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Modern Harvesting of Small Natural Biomolecules

August 23, 2011

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.

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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.

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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 use of natural product molecular scaffolds for the future design of antibiotics has been promoted…’     See Butler & Buss 2006; Singh & Barrett 2006.

‘  Natural products remain highly regarded…..’    See Koch et al. 2005; Newman & Cragg 2009.

‘…..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 continuing value of ethnopharmacology has been vigorously promoted…..’     See Heinrich & Gibbons 2001; Plotkin 2001.

‘……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.

‘…….if old written records exist they may possibly be tapped for such irreplaceable [ethnopharmacological] knowledge….’     See Buenz et al. 2004; Buenz et al. 2006.

‘…..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.

‘……clinical trials for the anti-cancer efficacy of certain marine bioproducts…’    See Jimeno et al. 2004; Provencio et al. 2009.

‘…..the true source may be commensal micro-organisms….’    See Schmidt 2005,  ‘…..and their ancient and robust variety….’    See Sogin et al. 2006.

‘…..the ‘Global Ocean Sampling’ voyage….’    See Rusch et al. 2007.

‘…..efforts have been made towards streamlining sample preparation and purification….’    See Grabley & Thiericke 1999; Koehn 2008.

‘…..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.

‘……complete genome sequences for an increasing number of key organisms….’    For example, see Bentley et al. 2002; Donadio et al. 2002.

‘……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.

This effort towards bioproduct improvement [modifications upon an existing molecular scaffold] is still a productive venture…..’     See Hamann 2003; Grant et al. 2004.

‘……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.

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