in the early 19th century by Dr. James Parkinson (Thomas & Beale 2007), who thus bequeathed his name to it. Although obviously an eponymous title, the “Parkinson” is often now rendered with a lower-case ‘P’.
‘ These neurons are also pigmented…..’ Melanocytes, the cells in the skin which produce the pigment melanin responsible for skin color (along with the related pigment pheomelanin) are derived from the same embryological origins as neurons, the neural crest.
‘….a type of melanin (‘neuromelanin’)….’ Neuromelanin is chemically similar, but not identical to, the black melanocyte pigment, which itself is often termed ‘eumelanin’. See Zecca et al. 2001.
‘…..the source of the problem [Parkinson-like illness] was tracked down…..’ See Langston et al. 1983.
‘….widely reported in the scientific literature….’ For example, see an article in 1984 by Roger Lewin in Science, whose title (‘Trail of Ironies to Parkinson’s Disease’) speaks for itself.
‘…even found their way into popular fiction quite quickly….’ The well-known ‘new wave’ science fiction novel Neuromancer by William Gibson (Ace Science Fiction, 1984) features a particular scene where an individual is deliberately victimized by means of the nasty aspects of MPTP neurotoxicity. Since the book was first published in 1984, this was at the time a very quick uptake on a scientific and medical development.
‘ Those unmistakably victimized by MPTP had varying fates…..’ See Langston’s popular book (co-authored with Jon Palfreman), The Case of the Frozen Addicts (Pantheon, 1995). Also see a Wired magazine article.
‘…..a relative latecomer in 1817…..’ See the above note about James Parkinson.
‘….‘natural’ PD …. a toxic condition…?’ See Calne & Langston 1983.
‘….exposure to insecticides ….as a potential agent of PD …. not been firmly nailed down…’ See Brown et al. 2006.
‘ Most cases of sporadic PD occur later in life….’ Only 1-3% of total PD cases can be attributable to direct genetic causes (See Lorinicz 2006).
‘….MPTP itself is acted upon by a specific enzyme with the brain, monoamine oxidase….’ See Herraiz 2011 (a).
‘…..inhibitors of MAO enzymes are protective against the effects of MPTP…..’ Herraiz 2011 (b).
‘….also explains the high selectivity of MPTP (the precursor to MPP+) in its toxic action…’ For an early report on MPP+ uptake, see Javitch et al. 1985.
‘….it [MPP+] acts as a primary toxic agent towards mitochondria….’ For a little more detail on mitochondrial activity, see a previous post. For more on Complex I in general, and with respect to MPTP / MPP+, see Schapira 2010.
‘….epidemiologists have noted an unusual incidence ….ALS-PDC…’ For an entertaining account of the history of this topic, see The Island of the Colour-blind (Picador, 1996; Book Two, Cycad Island) by the famous neurologist Oliver Sacks. For a general overview of ALS-PD, see Steele 2005.
‘ Cycad flour fed to experimental animals…..’ See Shen et al. 2010.
‘ A specific amino acid …BMAA….has been repeatedly linked with cycad-induced disease…’ For a review and disputation of this, see Snyder & Marler 2011.
‘ Another contender is methylazoxymethanol….’ See Kisby et al. 2011.
‘….a specific compound from this fruit (annonacin) has implicated….’ See Champy et al. 2004; Lannuzel et al. 2008. Other compounds chemically related to annonacin have also been implicated: See Alvarez Colom et al. 2009.
‘…one aspect of the neuropathy induced by annonacin is abnormal neuronal tau behavior…’ See Escobar-Khondiker et al. 2007.
Next Post: This is the last post for 2011; will be back early next year.
From time to time, it will be appropriate to offer updates (or upgrades) of previous posts when it seems appropriate. In late March, I looked at ‘paradigm shifts’ in biological science, particularly in the context of so-called biological ‘dark matter’. Here a Table was provided with a list of some developments in recent bio-history which could qualify as paradigm shifts, especially against the current background where the meaning of a scientific ‘paradigm’ has been diluted in much of the literature. While this Table was not originally intended to be completely comprehensive, after the fact I have noted that a particularly important case was inadvertently overlooked. That is the subject of the current post.
The Chemiosmotic Hypothesis
Cellular processes require energy, and a universal energy ‘currency’ is the molecule adenosine triphosphate (ATP). It has been long recognized that the hydrolysis of ATP to the corresponding diphosphate (ADP) provides the free energy for driving a host of biological reactions. The synthesis of ATP itself is therefore of crucial significance, and naturally requires an energy source in order for this to be accomplished.
In 1961, a British biochemist by the name of Peter Mitchell published a paper in Nature outlining a novel proposal for the mechanism of the generation of ATP through the electrochemical properties established in certain biological membranes. These are found in prokaryotes, and also eukaryotes via their mitochondria (the ubiquitous organelles concerned with energy production) or chloroplasts (the plant cellular organelles mediating photosynthesis). Mitchell’s ‘chemi-osmotic’ hypothesis postulated that, rather than relying on an energy-rich chemical intermediary, oxidative phosphorylation (the synthesis of ATP from ADP occurring during respiration) was dependent on proton (hydrogen ion) flow across membranes. In essence, respiratory processes pump protons across an enclosed membrane boundary such that an electrical potential is generated across the membrane. Mitchell termed the ‘pull’ of protons back across the membrane as the ‘proton motive force’, or a proton current. This flow of protons could be directed through protein-mediated channels for the purposes of performing useful work.
Although now enshrined within the modern biochemical world-view, in the early 1960s this notion was quite radical, and not at all in tune with many of the ideas of most major researchers in the field at that time. In fact, it took over a decade a half before enough evidence was garnered to convince most remaining doubters. But Mitchell certainly had the last laugh, being awarded a Nobel Prize for his innovative proposal in 1978.
ATP Synthase and the Chemiosmotic Hypothesis
A remarkable catalytic complex at the core of ATP generation, the membrane-associated ATP synthase (ATPase), has had a central role in the ultimate acceptance of the chemiosmotic hypothesis. This resulted from studies on purified components of the synthase complex and reconstitution experiments, where directed proton flow across sealed model membranes (liposomes) was shown to be crucial for ATPase activity. In some ingenious experiments, the required proton flow was produced by the introduction of a protein involved with prokaryotic photosynthesis (bacteriorhodopsin) as a light-driven proton pump. (Other proton pumps from diverse biochemical sources could also perform similar roles). Such findings were subsequently reinforced by numerous structural and functional studies.
The ATPase has been revealed as a molecular motor driven by proton flow directed through the transmembrane (‘Fo’) component of the catalytic complex. The proton current is harnessed to provide energy for driving the physical rotation of the soluble (‘F1’) ATPase component, resulting in ATP synthesis at three catalytic sites. In some amazing cases of experimental virtuosity, this molecular rotation has been visualized in real time using fluorescent tags, and the association of rotation with ATP synthesis demonstrated by magnetic bead attachment to the F1 subunit, followed by artificial rotation induced by appropriate magnets.
The striking nature of the membrane-associated ATPase as a rotary molecular motor has inspired many offshoot thoughts and speculations. As a demonstration of a ‘natural nanomotor’, it would come as no surprise to hear that that the nascent field of nanotechnology has paid particular notice.
Why a Paradigm Shift?
So, it might be immediately seen that the proposal, experimental testing, and ultimate support for the chemiosmotic hypothesis is of great scientific significance, but is it really meaningful to refer to it as a paradigm shift? Well, yes, it is. Firstly, the initial resistance to this idea in itself is consistent with the view of shift in a paradigm requiring the upheaval and dismantling of an earlier view – if not by the death of an aging cadre of reactionary biologists, at least via their eventual accession to the concept through the accumulated weight of evidence.
But perhaps the most fundamental novelty of Mitchell’s ideas came from the inherent aspect of spatial organization of cellular structures in determining function, as he explicitly stated. In his own words, from his 1961 Nature paper:
“the driving force on a given chemical reaction can be due to the spatially directed channelling of the diffusion of a chemical component or group along a pathway specified in space by the physical organization of the system”.
In other words, structures on a cellular scale (membranes, in this case) can serve as a basis for directing biochemical reactions in specific ways, and this general effect has also been termed ‘vectorial biochemistry’. This view was a radical proposal in the early 1960s – and accordingly met with considerable resistance. In fact, cells are not just ‘bags of enzymes’, but partitioned in complex ways into different compartments, and this partitioning is very significant for specific functioning. This is particularly so (as we have seen) for bioenergetics.
The development of some form of membrane compartmentalization of proto-cells during the early stages of the origin of life is recognized as a major evolutionary transition. Its importance can be inferred from simple logic, since an evolving molecular biosystem could never undergo progressive selection and functional advancement were its components not restricted into a bounded spatial compartment. Dilution of reactants would otherwise rapidly remove any useful molecular innovations, and bring in potentially interfering molecules. Included among the latter are likely parasitic systems, whose unchecked activities would be a permanent stumbling block. But the long-term implications of the chemiosmotic principle show us that biological membranes are much more than just phospholipid sacks demarcating collections of biological molecules from the external environment. They are integral and essential parts of biological operations in their own right. And their evolution into these roles is a very ancient event in the history of life. Leaping from early biogenesis to future human aspirations, the importance of membranes and higher-level structures for vectorial direction of function should not be forgotten when artificial cell design is contemplated.
So Mitchell’s contribution is duly inserted into the original ‘paradigm shift’ Table thus:
It is also notable that this year marks the 50th anniversary of the publication of Mitchell’s seminal paper.
And finally, a biopoly(verse) salute to the pioneer:
The hypothesis chemiosmotic
Made Mitchell seem quirky and quixotic
But opinions revise,
And then a Nobel Prize
Sealed the field as no longer exotic.
References & Details
(In order of citation, giving some key references where appropriate, but not an exhaustive coverage of the literature).
‘……a British biochemist by the name of Peter Mitchell published a paper in Nature…’ See Mitchell 1961.
‘….Mitchell ….. awarded a Nobel Prize for his innovative proposal in 1978.’ See Harold 1978; also the Nobel organization site for the 1978 Chemistry prize. See also a relevant piece in Larry Moran’s Sandwalk blog. Mitchell died in 1992.
‘…..studies on purified components of the synthase complex…..’. A major contributor to these studies was Efraim Racker (1913-1991), A biographical memoir by Gottfried Schatz (National Academies Press, online) provides an excellent background to this and numerous related areas. Paul Boyer and John Walker also were pivotal in structure-function studies regarding ATP synthase, for which they received the Nobel Prize for Chemistry in 1997. For a very recent and comprehensive review of the membrane-associated rotary ATPase family, see Muench et al. 2011.
‘…..the introduction of a protein involved with prokaryotic photosynthesis….’ See Racker et al. 1975.
‘…..nanotechnology has paid particular notice….’ See Block 1997 (Article title “Real Engines of Creation”, which refers to K. Erik Drexler’s book Engines of Creation, a pioneering manifesto of the potential for nanotechnology – Doubleday, 1986). Also see Knoblauch & Peters 2004.
‘…..artificial cell design…..’ See a previous post on synthetic genomes and cells for more on this cutting-edge topic.
Next Post: Regrettably, work commitments enforce a temporary hiatus on biopolyverse posts until early December. But will return then!!
A considerable number of the recent series of posts have been concerned with molecules that can be referred to as drugs. It seems useful here to take a look at this from a semantic point of view.
Drugs at Different Levels
Most people carry around in their minds more than one specific meaning ascribed to the small word ‘drug’. If you hear “He’s a drug dealer” or, “She’s on drugs”, the references are not likely to be to antibiotics or blood-pressure medication. Conversely, the sentence, “My doctor prescribed a drug for me” is most unlikely to refer to crystal meth. As a result, the great majority of people realize (even if only intuitively) that the illegal band of drugs are but a subset of a much larger group, that includes substances both universally approved and potentially truly life-saving. It would then be reasonable to assume the definition of a ‘drug’ in this larger sense should be fairly straightforward.
Some standard dictionary references are along the lines of: “A substance used in the diagnosis, treatment, or prevention of a disease or as a component of a medication” or, “a chemical substance that affects the processes of the mind or body”. The US Food & Drug Administration (FDA) defines drugs with wording introduced by the Food, Drug, & Cosmetic Act of 1938, as “articles (other than food) intended to affect the structure or any function of the body of man or other animals” [Sec. 201(g)(1)]. These definitions are quite broad, especially the latter. But speak of ‘drugs’ with a pharmacologist, and small molecules are most likely to be the topic of conversation, and not just any small molecules. Indeed, the term ‘drug-like’ is frequently used in the general field of drug discovery to encapsulate (so to speak) the special features which a successful medicinal drug should embody. Obviously, a drug must have definable function(s), which means that it must be directed to a specific molecular target or a limited set of targets (very often, but by no means exclusively, proteins). But a number of additional properties are very important if the drug is to successfully survive in an active form long enough to be useful, and to find its way to the desired target when administered to a patient. For example, a simple set of guidelines for evaluating a candidate compound formulated by Lipinski has been termed the ‘rule of five’, owing to the recurrence of five (or multiples of it) in the definition of the useful range of properties to look for. Getting a drug to where it needs to go is the preoccupation of the burgeoning field of drug delivery, which now intersects in many cases with advances in nanotechnology.
These rules were designed expressly with reference to small molecules, since increasing molecular size is often associated with diminishing returns in terms of delivery, and sometimes physical properties such as solubility. But that is certainly not to say that large molecules cannot be useful pharmacological and therapeutic agents. In the previous post, it was noted that it has only been in very recent times (historically speaking) that large proteins (especially antibodies) could be garnered from the biosphere for useful human applications. In fact, (as also noted), antibodies have become a billion-dollar industry, especially where monoclonal or specifically engineered antibody variants are concerned. And these antibodies are often referred to as drugs, especially in lay usage. Though obviously moving beyond a tight pharmaceutical definition of ‘drug-likeness’, this is perfectly consistent with the above broad definitions, including that from the FDA. But there are some gray areas…..
Drugs and Category Overlap
It is quite clear that some classes of substances or preparations may have members which have dual drug and nondrug characteristics. One such case noted by the FDA is the field of cosmetics, where even more narrow types of products differ in this kind of duality. For example, shampoos may be seen as primarily hair cleansing, or cosmetic, preparations, and indeed many are simply that and no more. But certainly some have additional functions, such as treating fungal (dandruff) or louse infestations. In this case, specific compounds are added for the indicated medicinal specifications. Although it is obviously of practical significance for product safety and efficacy requirements that some preparations should be regulated and licensed as both drugs and cosmetics, here the relevant products are mixtures, and specific molecules are not doing functional double-duty. For example, an anti-dandruff shampoo may have many different components, but the most significant are the detergent (usually sodium lauryl sulfate, for the cosmetic washing function) and the dandruff inhibitor (specific compounds such as zinc pyrithione). In other words, we cannot speak of either of these individual compounds as having an overlapping drug / cosmetic function; it is only as mixtures (along with various other materials) that the product as a whole acquires this status.
Yet there are certainly well-defined cases where specific molecules share categorization as a drug in combination with other properties. A prime case in point is a contender for the title of oldest drug used by humans: ethyl alcohol, or ethanol. Its psychoactive and other physical effects clearly indicate its drug status, but ethanol can also be metabolized to yield specific calorific value. As such, it is then a food as well as a drug. This is straightforward, but some other areas of ‘foods’ are less so. One definition of a ‘food’ might focus on the ability of a substance to be digestible, or act as source of energy, but clearly this is not sufficient for a healthy diet. There are numerous nutritional ‘cofactors’ which are essential for human health, included among which are a number of inorganic elements (principally metals, but also some other trace elements), and a group of vitamins.
Where do vitamins stand with respect to drug classification schemes? As small molecules which act as organic enzymatic cofactors for catalysis (coenzymes), the defined vitamins are an essential human dietary requirement, owing to our inability to synthesize them. But since they are not directly digestible themselves, they are classified by the FDA as ‘dietary supplements’, which are encompassed within the broader area of foods, and not drugs. Vitamins, then, fall into the this category and therefore would escape labeling as drug materials, unless they were chemically altered from the natural forms. Most dictionary definitions of ‘food’ also include vitamins. Even so, in other quarters vitamins have been clearly depicted as drugs. One basis for doing so is that vitamins can clearly cure diseases – but since the relevant diseases are deficiencies of the vitamins themselves, this would seem to be a special case. As we have seen with alcohol, assignment of a specific compound as a food does not mean it cannot also be a drug. But clearly there is a difference here: vitamins are essential for life, while alcohol (whatever some people might say) is not.
The figure below depicts two separate classifications where the vitamins are considered either as drugs (A) or not (B):
Two depictions of drug categorization and its overlapping areas. These are not to scale in terms of the relative sizes of the respective groups, and are schematic only. ‘Food / nutrients’: This refers to subtances which directly provide energy, structural building blocks, or essential assistance with normal metabolic functioning. ‘Dietary cofactors’ in general include both vitamins and inorganic substances (such as essential metals). ‘Proteins / macromolecules’: Not all macromolecular therapeutic agents are proteins, as for example nucleic acid aptamers. A, Vitamins considered as drugs. Some cosmetic preparations contain vitamins, so vitamins are shown to intersect with the ‘drug-overlap’ region of all cosmetics. B, Vitamins excluded from classification as drugs.
Does this cover everything? Well, there is an additional broad grouping of substances termed ‘nutraceuticals’, a hybrid term from ‘nutrient’ and ‘pharmaceuticals’. A nutraceutical in principle can be any food source component with biological properties outside of direct nutrition, but many of the best-known examples are antioxidants. Included among these are phytoestrogen compounds, considered in an earlier post. Resveratrol in particular (see the relevant Figure from this same post ) has generated enormous interest for its observed anti-ageing effects.
Where do these compounds reside in the above figure? Although they are by definition associated with some kinds of foodstuffs, they are neither directly digested (as for proteins, and digestible carbohydrates and fats), nor required for essential metabolism (as for vitamins). Therefore, it is logical that they be considered a subset of the large drug category, outside of the macromolecular subregion, as shown above. These compounds can be identified, purified, synthesized, and administered independently of their original sources. In this respect, they are no different from any other small molecule natural products derived from the biosphere.
Drugs as Foreign?
Can drugs be thought of as molecules which are ‘foreign’ to the body to which they are administered? (In other words, compounds which are not synthesized by the human or animal which receives them). In a strict sense, this would include vitamins which are dietary essentials through the lack of synthetic machinery for their production by a host animal or person. But there are problems with this proposal, and vitamins themselves are a case in point. For example, although Vitamin C is essential for human health (scurvy resulting in its absence), rats, mice and numerous other species have no problem making their own. Is Vitamin C then a drug for humans (capable of curing scurvy) but not for rats?
And numerous human proteins can be administered under circumstances where they can be considered drugs. Antibodies are an interesting case in point. Originally, monoclonal antibodies were of murine origin owing to the technological requirements of their production. The xenogeneic (foreign) nature of these proteins resulted in the induction of immune responses against the monoclonal antibodies themselves, when they were given to patients. In more recent times, fully human monoclonal antibodies have been developed, in order to circumvent this very significant problem. Yet an antibody of this type is still not literally and totally ‘self’, since its specific combining site is generated by recombinational and mutational mechanisms such that its exact sequence is not directly encoded in the human germline.
But non-variable molecules both large and small also come into this picture. Think of human growth hormone, of value for treating some forms of dwarfism – and sometimes abused for the purposes of body-building. Numerous other proteins and small molecule hormones can also be cited – so the notion of ‘foreign-ness’ for drugs in general becomes untenable.
Drugs, Poisons, and Doses
Drugs have been termed ‘poisons that save lives’, which carries the implicit message of the importance of dosage. But stating that ‘all drugs are poisons’ may be technically correct at a broad enough level, yet not particularly useful, given the vast differences in dosage ranges for efficacy vs. safety seen with different therapeutic compounds. Here a balance or ‘window’ must be found between the two poles of beneficial drug activity and unacceptable toxicity. The old saying ‘the treatment was successful, but the patient died’ provides an ironic testament to this inherent dilemma of drug pharmacology.
As an example of the great range of drug therapeutic windows which can exist, consider the treatment of syphilis. The pioneer of chemotherapy, Paul Ehrlich, found an arsenical compound (Salvarsan) which became an effective treatment for syphilis through its activity against the bacterial causative agent Treponema pallidum. But its toxicity at therapeutic doses was a major problem encountered in a high percentage of patients, so it was clearly not ideal. When penicillin became available in the 1940s, it was not only highly effective but also associated with very low toxic side effects. Indeed, the rising problem of bacterial resistance was initially countered by simply increasing the dosage of penicillin (or its many derivatives) without problems – but of course this soon becomes ineffectual as resistance increases. (Penicillins can actually induce serious problems through allergies in a minority of people, but this is quite distinct from direct toxicity).
There is also a piece of folk-wisdom along the lines of ‘too much of anything can hurt you’, which is certainly true for some natural nutritional requirements as well as drugs. In a general sense, too much food is clearly bad through the development of obesity, but the ‘dosage’ effects of nutrients can be observed in a much more specific manner. We can look within the set of vitamins once more for useful comparisons, which also demonstrate similar variation in the ‘safety’ windows of dosage as seen within artificial drugs. Vitamin C has exceedingly low (if any) toxicity, for example, and some people have routinely taken very high doses of it for long periods as part of ‘megavitamin’ therapy. On the other hand, the fat-soluble Vitamins A and D are unquestionably highly toxic when taken in excess of recommended daily requirements.
The dosage effect can also be related to the above observation that drugs need not be alien to the biochemistry and physiology of the patient (or animal) undergoing treatment. A pathology caused by a deficiency in a specific molecule can be corrected through artificial intervention. Conversely, certain pathological states may benefit from the provision of ‘unnatural’ administration of normal bodily proteins, such that the circulating amounts of the factor of interest are maintained for therapeutic purposes at higher levels than would normally be the case.
After this short foray into some issues surrounding the meaning of drugs, I’ll conclude with references to ‘nutraceuticals’ once more, by means of a biopoly(verse) note.
By analyses really quite shrewd
On mixtures both complex and crude
Smart chemists have shown
(And now it is known)
Natural drugs exist in some food.
References & Details
‘….. (FDA) defines drugs….’ For FDA definitions of both drugs and cosmetics, see the relevant page of the FDA site.
‘….set of guidelines for evaluating a candidate compound formulated by Lipinski…’ For a discussion of the basis of the Rule-of-five and moving beyond it, see Zhang & Wilkinson 2007.
‘…..field of drug delivery, which now overlaps with advances in nanotechnology.’ For a recent review of this topic in the cancer field, see Chidambaram et al. 2011.
‘….sodium lauryl sulfate….’ Also known as sodium dodecyl sulfate, this detergent also has wide application in laboratories as well as cosmetics.
‘…..a contender for the title of the oldest drug used by humans….’ Often alcohol is definitively cited as the oldest drug. I call it ‘a contender’ here since (as noted in an earlier post), the use of botanical medicines is also very old, and can even be linked with primate behavior (see a previous post on zoopharmacognosy). On the other hand, the use of alcohol (or abuse, depending on one’s views) is possible simply from natural cases of fruit fermentation, also seen with animals. So the origins of alcohol sampling by humans need not require any technology, and is undoubtedly of great antiquity.
‘……ethanol can also be metabolized……’ For an example of the differing influences of food vs. drug effects in an animal system, see Dole et al. 1985.
‘…..classified by the FDA as ‘dietary supplements’….’ For the FDA definitions of dietary supplements, see the relevant page of the FDA site.
‘…..vitamins can clearly cure diseases…..’ This was noted by Tulp et al. 2006, and that vitamins were thus ‘drugs by any definition’. Taken literally, this is clearly incorrect (one can simply exclude vitamins from a drug definition as dietary supplements, as for the FDA).
‘…..nucleic acid aptamers….’ (Figure footnotes). See a previous post for a brief consideration of RNA aptamers. From large libraries of variants, RNA molecules can be selected to bind desired ligands, and this can be used therapeutically. The first therapeutic aptamer (‘pegaptanib’) was directed against a specific form of vascular endothelial growth factor, for the treatment of ocular vascular diseases. See Ng & Adamis 2006.
‘……an additional broad grouping of substances termed ‘nutraceuticals’……’ For example, see Tulp et al. 2006.
‘……Resveratrol in particular …… has generated enormous interest for its observed anti-aging effects……’ See Pezzuto 2011.
‘……Vitamin C is essential for human health (scurvy resulting in its absence), rats, mice and numerous other species have no problem making their own.’ See Martí et al. 2009 for more information. The production of Vitamin C from glucose requires the enzyme L-gulonolactone oxidase, which humans, primates, and guinea pigs lack. Lachapelle & Drouin 2011 look at when this occurred in evolutionary time.
‘…..its exact sequence is not directly encoded in the human germline.’ Antibodies are composed of constant and variable regions, where the variation of the latter accounts for the vast range of different antibody binding specificities which can be induced by immunization. Particular variable region sequences allowing antigen recognition are specific to that immunoglobulin molecule, and are termed an ‘idiotype’. See Searching for Molecular Solutions Chapter 7 for a more detailed discussion of this.
‘ Numerous other proteins and small molecule hormones can also be noted…..’ Proteins such as interferons were noted in the previous post. Small molecules include adrenalin, thyroid hormones, and natural steroids. In all such cases, though, there is the potential (realized in many cases) for rendering such molecules ‘non-natural’ by various forms of artificial tinkering to improve their performances as drugs.
‘….pioneer of chemotherapy, Paul Ehrlich…’ See Thorburn 1983 for some biographical and other relevant information.
‘……certain pathological states may benefit from the provision of ‘unnatural’ administration of normal bodily proteins…..’ Again, see the reference to the example of interferons in the previous post.
‘ Vitamin C ….. ‘megavitamin’ therapy.’ The Nobel Prize-winning chemist Linus Pauling was a notable proponent for the efficacy of large Vitamin C (ascorbate) doses for conditions ranging from viral infections to cancer. (For example, see Pauling & Moertel 1986). However, no experimental evidence has validated these claims.
‘…..fat-soluble Vitamins A and D are unquestionably highly toxic….’ It is notable that the livers of certain polar animals (including bears and seals) are very rich in Vitamin A, and the eating of such livers by polar explorers has resulted in Vitamin A poisoning (hypervitaminosis A). See Rodahl & Moore 1943.
Next Post: Two Weeks from now.
It was noted in the previous post that most natural molecules of use to humans (‘chrestomolecules’ ) have traditionally been of low molecular weight, with the exception of simple biopolymers. Here it is time to have a brief look at the scope of large molecules and even larger biological systems which can be sampled from the biosphere and usefully applied to human activities.
Big Uses for Big Molecules….
Large biomolecules such as proteins can obviously act as nutrients, but this special category of ‘usefulness’ is not the focus of interest here. Indeed, in this context it should be emphasized that the benefits of consuming a protein as a foodstuff for any organism (humans included) result from the acquisition of its amino acid building blocks. ‘Digestion’ is intimately associated with breaking down consumed nutrients into components which an organism can employ for its own biosynthetic requirements. The original three-dimensional structure and function of the consumed protein is clearly irrelevant for such a nutritive purpose. But when we are considering the utility of large biomolecules for other human needs, the protein shape and function is crucial.
Biotechnological developments in the past century or so have extended the field of molecular utility to include a diversity of proteins (noted in part of in the ‘chrestomolecule’ Table of a previous post). The great majority of these can be grouped into the functional categories of natural biocatalysts (enzymes) and natural specific binding molecules (antibodies), but some additional cases can be noted:
Let’s take a quick look at some specific examples among these large protein sets:
Looking for Enzymes in All the Right Places
Many enzymes from a variety of biological sources are used in industrial processes, and several examples are of particular interest from a molecular biological point of view. There is at this time considerable interest in biological resources obtainable from ‘extremophile’ micro-organisms, or literally ‘lovers of extreme environments’. Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure, and bacterial biosystems mediating metabolic processes and growth must in consequence be selected for tolerance of each type of extreme conditions. Only in quite recent times has it become recognized that the earth harbors an entire thermoresistant prokaryotic ecosystem which lives by chemical energy independent of solar radiation. These organisms may be found in geothermal hot springs (as in Iceland or Yellowstone National Park), at deep underground sites or at thermal vents in the deep oceans.
Thermophilic (heat-stable) enzymes in general have many applications, but probably the most famous thermophile application in molecular biology is the use of the DNA polymerase from the hot-spring bacterium Thermus aquaticus, or Taq polymerase. (DNA polymerases are of fundamental importance for the copying and replicating of an organism’s DNA during cellular growth). The hardy Taq polymerase has made the polymerase chain reaction (PCR) a practical proposition, which has had an enormous impact in all areas of this and related sciences and gained its originator (Kary Mullis) a Nobel Prize. PCR involves replication and amplification of a DNA duplex in the laboratory through successive cycles of strand denaturation, association on each strand of specific primer oligonucleotides (short DNA segments), and primer extension (strand replication) with the DNA polymerase. Since strand denaturation is achieved by heating to 95˚ C or greater, a conventional polymerase present in the reaction is ‘killed’ and must be (very tediously) re-added for each cycle. A thermostable DNA polymerase, however, can be maintained throughout the course of the reaction. Although many other such heat-resistant polymerases are compatible with PCR, Taq polymerase is still the most widely used. At the other extreme from thermophilic organisms are psychrophiles, which thrive at very low polar temperatures, and are also potential sources of novel enzymes for novel applications.
In the ‘chrestomolecule’ Table of a previous post, antibodies (immunoglobulins) were featured. These important effector molecules of the mammalian adaptive immune system can be regarded as natural binding molecules produced ‘to order’ in response to specific stimuli (usually threats from invading external organisms), by diversification and selection mechanisms. Their applications are many, but can be divided into in vivo tasks which relect the original biological role for antibodies, or in vitro roles. Even naturally-formed antibodies within an individual are obviously useful if they mediate recovery from microbial infections, and the production of antibodies within a host has long been artificially directed by means of vaccines. Relatively recently compared to the first use of vaccination (attributable to Edward Jenner and cowpox immunization against smallpox), passive immunity has emerged as a therapeutic tool. This can be effected by transfer of whole serum or serum components rich in immunoglobulins from immune animals or humans to non-immune recipients, for the purposes of their protection from sudden exposure to a toxic or infectious environmental agent. With passive immunization even with crude serum fractions, antibodies qualify as examples of large natural ‘chrestomolecules’. But in modern times, passive immunization goes far beyond this.
This essentially began with the discovery of monoclonal antibodies in 1975, and has led to the vast field of antibody engineering. The manipulation of antibodies has wide therapeutic applications, and products based on antibodies have become a billion-dollar earner for the biotechnology industry. But engineered (and conventional) antibodies have many uses in vitro as well, as noted above. These include diagnostics and sensors, and for a variety of purification purposes in research and industry. In its range, this topic really requires further attention in a later post. But before moving on, it’s worth noting that the fields of antibodies and enzymes are not mutually exclusive. For a couple of decades, it has been known that antibodies can be raised such that they catalyze specific chemical reactions. This is possible through clever immunization strategies, where host animals are immunized with analogs of chemical transition-state intermediates of enzymatic catalysis. Even so, the best artificially-generated catalytic antibodies fall short of the efficiencies observed with natural protein enzymes.
And Other Proteins….
Although antibodies remain the star players, the adaptive immune system can contribute other useful large molecules. The key adaptive receptor of the cellular arm of the vertebrate immune system is the T cell receptor, signaling through which activates various different classes of effector T cells. Engineered T cell receptors in a number of forms have application in some cutting-edge technologies for human therapies. Some other proteins which modulate the functions of immune and other cells also have therapeutic and commercial significance. Included among these are interferons, known initially for their antiviral properties, but also for their immunomodulatory activities.
Beyond the immune system, various other proteins are mostly important through their contributions to knowledge, which have enabled the subsequent design of other useful structures. For example, the zinc-finger protein motif mediating DNA-binding has been intensively studied, allowing the derivation of a ‘code’ for binding and the generation of artificial binding motifs towards virtually any desired DNA sequence of 18 base pairs. When teamed with a nuclease domain, such tailored ‘zinc finger nucleases’ have tremendous potential in a number of clinical contexts. In different areas, numerous proteins have contributed their structural frameworks themselves for useful applications. This principle has been used for development of protein frameworks which can replace immunoglobulins for the specific recognition of desired binding targets.
….and even Bigger Higher-Level Useful Systems
By analogy with the introduced term ‘chrestomolecule’, an economically useful biological system (involving numerous mutually interactive molecules) could be termed a ‘chrestosystem’. But if we introduce biosystems into this picture, there are many levels of complexity that might apply. Does a whole organism thus qualify? Is even a domestic mammal a high-level ‘chrestosystem’? (After all, such animals have had many uses over the millennia in addition to being raised as food sources). And downward from there, certain fungi, yeast, and bacteria have long had uses in the modification of foodstuffs, long before they were even recognized as living organisms.
Perhaps cows (and so on) might indeed be viewed as simply highly complex organized systems in the service of humanity, but for the present purposes it will be most constructive to restrict our focus onto viruses. Viruses are usually considered as life-forms, and often have sophisticated regulatory controls, but are nevertheless many orders of magnitude less complex than a large multicellular organism. In their simplest forms, they can be regarded as small nucleic acid genomes packaged within a protein coat, and thus have a relatively small set of macromolecules which collectively allow them to infect their target cells and reproduce. So it is worth considering how some viral systems have useful properties for artificial exploitation. These will be grouped into bacterial viruses (bacteriophages, often referred to simply as ‘phage’) and a select group of animal viruses:
The ‘other viral’ categories are relevant examples, and not intended to be exhaustively comprehensive. (Many other cases exist).
Phage For This Age
Viral parasites of bacteria and other prokaryotic organisms, phage in general are extremely diverse in structure and genetic organization. Phage must attach to their specific prokaryotic host and ensure that their genomes (single or double-stranded DNA or RNA) enter the host cell. Usually the result of this is viral hijacking of the host and its conversion into a phage factory, ultimately leading to host cell destruction (lysis) and liberation of a large number of new phage particles. In broad outline, or course, this is a universal propagation strategy used by viral parasites across all kingdoms of life. Sometimes phage can be carried by their hosts indefinitely by a process termed lysogeny, where the phage integrates into the host chromosome or is carried as an independent replicating genome (plasmid replicon). Study of these and other features of phage biology has yielded a wealth of productive information. Owing to the relative simplicity of phage genomes, it is in fact no exaggeration to state that the rise of molecular biology itself derives largely from phage studies.
Phage have been major contributors to numerous biotechnological advances, and providers of information of general molecular biological significance. These viruses of prokaryotes can also be used as specific information sources potentially leading to novel therapeutics. In the case of pathogenic bacteria, systematic study of their phage has been shown to be a useful approach towards identifying phage-expressed proteins or peptides directed against bacterial host cell components. Identification of both the phage products and their host targets allows screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves. Owing to the specificity of phage interactions with bacterial hosts, drug-modified phage have also been considered as vehicles for drug delivery to desired target cells. Modified phage have also been tested as vectors for transfection of mammalian cells with protein or nucleic acid ‘payloads’.
The use of bacteriophage in the never-ending war against bacterial pathogens does not stop with phage protein or nucleic acid components. Perhaps inspired by the dictum, ‘the enemy of my enemy is my friend’, phage themselves have long been considered as therapies for a variety of bacterial diseases. The idea is simple enough: kill infecting bacteria with their own parasites. Since phage are specific to their bacterial hosts and cannot replicate in eukaryotes, safety also is not likely to be a problem. Yet the history of ‘phage therapy’ has seen conflicting claims of efficacy, although many successful treatments have been reported. In recent times there has been renewed interest in the potential of phage therapy, accompanied by the rise in antibiotic-resistant bacteria and a slow-down in the pipeline of new replacement drugs. The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves. The unique feature of phage used in this manner is that they are self-replicating, indeed the first therapeutic agents in this category. Their capacity for self-replication in turn is directly linked with the population of their target hosts, which gives the pharmacokinetics of phage therapy some unusual aspects. It has been suggested that inadequate understanding of these novel pharmacokinetics is a major source of the mixed results previously seen with trials of phage therapy, and due attention to this detail may well shift phage therapy into a mainstream alternative to antibiotics. This is especially likely in cases of bacterial infections which are resistant to the current antibiotic armamentarium. Apart from direct therapeutic applications, phage have also been considered for environmental use in the reduction of bacterial loads in human-derived wastewater.
Another interesting feature of the exploitation of phage as whole viruses is that they are not a one-molecule ‘magic bullet’, and are not categorizable as ‘molecular solutions’ in a the sense of involving a single molecular entity. (Certainly a phage could not be deemed a ‘chrestomolecule’, although its subunit molecules could if they were of value in isolation). Phage often use elegant genetic systems for their regulation, and also show highly efficient and compact biological organization. (For example, the phage ΦX174 provided the first natural demonstration of overlapping genes). But despite such layers of underlying organizational complexity, phage have simplicity at the level of the types of their molecular constituents, all consisting of their informational nucleic acids (genomes) protected by a protein coat (capsid), combined with an entry system into their specific hosts. It then follows that the use of phage (whether natural or engineered) as therapeutic agents is an early example of exploitation of a (replicating) supramolecular system.
A huge abundance of as-yet uncharacterized phage are still to be found in the natural environment, and if past experience is anything to go by, such phage will provide rich fields of novel biodiversity. In an extension of the earlier overview (in the previous post) of the bounty of marine bioproducts, marine bacteriophages have been cited as the largest reserve of untapped genomic information. In support of this contention is the observation that such phage have a very high proportion of unassignable open reading frames (predicted proteins which cannot be recognized as belonging to current known protein families). Current high-throughput sequencing technologies not only rapidly reveal new phage genes but allow high-altitude phage genomic comparisons, which are greatly facilitating studies of phage origins and evolutionary processes.
Other Viruses, or: What’s HIV Ever Done For Us?
Many viruses afflicting humans are infamous for the terrible toll they take on human lives, and for the resulting human suffering. Yet certain features of animal viruses can be harnessed towards desirable ends, especially their proficiency for targeting and invading specific host cells. As weapons against cancer, specific viruses have been modified and used to promote beneficial immune responses against cancers or infectious agents. Alphaviruses (a viral family which includes human encephalitis viruses) have been used as vectors to promote directed anti-tumor immune responses. Alternatively, viruses can be designed to directly kill (lyse) tumor targets, with viral examples including herpesviruses and poxviruses. (Obviously, in all such cases, it is of great importance that the introduced genetic modifications inactivate the ability of the virus to damage normal cells).
Even the human immunodeficiency virus (HIV), the causative agent of AIDS, has had something to offer. As a member of the lentivirus family, HIV has the useful property of infecting non-dividing cells, which many other viruses cannot accomplish. Vectors have been derived from ‘gutted’ versions of the HIV genome which are unable to complete the usual replication cycle, but retain the ability to insert a copy of the viral RNA as a DNA sequence in the host genome. These features render them an attractive means for transferring desirable gene sequences to recipient cells of interest. For example, such lentiviral vectors have been used to transfer specific T cell receptors to T cells from human cancer patients. In such cases, the novel engineered T cell receptors are designed to recognize tumor-specific antigens, and allow the transduced T cells to then recognize and kill tumor targets. The lentiviral vector-mediated gene transfer process is also useful for gene therapy applications.
It is interesting that a pathogen as serious as HIV, the destroyer of tens of millions of human lives since its full-scale emergence in the early 1980s, can ‘give something back’ by way of contributing a useful system for human application. Not that this would compensate its victims, of course, but the very fact that anything positive can be gleaned from the HIV experience says something about the human ability to recognize natural opportunities when they arise. There are many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease, but these have only been made possible through technology. In many forms, modern biotechnology provides both the means to perceive useful biological opportunities (whether from pathogens or otherwise), and also pathways towards their beneficial application.
Homage to Nature, and then Moving Beyond It
In the molecular domain, Nature’s bounty is translatable into human benefit at several levels, as we have seen. There is firstly of course the direct discovery and application of low molecular weight bioproducts (detailed in the previous post), which has been done for thousands of years. The data provided by specific chrestobiomolecules can be used as a stepping-stone to even more effective agents, through successive rounds of human design intervention.
So, in the long run, the most important thing that large natural biomolecules and biosystems provide to us is information – information that acts as a spring-board towards the development of our own complex molecules and biosystems which are tailor-made towards specific applications. When this process is taken to its logical conclusion, we will reach a stage when we supersede the need to use such incredibly useful biological resources.
The use of higher-level systems in the future will thus inevitably become less a slavish derivation or copying of a natural precedent, and more and more an artificial improvement for specific design ends. This trend then increasingly overlaps with the field of synthetic biology, considered in several previous posts (30th May, 7th June, 20th June ).
Finally, a little comment on big molecules:
About natural products, one should recall:
They go far beyond molecules that are small
And think, if you please
That included in these
Are some considered the most useful of all.
References & Details
‘ Certain bacteria thrive under amazing extremes of temperature, salinity, pH or pressure…..’ See Van den Burg 2003.
‘…..earth harbors an entire thermoresistant prokaryotic ecosystem…….’ See Gold 1992.
‘ Thermophilic (heat-stable) enzymes in general have many applications….’ See Haki & Rakshit 2003.
‘….the polymerase chain reaction…’ For more details, see the accompanying ftp site for Searching for Molecular Solutions, for Chapter 4, Section No. 6 (‘PCR’).
‘….natural binding molecules produced ‘to order’ …….by diversification and selection mechanisms…..’ In brief, somatic diversification within immune cells of an vertebrate with an adaptive immune system results from combinatorial genomic rearrangements and somatic hypermutation, coupled with selection and amplification processes for cells uniquely expressing receptors recognizing a target antigen. This is also described (and referenced) in Chapter 3 of Searching for Molecular Solutions.
‘…..passive immunity has emerged as a therapeutic tool…..’ Among a vast number of examples can be found ‘antivenoms’ produced in horses or goats against various natural toxins from snakes, spiders, jellyfish, and other toxic organisms.
‘…..discovery of monoclonal antibodies…..’ See Köhler & Milstein 1975.
‘…….products based on antibodies have become a billion-dollar earner…..’ See Reichert et al. 2005.
‘……Engineered T cell receptors in a number of forms have application….’ More details (and references) and provided in the Cited Notes for Searching for Molecular Solutions in the accompanying ftp site, for Chapter 7, Section 18, T Cell Receptor Technology and Applications.
‘….interferons, known initially for their antiviral properties, but also for their immunomodulatory activities….’ Modified isoforms of interferon-alpha have been widely used (in conjunction with antiviral drugs) for treatment of Hepatitis C (See Tsubota et al. 2011); interferon-beta has been used as a therapy for the autoimmune disease multiple sclerosis (See Rudick & Goelz 2011).
‘…..development of protein frameworks……’ This is considered in Chapter 7 of Searching for Molecular Solutions.
‘……the rise of molecular biology itself derives largely from phage studies…..’ See Cairns, J., Stent, G. & Watson, J. Phage and the Origins of Molecular Biology. (Cold Spring Harbor Laboratory Press, 40th Anniversary Edition, 2007).
‘…….screening systems to be established for finding low molecular weight chemical inhibitors of the host cell targets themselves…..’ See Liu et al. 2004.
‘….drug-modified phage have also been considered as vehicles for drug delivery….’ See Yacoby et al. 2007.
‘……the history of ‘phage therapy’ has seen conflicting claims of efficacy…..’ See Sulakvelidze 2005.
‘……The great diversity of phage has been suggested to be superior to the diversity of natural antibiotics themselves…..’ See Schnaitman 2002.
‘…..the pharmacokinetics of phage therapy [has] some unusual aspects…….’; ‘……inadequate understanding of these novel pharmacokinetics is a major source of the mixed results….’ See Payne & Jansen 2003; Levin & Bull 2004.
‘…….phage have also been considered for environmental use…….’ See Withey et al. 2005.
‘…….the phage ΦX174 provided the first natural demonstration of overlapping genes…..’ When genes ‘overlap’, portions of their coding sequence occupy the same DNA tract, but in different reading frames. For the ΦX174 precedent, see Barrell et al. 1976.
‘…….marine bacteriophages have been cited as the largest reserve of untapped genomic information…….’ See Paul & Sullivan 2005.
‘……Alphaviruses …… have been used as vectors to promote directed anti-tumor immune responses.…..’ See Avogadri et al. 2010; ‘…..viruses can be designed to directly kill (lyse) tumor targets (including herpesviruses…..’ See Todo 2008; ‘……and poxviruses…..’ See Kirn & Thorn 2009.
‘…..many other examples of ‘positives’ accruing from dangerous viral and bacterial agents of disease…..’ This general issue was discussed in the Searching for Molecular Solutions associated ftp site, in the Cited Notes for Chapter 3, Section No. 4 (DNA-binding protein design).
Note: If problems are encountered in directly accessing the above ftp site, go here, and then click on molecular-solutions.zip (8.11 Mb).
This post and the succeeding one are the culmination of a recent theme dealing with ‘Natural Molecular Space’ . This series has looked at the biology of natural products, and also their use by both animals and humans from the earliest of times. Here the same activity in the modern world is considered.
Natural ‘Chrestomolecules’ Now vs. Then
An earlier post discussed the significance of ‘chrestomolecules’, or useful molecules of economic significance (in its broadest sense). This survey did not discriminate on the basis of any molecular properties, and a range of different categories of such molecules was listed. Yet ‘natural molecular space’ as considered in several previous posts (19 July, 26 July, 9 Aug, 16 Aug) has been preoccupied with small molecules. How does the “old” (traditional) vs. “new” (modern) use of all molecules from the biosphere stack up if viewed through a size-based lens?
Notes for diagram: This compares traditional and modern use of all bioproducts. The traditional group is encompassed as a subset of the wider modern group, with Nutrients as a special category. Sources based exclusively on small molecules are shown in blue; those based on either large or small molecules are shown in red, and those based on large biomacromolecules (mostly proteins and nucleic acids) shown in orange. ‘Repetitive biopolymers’ refer to molecules of biological origin whose structures are usually highly repetitive polymers based on a limited number of subunits, such as certain polysaccharides. ‘Therapeutics’ is placed in both Traditional and Modern groups, owing to the use of many small natural biomolecules within each, but therapeutic antibodies only within modern times.
The above diagram uses the categories found in the chrestomolecule Table of a previous post, and sorts them into two broad levels based on their histories of usage. The main message is the preponderance of small molecules in traditional applications. With the exception of certain biopolymers of relatively regular (and often linear) structures, the traditional group does not feature complex proteins and other macromolecules which are well-represented in the modern set. Why should this be the case? Clearly, knowledge and technology (only available from relatively recent times) make a big difference, in allowing the exploitation of the properties of large and complex molecules. Also many small molecules have an inherent major advantage over potential protein therapeutics in terms of their oral delivery potential. Traditional societies could not provide intravenous delivery by hypodermic syringes, and protein or peptide-based substances usually fare poorly in making transit through the acidic digestive barrier of the stomach.
So, this size-wise breakdown will be used as a divide for the coverage the modern use of the biosphere: This post will feature the modern harvesting of natural small molecules, and the next will concentrate on the modern use of natural large molecules, and indeed entire useful molecular systems.
What other general differences might then exist between the (very) old and the (relatively) new, in this context? As a broad principle, one could note that modern science, technology, communications and mobility provide the ability to initiate systematic screens of a wide variety, with increasing refinements as to exactly how they are performed. One consequence of this is the ongoing exploitation of marine environments for natural products, which were almost untouched during ancient times. This theme is looked at in a little more detail below. Another area unique to modern times might be summed up by the phrase ‘getting more from what’s on offer’, or using natural drug precedents to the best advantage through technological developments.
Harvesting of Small-Molecule Natural Molecular Biodiversity in Modern Times
At this point we have considered natural molecular space from a number of viewpoints, including its functions, evolution, classification, and empirical exploitation by traditional tribal medical lore. This leads us to a central issue directly relevant to the theme of this post: How important do natural products as a whole remain for human needs, to what extent have they been replaced by other technologies, and what are future trends in this area of biotechnology? Or using a previously-introduced terminology, what fraction of chrestomolecules now and in the foreseeable future will derive from natural biomolecules?
Beyond two decades ago, a majority of pharmaceutical drugs derived from natural product sources, but by the 1990s the fraction of drugs directly or indirectly originating as natural biomolecules stood at about 50%, owing to advances in synthetic chemistry and combinatorial screening. Nevertheless, the fraction of naturally-derived pharmaceuticals varies considerably if specific therapeutic applications are considered. Over the time-frame 1981-2006, over 70% of cancer drugs have been cited as non-synthetic in origin, and naturally-derived drug percentages increase in all therapeutic categories if one includes semisynthetic analogs based on the original core structure of the biomolecule. In this area, the use of natural product molecular scaffolds for the future design of antibiotics has been promoted. Natural products remain highly regarded for their diversity and as source of novel structural motifs. Generalizations of the properties of natural product molecules have shown significant average differences to synthetic molecular libraries, including steric complexities, atomic contents, and ring structures. As considered in a previous post, the evolutionary origins of natural products may provide a positive bias in favor of their utility, at least as a source of novel molecular scaffolds. Even among plant sources alone there is still a huge range of material remaining to be investigated by systematic ‘bioprospecting’.
In modern times, conscious attempts have been made to harness traditional ethnic knowledge of therapeutic natural products, with the aim of accelerating drug discovery. As noted previously, these kinds of studies have been termed ‘ethnobotany’ (given the preponderance of plant products involved) or more generally ‘ethnopharmacology’, and the advent of a number of important drugs have been attributed to the transfer of ethnopharmacologic knowledge. (The example of quinine, and the complexities associated with Europeans’ awareness of it, was considered in the previous post). The continuing value of ethnopharmacology has been vigorously promoted by certain researchers. Throwing cold water on this enthusiasm to some extent, over a twenty year period the US National Cancer Institute (in the course of systematically screening very large numbers of plant extracts) did not find useful anti-cancer drugs specifically from ethnobotanical information, with the possible exception of the anti-cancer drug taxol.
Commercial interest in ethnopharmacology in recent times has led to the formation of companies dedicated to mining such potentially valuable knowledge as the basis for a drug discovery platform. One much-publicized example is the now defunct Shaman Pharmaceuticals, but numerous companies have had at least a passing interesting in drug acquisition by such means. A highly contentious issue has been the rights of indigenous peoples to compensation if their information led to a successful profit-making venture, and accusations of ‘biopiracy’ have been made towards many Western drug discovery activities in environments used by native people. These kinds of political, legal and ethical issues have clouded or retarded a number of relatively recent bioprospecting ventures. Despite the importance of these considerations, a more fundamental problem is the continuing destruction of rainforests and other natural habitats, which threaten to result in irreversible losses in biodiversity and rich sources of novel biomolecules. Associated with this, and the forces of cultural homogenization, loss of tribal languages and lore are also lamentable outcomes not only in their own right but as a potential source of ethnopharmacological information. This is an absolute loss if the ethnic group is pre-literate, but if old written records exist they may possibly be tapped for such irreplaceable knowledge.
A Natural Frontier in the Sea
In contrast to terrestrial environments, the seas and oceans have not yielded a large number of notable traditional medicines, or an associated rich ethnopharmacologic folklore. This is undoubtedly due to the relative inaccessibility of most marine environments without comparatively recent technological back-up, and marine bioproducts are thus greatly under-represented in traditional medicinal tool-kits. Even peoples with close access to the sea, such as the Samoans, appear to have derived most of their traditional ethnopharmacological lore from land plants. The environmental ‘diversity factor’ D noted in the previous post for traditional drug discovery is in turn reduced in practical terms by the inability to recognize and ‘fish’ the oceans for useful molecules. Thus, the marine environment has been poorly exploited as a source of drug discovery until recent times, despite it bearing a plethora of a potentially useful and highly diverse organisms with equally diverse biosynthetic capabilities. Or possibly even more; it has been claimed that at this juncture in history, searching marine natural molecular space is much more likely to yield novel biodiversity than land ecosystems.
Sponges alone have proven to be rich in a variety of bioproducts with promising applications. Useful metabolites, possibly with antimicrobial properties, may be obtained from seaweeds or marine cyanobacteria. Conotoxins (from cone shell molluscs) from approximately 500-700 Conus species are a highly diverse family of peptides with neurotoxic activities. These find many important applications in neurological research and possibly in a number of therapeutic contexts, at least as prototype molecules pointing towards pathways for future drug development. Sea hares, marine molluscs which have found productive application in research on memory mechanisms, are yet another source of useful products, including antibacterial proteins. Tunicates (sessile marine invertebrates) have yielded chemically diverse cytostatic and cytotoxic drugs with potential applicability in clinical oncology. Given the continuing unmet need for effective anti-cancer treatments, and the very large international market which successful cancer therapies can fill, it is not surprising that commercial interest has been stimulated towards marine natural products with potential for this kind of activity. Numerous clinical trials for the anti-cancer efficacy of certain marine bioproducts have been conducted and are continuing.
In a number of cases of apparent production of useful compounds by marine invertebrates, the true source may be commensal micro-organisms carried by the invertebrate organism. Combining this observation with the relatively poor knowledge base concerning marine microbes, and their ancient and robust variety, assessment of marine microbial populations accordingly deserves high priority for the analysis of oceanic biodiversity. A high-profile expedition launched with this end in mind has been the ‘Global Ocean Sampling’ voyage, under the aegis of J. Craig Venter, his eponymous Institute, and other participants.
More Bang for Your Natural Product Buck
At the present time conventional means for identifying and optimizing natural products has been supplemented by a number of different approaches, with some major examples shown in the Table below. Let’s now examine these in a little more detail.
Recent and ongoing advances in identifying, screening, processing, and developing natural products.
The first six categories of this ‘improvements’ Table deal with better ways for finding natural products of interest in the first place; categories 7 and 8 are more concerned with modifications and betterment of candidate molecules in hand.
Natural products are initially encountered as complex mixtures, and thus efforts have been made towards streamlining sample preparation and purification as much as practicable (No. 1 of the above Table) in advance of screening. Included within this category are important advances in the determination of the molecular structures of natural products from extremely small sample sizes. The capability of evaluating candidate samples for specific properties in very high volumes and with great rapidity (high-throughput screening) is also an important issue in modern natural product evaluation.
With the rise in bioinformatics and computer modeling, ‘virtual screening’ (No. 2 of the above Table; equivalent to computer-aided evaluation of possible candidate drugs by modeling their interactions with target receptors) has become an important adjunct in bioproduct testing, as well as general drug identification. Bioinformatics is also applied in computational searching for new members of specific gene families (such as novel biosynthetic genes), which may act as drug targets, and an important pre-requisite for this is the availability of complete genome sequences for an increasing number of key organisms which produce secondary metabolites.
Empirical screening of any bioproducts is fundamentally dependent on a specific assay and its read-out, whether it is high-throughput or not. If one is seeking a compound which can usefully modify a particular cellular system, a good understanding of the underlying biology of the system is likely to identify the specific molecule(s) which should be targeted. This in turn is a clear advantage for screening itself. Refined understanding of fundamental cellular processes involved with carcinogenesis, for example, will in general lead to assay improvement (No. 3 of the ‘improvements’ Table above). Ultimately, with a single-molecule target and structural information, a rational design strategy may become possible. Prior to such a point, a natural product (or any other) molecular library will be best put to use if it is well-focused on the appropriate target. While less efficient, a more complex assay system (such as whole cells) may on the other hand provide additional information about other side-effects of the tested compounds. The utility of therapeutic bioproducts is critically dependent on their abilities to approach the ideal of ‘magic bullets’ in complex biosystems, without deleterious side-effects.
The next item (No. 4) in the above Table refers to metagenomics, which has been briefly discussed in a previous post. In this context, the relevance of metagenomics centers on the large fraction of environmental bacterial species which are non-cultivatable in the laboratory, but which could potentially yield useful drugs. Amplification and assembly of environmental DNA samples into discrete genomes (in essence, the ambit of metagenomics) has the potential to accelerate the analysis and manipulation of important and novel metabolic pathways, through which new small molecules are synthesized. Accordingly, rapid high-throughput genomic sequencing technologies (No. 5) available only in past few years feed into this process. Further manipulation of whole pathways, ultimately with entire synthetic genomes (the domain of synthetic biology, item No. 6 of the ‘improvements’ Table; and discussed generally in a previous post) in turn will provide great control over both the production of new active secondary metabolites, and their specific chemical modifications as desired. These ambitions require an integrated ‘systems-level’ understanding of the pathways involved in the production of all small molecules by an organism of interest, or its metabolome. High-level metabolomic input thus has great potential for the engineering of organisms for either increased yields of specific secondary metabolites, or the production of novel ones.
Since early times in antibiotic research, natural products have contributed core molecular designs (‘scaffolds’) which have been modified artificially, either by complete or partial synthesis (No. 7 of the above Table). This effort towards bioproduct improvement is still a productive venture. The final (eighth) innovation of the ‘improvements’ Table above concerns attempts to improve yields of metabolites by chemical ‘elicitors’, and also efforts towards boosting metabolite chemical diversity by modifying culture conditions.
These modern technological developments, combined with the well-noted evolutionary advantages of ‘consulting’ the natural compendium of biological small molecules, suggests that there is still much practical value to be gained from them. The question of the ongoing future of the exploration of natural molecular space will be picked up again in the next post, particularly insofar as it may be eventually superseded (at least in part) by wholly artificial alternatives.
And finally, a general comment on the importance of technology in the modern science of natural bioproduct discovery and development, in a biopolyverse-like manner:
Bioprospectors don’t need to carry picks
Since they have a range of techniques in the mix
Using various means
And comprehensive screens
They can rely on a complete bag of tricks.
References & Details
(In order of citation, giving some key references where appropriate, but far from an exhaustive coverage of the literature).
‘…..small molecules have an inherent major advantage over potential protein therapeutics in terms of their oral delivery potential….’ It may be noted that this general issue raises the whole field of drug delivery, the science of devising the means for ensuring that useful drugs reach their intended in vivo targets in an efficient manner while retaining their functional properties. This is a huge modern field, of the most fundamental significance to pharmaceutical companies.
‘…..the fraction of drugs ….. originating as natural biomolecules stood at about 50%…’ See Li & Vederas 2009.
‘….over 70% of cancer drugs have been cited as non-synthetic in origin….’ See Newman & Cragg 2007.
‘…..the properties of natural product molecules have shown significant average differences to synthetic molecular libraries….’ See Koehn & Carter 2005.
‘…..Even among plant sources alone there is still a huge range of material…..’ See Phillipson 2003.
‘..…the advent of a number of important drugs have been attributed to the transfer of ethnopharmacologic knowledge….’ See Cox 1990.
‘……the US National Cancer Institute ….. has not found useful anti-cancer drugs specifically from ethnobotanical information…..’ See Cragg et al. 1994; ‘….with the possible exception of the anti-cancer drug taxol…’ See Cragg 1998.
‘…..accusations of ‘biopiracy’……..’ See Shiva, S. Biopiracy: the plunder of nature and knowledge (South End Press, Cambridge MA, 1997); ‘……..have clouded or retarded a number of relatively recent bioprospecting ventures……’ See Rosenthal 2002.
‘…..peoples with close access to the sea….appear to have derived most of their traditional ethnopharmacological lore from land plants…..’ See Cox 1993.
‘……marine natural molecular space is much more likely to yield novel biodiversity than land ecosystems….’ See O’Hanlon 2006.
‘ Sponges alone have proven to be rich in a variety of bioproducts with promising applications….’ Sample references for modern harvesting of marine bioproducts: From sponges, see Sipkema et al. 2005; seaweeds, see Kubanek et al. 2003; marine cyanobacteria, see Burns et al. 2005; conotoxins, see Alonso et al. 2003; sea hares, see Barsby 2006; tunicates, see O’Hanlon 2006, Simmons et al. 2005.
‘…..the ‘Global Ocean Sampling’ voyage….’ See Rusch et al. 2007.
‘…..advances in the determination of the molecular structures of natural products from extremely small sample sizes….’ See Murata et al. 2006.
‘……(high-throughput screening) is also an important issue…..’ See Koehn & Carter 2005.
‘…….‘virtual screening’ …..has become an important adjunct in bioproduct testing……’ See Rollinger et al. 2008.
‘……an integrated ‘systems-level’ understanding of the pathways involved in the ….. metabolome….’ See Weckwerth 2010. A good example of the economic significance and challenges faced with metabolic pathway manipulation can be found with efforts to engineer the synthesis of the anti-malarial drug artemisinin in microbes for large-scale production. Of Chinese origin, this compound has been effective against the deadly Plasmodium falciparum malaria species, but supplies of its natural plant source (Artemisia annua) are often limiting. To engineer ‘heterologous expression’ of the drug in microbial cells, an entire pathway of enzymes must be provided within the foreign host cells. To date, successes with producing artemisinin precursors in yeast and E. coli cells have been reported. See Arsenault et al. 2008 for an overview, and Tsuruta et al. 2009 for details on an E. coli expression system.
‘……to improve yields of metabolites by chemical ‘elicitors’…’ See Poulev et al. 2003.
‘…..efforts towards boosting metabolite chemical diversity by modifying culture conditions….’ See Bode et al. 2002.
‘…..suggests that there is still much practical value to be gained from them [small natural bioproducts]’ See Li & Vederas 2009.
Next Post: Two Weeks from now.
In the previous post, the current theme of ‘Natural Molecular Space’ was considered from the point of view of its sampling by animals for the purposes of self-medication, or zoopharmacognosy. Now, it is time to move beyond this, to the human use of the huge and highly diverse resource of natural biomolecules. Unlike other mammals, humans can use their intelligence to greatly expedite the profitable sampling of Natural Molecular Space, and in one form or another, this has been practiced from the earliest times across all cultures. The manner and nature of this ‘harvesting’ is the topic of the present post.
Early human pharmacognosy, and continuing
It is clear that since sampling and application of natural bioproducts do not require any awareness of the physical nature of drugs themselves, the process of useful molecular discovery can (in effect) take place long before the definition of a molecule. ‘Discovery’ here is thus defined in a loose sense as the identification of a natural source which contains a useful molecular species. Only within the last century can we apply the more rigorous definition which includes the purification and full characterization of the active constituent(s) in molecular terms. Obviously, early humans had no accurate picture of the nature of material reality, and even more obviously this applies as well to animals involved in zoopharmacognosy (as discussed in the previous post). Indeed, the very concept of a molecule as a precise group of atoms (held together in a very specific manner by the rules of chemical bonding) is of extremely recent vintage in historical terms.
It is unlikely that any human cultures have never used accessible environmental materials for health-related reasons, and many such natural sources contained active constituents which proved enduringly worthwhile. Examples of these are not hard to come by, particularly from local plant sources. Some well-known cases are the anti-malarial drug quinine, derived from the bark of South American trees of Cinchona species, anti-pyretic / analgesic salicylates from willow bark (related to aspirin), cardiotonic digitalis (digoxin) from foxglove, narcotics (morphine and derivatives) from the opium poppy, and anti-psychotics (such as reserpine) from Rauwolfia species. Apart from human disease management, other traditional uses for environmental pharmaceuticals include preparations for control of insect pests and animal diseases. This extensive fund of traditional knowledge about the usefulness of local biotas has been termed (in the most general terms) ethnobiology, or (when focusing on the active compounds found within traditional medicines), ethnopharmacology. In a majority of cases (albeit not exclusively), the sources of such medicines are plants, and the fund of long-standing plant-derived cultural medicinal lore is accordingly termed ethnobotany.
Obviously the biodiversity of the environment within reach of a specific culture will have a bearing on the range of potential tribal medicines, especially since most such treatments derive from plant sources. At extremes one can compare the scope of botanical sources available to polar Inuit peoples with rain forest dwellers; indeed jungle regions such as the Amazon have been particularly rich sources of biopharmaceuticals. So there is obviously no question that humans across the board have been skilled at finding medically useful materials from their environments. But how systematic has this been, and how comprehensive?
When it comes to finding edible and nutritious foodstuffs, it has been noted that human cultures which have long inhabited specific geographic regions have identified essentially all plants which can become dietary items. Apart from much practical field experience and observations in support of this proposition, if a tribal group have lived in a relatively small region for thousands of years, it would seem quite reasonable to accept ipso facto that their knowledge of the food value of local plants would be highly advanced, and close to completion, if not quite at a literal 100% level. (One factor complicating the sampling of foods is where certain plant materials possess initial toxicity, which can be removed by specific processing steps. Clearly, it will take longer to gain such knowledge than by simply tasting plant products for their agreeability).
But the acquisition of plants with medicinal value, as opposed to worthiness as foods, is not the same thing. Could we expect that a tribal people in a resource-rich environmental (such as a tropical rain forest) would have identified all potentially useful medical plant (or other biological) sources after thousands of years in a similar location? If ‘all potentially useful’ is taken literally to mean beneficial to all humanity, then the answer is clearly in the negative. This is obviously the case, given the simple observation that human medical afflictions are far from geographically homogeneous. While modern transport systems can now spread infectious diseases rapidly around the globe, infections and parasites were historically often relatively local in the extent of their reach. Although this is patently obvious, even potential treatments for genetic conditions would not be sought if such problems did not exist within a relatively small tribal population. (Genetic diseases occurring at a low frequency would only tend to be noted on average within large population groups). It then goes without saying that no tribal people could search for a treatment for any pathological condition outside their frame of reference, any more than even the most sophisticated modern screening and drug design methods can be used against a novel disease which has not yet ‘emerged’. So, natural landscapes may harbor drugs whose usefulness is not yet definable even in the present day.
But one can put this high-level issue of generality to one side, and restrict tribal medicine to matters which would be of direct concern to them. Yet here, too, there are problems. From first principles, it could not be automatically assumed that a molecular solution to a given medical problem will necessarily exist anywhere on Earth. And by the same token, a useful molecule within Natural Molecular Space might indeed exist, but not necessarily within the accessible environment of any given tribal group. (If it ain’t there, you can’t find it). This is a kind of counterpoint to the above point noting that a local biota might harbor drugs for as yet undefined purposes – it might also lack useful drugs for specific applications, which are naturally available elsewhere. Consider an example with respect to the ancient enemy malaria: Long endemic in Africa, one of the best natural solutions (at least until the acquisition of parasite resistance) was native to South America in the form of the Cinchona plant (more of which below). Thus, early African peoples obviously had no means for arming themselves against malaria with this particular weapon.
The other problem with identification of useful biological medications is the complexity of doing so in a systematic manner, when faced with knowledge limitations, a vast array of potential environmental sources, and the range and variation within human medical problems. Despite their familiarity with their biological surroundings gained over very long time periods, many tribal peoples, especially in tropical environments, certainly suffered from a high burden of infectious and parasitic disease before access to modern medicine. It can be accordingly inferred that evidently either no solutions to such pathologies existed in their environments, or potential solutions existed, but they had not yet found them (or they had not discovered how to use them in a productive way).
Sifting the Environment for Medicinals
What other factors might be involved in the growth of tribal medical lore? Is it possible to come up with some kind of formula as a rough guideline for the likelihood that a particular culture would develop, purely by trial-and error sampling from its available environment, a useful treatment for a specific medical condition? This question can be rephrased in the following manner: An early tribal people have lived in a forest area for many generations. During this time, some of them have become ill from a previously unknown disease. Their shamans try treating their sick patients with a whole range of available plant materials. What are the chances that they might find something genuinely useful? What factors influence whether there is no hope at all, or whether there is a real prospect of success, perhaps given an element of good luck? For the present purposes, ‘genuinely useful’ means a bioproduct which directly or indirectly alters the pathogenic state in a favorable manner. And such beneficial effects need to be objectively measurable.
Since time immemorial, human patients have been given real comfort from healers of any description who could deliver a placebo effect through their ministrations. Yet although shamanic medicine has a radically different view of reality to modern chemical and pharmaceutical science, there is no question that traditional healing practices have found many therapeutically useful biological materials. Indeed, this very fact has led to the development of ethnobotany as a science in its own right.
While it is not possible to produce a quantitative formula for the likelihood of finding useful local environmental bioproducts by traditional medicines, we can think about the relevant factors involved, and for convenience represent them as R, D, B, C, and t; explained as follows:
R denotes the ‘druggability’ of the specific biological need in question, in terms of the probability of ‘solutions’ available within Natural Molecular Space. In brief, this refers to how well-suited are target molecules (relevant to a specific disease process) to interaction with drug-like compounds found among the gamut of natural biomolecules. This is influenced by the complexity of the illness or disease symptom itself, and what kind of molecular targets it offers for outside intervention. A complex multicellular parasite will contain a large number of proteins vital to its own functioning (but foreign to the host) which are potential sites of action for therapeutic drugs, while a viral infection which hijacks the machinery of the host cell presents fewer targeting options. If the disease stems from some internal physiological malfunctioning, the susceptibility of relevant target molecules to drug action is uncertain in the absence of specific information or empirical evidence. For example, perturbation of protein-protein interaction surfaces has historically been regarded as a difficult proposition, in contrast to the ‘drugging’ of protein clefts and pockets (which usually include enzymatic active sites).
This ‘R factor’ can be viewed as a kind of general bias factor for small molecules in Natural Molecular Space towards the common range of protein folds. (This aspect of natural bioproducts as a very non-random selection of general chemical space has been considered in a previous post). But on the other hand, compounds modulating protein-protein interactions (as opposed to intraprotein folds) appear to be harder to find within the same natural set. To illustrate this further, consider the following hypothetical situation:
A series of human medical needs are compared, assuming complete knowledge of the underlying problem and the best protein molecule(s) which should be targeted for therapeutic improvement. These problems are grouped into two broad classes: In one type, the best target solutions involve clefts or pocket-like regions on a single protein; in the other, relatively flat protein-protein interaction surfaces present the best possible targets. Humans undertake a search of available biotas for natural products with therapeutic value for both types of pathological conditions. In principle, it does not matter whether tribal shamans or biotechnological operatives are involved, although of course the former act in complete ignorance of the underlying reality (biotechnologists may or may not possess full detailed target information in advance of their search). It can be then predicted that a preponderance of useful ‘hits’ for the cleft/pocket targets would be obtained from Natural Molecular Space, over the protein-protein surface type.
It should be noted though, that the R factor here is not at all intended to represent an absolute measure of druggability, only what Natural Molecular Space tends to define as ‘druggable’. Thus, the druggability of protein-protein interactions, once believed to be intractable, is now seen as solvable in many (if not all) cases through ingenious artificial design approaches. The central ‘take-home’ message is that not all diseases and not all molecular targets are created equal, and as a consequence some have a higher likelihood of being vulnerable to a therapy based on a naturally available low-molecular weight drug than others.
D refers to the diversity of the total potential set of biomolecules accessible in the human environment. (‘Accessible’ here means that which is available for the normal geographical range of the human group. Nomadic groups may often cover different environments and thereby increase their exposure to diversity, but possibly at the expense of the time factor as below). The assumption here is that the higher the diversity, the higher the probability that beneficial biomolecules will exist in the environment, and indeed a rainforest has far more potential as a pharmaceutical source than an arctic tundra. (But note that although arctic and other environments with extreme conditions have lower biodiversity, they are nonetheless useful for bioprospecting in specialist roles. The adverse conditions which restrict diversity in the first place also mean that life in these regions has special adaptations which can be useful for biotechnology, such as ‘antifreeze’ proteins. The same observations also apply to other extreme environments, including the opposite pole of very high temperatures.) How does one define and measure diversity, anyway? In molecular terms, one must have yardsticks for comparing molecules with each other, in order to assess their relative dissimilarities. A large number of such ‘descriptors’ have been devised, including molecular structures, shapes, and chemical and physical properties, which must be rendered into mathematical representations for formal modeling purposes. But a working definition of a diverse library of molecules is one which spans a chemical space in a non-redundant fashion, by means of covering a wide and continuous range of properties. A large group of molecules can thus be less diverse than a smaller set, and a set which only included members at extreme ends of property values would have reduced diversity in comparison with a set whose members possessed properties covering a wide dynamic range. Even so, no absolute standard for assessing diversity exists, although many sophisticated approaches have been developed.
But to return to out environmental considerations, an implicit assumption is that the higher the D value, the greater the odds that the total collection of environmental compounds will include one which is fortuitously useful for a human requirement, even if this is totally unrelated to the function for which the compound was originally derived by natural selection. But this D value is clearly not a random portion of chemical space, since the environmental biomolecules of inherent value are far from randomly generated (as discussed in a previous post). The ‘druggability’ factor R noted above already addresses a general bias of Natural Molecular Space towards a relatively limited set of protein folds found in nature (compared to the huge potential size of protein sequence space). But there are more direct factors which may also bias the prospect of success towards a given biomolecular target, and this is the basis for considering a specific bias element (the B factor raised above).
A full discussion of this would become complex and very lengthy, but for the present purposes, consider the notion of functional bias within Natural Molecular Space towards a human need, and consequently specific targets. ‘Secondary metabolites’ (raised in a previous post) are present as a result of a long history of natural selection processes in response to environmental pressures on the organism in question. In certain cases these influences may strongly bias the repertoire of available biomolecules in a positive manner from the point of view of human needs and desires. For example, a search for an anti-bacterial drug may have far greater prospects for success from sampling natural sources than from a random collection of molecules of comparable size. (Selective pressures have resulted in the generation of anti-bacterial products by a variety of organisms, and in this sense the ‘aims’ of the producer organisms and the human biologist coincide). Conversely, a search in a natural environment for a molecular function absent from the totality of environment’s biology (Such as a low-molecular weight organic catalyst of a non-natural chemical reaction ) will depend on chance alone for its success. To take this kind of functional bias into account, the B factor is therefore needed. (The higher this bias factor, the higher the probability that molecules of the type sought after exist in the natural environment).
Nevertheless, this ‘bias factor’ caveat is not necessarily as limiting as it may first seem, and can actually work in favor of the bioproduct ‘ocean’ as a source of useful molecules. The shared evolutionary origins of life on Earth means that many biochemical pathways of humans and their domestic animals also have analogs in environmental organisms. In some cases receptor proteins between widely evolutionarily separated organisms may have divergent functions but recognizably homologous structures and primary sequences. This means that a search for a biological modifier within a diverse natural environment in many cases may have a higher probability of success than within a random molecular collection of comparable size, and in turn stresses the worth and value of screening for active natural products.
As noted in a previous post, if it were possible to screen an utterly alien biological world, this positive bias factor would disappear, and such evaluation of the alien natural environment would be equivalent to a random molecular library screen. (Alien micro-organisms might generate secondary metabolites for similar selective reasons as for terrestrial organisms, but the chances of an alien ‘penicillin’ affecting Earth bacteria would be very low). Obviously the ‘molecular bias factor’ was not of great concern for tribal peoples, and in any case will often be difficult to predict in advance. But however one defines the chemical diversity of the total available environmental resources of biomolecules, in the context of ‘natural bioproduct space’ diversity alone is an insufficient guideline.
To return the above factors determining the likelihood of a human culture finding useful environmental molecules, C denotes ‘cultural factors’, probably impossible to quantitate but nonetheless real and important in the present context. Not all human groups will find and use biopharmaceuticals at the same rate, and the determining factor here is the shared set of social values referred to as ‘culture’. Within this set of values we will find such qualities as openness to enquiry and resistance to change. All human beings have the same fundamental genetic endowment, and it has even been suggested that the human talent for ‘folk biology’ may be based on an innate neural module facilitating recognition of differing plant forms or other biodiversity. These considerations aside, clearly human societies vary in their tolerance for behavioral experimentation and their willingness to implement new procedures, both of which can influence the rate of identification and adoption of pharmaceuticals from the bioenvironment. It was noted above that we must take care to define utility of a treatment at the outset, since it is also clear that some tribal medicines have little or no real effects on their supposed target diseases. No early pharmacognosy could pass judgements based on proper clinical trials of biomaterials, and the placebo effect would routinely be present on top of any real benefits deriving from administration of candidate preparations to ill individuals. (Given the demonstrated power of placebos in medicine, it is to the credit of tribal human groups that treatments of real effectiveness could nonetheless be identified). The cultural factor of passing on a time-honored tradition can effectively ensure that knowledge of genuinely useful medicines is retained by a group over time, but unfortunately bad memes can be just as transmissible as good ones. A case in point is the continued use of rhinoceros horns for medicinal purposes in some parts of the world, a false supposition whether for fever reduction or as an aphrodisiac.
Finally, t of the formula above is the time factor, on the logical assumption that the longer a group inhabits a particular environment, the greater the opportunities for biomaterial trial-and-error experimentation. We have already noted the potential for duration to have an impact on the diversity factor for nomadic groups, but time is also relevant to the C (cultural) factor, since even the most hidebound and anti-innovative group may eventually stumble on and adopt useful materials if they dwell for a long enough period in the same environment. And cultures, of course, are not static systems but are themselves mutable with time.
An Interesting Case Study – Cinchona and Malaria
The pathway towards finding that cinchona bark is an effective treatment for malaria serves to demonstrate the complexities that can impinge upon the time / culture factors, and an interesting example to consider in a little more detail. In historical terms, cinchona was identified as a malarial cure relatively soon after the arrival of Europeans in South America. Since it is generally accepted that the malarial parasite did not exist in the New World prior to the European invasions, cinchona may be viewed as an example of rapid acquisition of a natural medicine. Although Peruvian Indians are often credited with both the discovery of the anti-malarial effects of cinchona and its transmission to Jesuit or other European visitors, most available evidence suggests that it is more likely that Europeans themselves first came to this knowledge. But upon reflection, it is not probable that newcomers would have tested cinchona bark so rapidly without benefiting from the accumulated lore of the original inhabitants. Some sources indicate that native Peruvians used cinchona bark to relieve shivering, which can occur through pharmacological effects of active constituents of the crude bark. Quinine itself can act as a muscle relaxant, and also has inherent anti-pyretic activity, both of which could alleviate unwanted shivering depending on the cause of the problem. In any case, awareness of this on the part of Europeans could easily act as a prompt for testing the bark in malaria sufferers, owing to the intense fevered shivering occurring at certain stages during the disease cycle. Thus in this particular set of circumstances, the relatively rapid identification of the utility of cinchona bark (and thence quinine) is ironically likely to have resulted from a side-effect of the bark not directly related to its ability to kill malaria parasites. Also, it is most likely (though not provable) that it developed from interplay between traditional native Peruvian empirical lore and European activities. While the story of cinchona may be a special case (and it is certainly noteworthy in that it has occurred during historical times, even though much of the specific detail is obscure), the adoption of many other traditional pharmaceuticals may have similarly tangled origins.
All of the factors except C (‘cultural’ factors) are also relevant to modern empirical screening of natural sources. At least, cultural factors are not operative in the same sense as for isolated independent tribal groups. Potentially inhibitory cultural factors no longer apply since the acquisition of new drugs has become effectively a global enterprise, transcending national barriers. On the other hand, the uptake of a new drug in the modern world depends on regulatory agencies, marketing, and commercial competition with rival products, all of which can be considered as ‘cultural’ forces under a broad definition of the word.
By the arguments presented here, a traditional society would have maximal success for finding useful natural molecules for a specific need if all of the above relevant factors were optimal: Highly druggable ultimate target(s) • Rich natural local environment, (such as rainforest) • Highly positive specific functional bias (for example, sought-after treatment could be satisfied by metabolites of diverse fungi or other organisms) • Highly acquisitive culture, highly receptive to innovation • Extensive duration of time for conducting ad hoc empirical testing of environmental resources.
To finish up, a double-barreled offering of biopoly(verse) of relevant note:
Are shamans biology sages
Possessing the wisdom of ages?
Some shamanic insight
In a drug textbook write
Might add some significant pages
Some chemists consult tribal lore
Seeking botanical info, and more
From suitable plants
They’ll thus take a chance
That good drugs will pay them a score
But where Westerners (especially those with commercial motivations) “consult tribal lore”, the outcome may be an exploitative one-sided affair sometimes termed ‘biopiracy’ – and that will be taken up in the next post, among other things.
References & Details
‘ Examples of these [natural bioproducts as medicinals] ….particularly from local plant sources’ See Rates 2001 for a general review, with specific examples.
‘…..jungle regions such as the Amazon have been rich sources of biopharmaceuticals…..’ See Schultes 1994. This author, the late Richard E. Schultes of Harvard University, was renowned for his ethnobotanical studies, including works on plant-derived hallucinogens. In the latter vein, he co-authored a well-known book in 1979 (Plants of the Gods: Origins of Hallucinogenic Use. New York: McGraw-Hill; with revised editions in 1987 and 2001) with Albert Hofmann, the discoverer of LSD.
‘…….human cultures …… have identified essentially all plants which can become dietary items.’ This point has been discussed by Jared Diamond in his well-known book, Guns, Germs and Steel (Vintage Books, 1997).
‘……where certain plant materials possess initial toxicity, but which can be removed by specific processing steps….’ A good example of this is the case of cycads of certain Pacific islands. The seeds of these ancient plants carry both nutrients and toxins, requiring pre-treatment by extensive washings and soakings in order to render the seeds edible for humans. This same example also raises another interesting general issue: humans screening their environments for edible plants will reject those that obviously poisonous (or finds ways to de-toxify them), but some plant sources may have toxins which produce subtle damage that is far more difficult to detect. Peoples of Guam and several other Pacific locales have suffered high rates of neurodegenerative diseases (with features of amyotrophic lateral sclerosis, Parkinson’s disease, and dementia, in varying combinations), and a long-standing hypothesis has attributed this disease focus to the local habits of cycad product consumption. (See the book The Island of the Colour Blind, by Oliver Sacks, Picador 1996, for an extended account of these issues). Yet this has been highly controversial, with varying forms of the ‘cycad hypothesis’ proposed and then discounted. It has become clear, though, that an environmental contribution is highly likely, as the disease incidence is in decline, in parallel with dietary changes (see Steele 2005; Steele & McGeer 2008).
‘…..historically infections and parasites were often relatively local….’ For more on this, one can refer to the same Jared Diamond book again as above (Guns, Germs and Steel ; Vintage Books, 1997).
‘…..even potential treatments for genetic conditions…’ It might be thought surprising that solutions to genetic problems could be found within low-molecular weight natural bioproducts, even in principle. A simple answer to this would be, ‘It depends on the nature of the genetic problem’. If a genetic lesion causes the loss of an entire genomic coding sequence for an essential large protein, clearly it is extremely unlikely that this defect will be ‘fixed’ by ingestion of a small natural molecule. But on the other hand, many genetic problems result from premature termination of protein synthesis, or mutations which cause protein misfolding. In the former case, mutations which result in the formation of abnormal stop codons in an mRNA molecule can be suppressed by drugs which induce translational ‘readthrough’, and thereby enable production of the formerly abnormally truncated protein. Although recent clinical successes have used artificially-obtained compounds for this purpose (see Welch et al. 2007), the original definition of the effect was provided by naturally-derived aminoglycoside antibiotics (see Howard et al. 2004). In the case of protein misfolding, certain compounds can interact with and stabilize misfolded proteins in their correct conformations. Natural products (and artificial compounds) have been found with this kind of ‘chemical chaperone’ potential in treating certain specific genetic diseases (for example, see Brumshtein et al. 2007)
‘……natural landscapes may harbor drugs whose usefulness is not definable even in the present day.’ This is a potent argument for the retention of natural biodiversity, and to resist increasing destruction of natural habitats – especially those within resource-rich tropical zones.
‘…..compounds modulating protein-protein interactions (as opposed to intraprotein folds) appear to be harder to find within the same natural set.’ Note that this is not stating that such compounds are entirely absent in Natural Molecular Space. A notable case in point are natural immunosuppressants: cyclosporins, rapamycin, and FK506. These molecules act by promoting the formation of ternary complexes which inhibit the activation of a key transcription factor for T cell activation. For some details, see Mann 2001; Lee & Park 2006.
‘……the druggability of protein-protein interactions, once believed to be intractable, is now seen as solvable.’ For progress in this area, see Dömling 2008.
‘…..a diverse library of molecules is one which spans a chemical space in a non-redundant fashion….’ For background on medicinal chemical diversity, see Gorse 2006.
‘…..no absolute standard for assessing diversity exists, although many sophisticated approaches have been developed….’ See Maggiora & Shanmugasundarum 2011, for a recent mathematical treatment of molecular similarity, which measures the flip side of diversity.
‘…….it has even been suggested that the human talent for ‘folk biology’ may be based on an innate neural module…..’ This was made by Steven Pinker in The Language Instinct (William Morrow & Co., 1994).
‘…..Peruvian Indians are often credited with both the discovery of the anti-malarial effects of cinchona and its transmission to Jesuit or other European visitors, most available evidence suggests that it is more likely that Europeans themselves first came to this knowledge….’ For much information on this on related topics, see Fiammetta Rocco’s excellent book; The Miraculous Fever Tree (HarperCollins, London, 2003).