《天然药物化学》课程参考文献（海洋天然产物）Lessons from the past and charting the future of marine natural products drug discovery and chemical biology.pdf

Chemistry & Biology Review Lessons from the Past and Charting the Future of Marine Natural Products Drug Discovery and Chemical Biology William H. Gerwick1, * and Bradley S. Moore1, * 1Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Science, University of California San Diego, La Jolla, CA 92037, USA *Correspondence: wgerwick@ucsd.edu (W.H.G.), bsmoore@ucsd.edu (B.S.M.) DOI 10.1016/j.chembiol.2011.12.014 Marine life forms are an important source of structurally diverse and biologically active secondary metabolites, several of which have inspired the development of new classes of therapeutic agents. These success stories have had to overcome difficulties inherent to natural products-derived drugs, such as adequate sourcing of the agent and issues related to structural complexity. Nevertheless, several marine-derived agents are now approved, most as ‘‘first-in-class’’ drugs, with five of seven appearing in the past few years. Additionally, there is a rich pipeline of clinical and preclinical marine compounds to suggest their continued application in human medicine. Understanding of how these agents are biosynthetically assembled has accelerated in recent years, especially through interdisciplinary approaches, and innovative manipulations and re-engineering of some of these gene clusters are yielding novel agents of enhanced pharmaceutical properties compared with the natural product. Introduction Familiar to all, the coastal margin of the world’s oceans are extraordinarily rich in species diversity, especially in tropical environments. The last 40 years has seen a remarkable exploration of these unique organisms for their structurally diverse natural products. Early on, this was motivated by a need to detect and understand the chemical basis of various toxins with which people came into contact, such as saxitoxin, tetrodotoxin, and the brevetoxins (Grindberg et al., 2008), and subsequently from a fundamental interest to describe the unique adaptations that marine life possess in this regard. For example, a distinctive and pervasive feature of these is the incorporation of halogen atoms, bromide or chloride, covalently attached to organic compounds (Molinski et al., 2009; Villa and Gerwick, 2010; Sashidhara et al., 2009). However, as the field has progressed, efforts have focused more on biomedical screening programs, and now several drugs have been developed from leads generated from marine natural products (Newman and Cragg, 2010). More recently, the enzymatic and genetic basis by which these unique secondary metabolites arise has become a focal point with an ultimate vision of capturing and harnessing these unique biochemical catalysts so as to create molecular diversity of biomedical and agrochemical utility (Gulder and Moore, 2009). In this perspective review, we trace these developments in the field of marine natural products drug discovery and chemical biology, describe some of the success stories of these efforts, and then provide some vision of the future for this area of investigation. Tracing the History of Marine Natural Products Drug Discovery The early days of marine natural products discovery efforts focused on those organisms most conspicuous and easily collected. Indeed, before the advent of SCUBA, exploration of the marine world languished behind that of the terrestrial world for exactly this cause; an inability to access marine life beyond the intertidal. Thus, the large and showy creatures of the sea, such as red algae, sponges, and soft corals, were early on shown to produce a multitude of quite unique molecular species, such as highly halogenated terpenes and acetogenins from Rhodophytes such as Laurencia (Suzuki and Vairappan, 2005), the highly toxic polyketide ‘‘tedanolide’’ from the fire sponge Tedania ignis (Taylor, 2008), and prostaglandins from the gorgonian coral Plexaura homomalla (Weinheimer, 1974). With continued exploration, other groups of organisms, some more cryptic in space and time, were studied, and the repertoire of unique molecular species grew. By the turn of the century, marine natural products had become an established subdiscipline of natural products chemistry, and several thousand compounds had been described. Some groups of marine organisms were fairly well characterized for their major metabolites, and more often known compounds or their close relatives were being re-encountered (Faulkner, 2002). Thus, attention turned to smaller and smaller creatures that had previously escaped collection and examination, such as marine cyanobacteria, marine fungi, and diverse other groups of marine eubacteria. This emphasis on microorganisms has been rewarded with a wealth of new natural products chemistry, as well as the realization that many compounds previously isolated from macroorganisms, such as sponges and tunicates, are actually metabolic products of associated microbes (Piel, 2009). As a result, a number of groups around the world have sought to culture marine bacteria from various sources, including shallow and deep water sediments, animate as well as inanimate surfaces, and from within the tissues of other macroorganisms (Williams, 2009). Productive as this has been, it has still only begun to sample the phylogenetic richness present within microbial groups in the sea. From seawater alone, it is estimated that Chemistry & Biology 19, January 27, 2012 ª2012 Elsevier Ltd All rights reserved 85

only 1% of bacteria present have been cultured, and from culture independent methodologies, many major lineages remain without any or only a few cultured members. From another perspective, while Actinomycetes and Myxobacteria are the richest terrestrial groups of bacteria in terms of number and diversity of natural product structures, these microbes were largely unrecognized in the world’s oceans until recently, and exploration of the extent and diversity of these two groups in the sea is ongoing (Fenical and Jensen, 2006; Scha¨ berle et al., 2010). Combined with the exploration of marine microbial life forms, largely by fermentation methods, have been improvements in various approaches and strategies for interfacing natural products with modern biology and screening platforms. Automated extraction protocols coupled to prefractionation strategies have enhanced the quality and number of materials being evaluated in biological assays (Johnson et al., 2010; Bugni et al., 2008). In particular, the prefractionation of extracts to single compounds or reduced complexity mixtures enhances hit rates, due to the concentration of low abundance actives, and accelerates the identification process as the active compound is already pure or nearly so. However, there are two long-standing schools of thought on natural products discovery: ‘‘isolate and then test’’ versus ‘‘test and then isolate,’’ and both have a historical track record of success. Thus, a fusion approach is commonly employed wherein extracts or fractions are tested for bioactive compounds, and if a ‘‘strong’’ activity is detected, then bioassay-guided approaches are used. On the other hand, profiling of the material by 1 H-NMR and/or LCESIMS for unique chemical constituents can be followed by their isolation and broad evaluation in diverse biological assays. The strategies in biological assays have similarly shifted, from at one time in vivo screens to in vitro cell assays to isolated protein biochemical screens, and most recently, to high content phenotypic assays (Swinney and Anthony, 2011). The great gains achieved in throughput have resulted in large part from advances in automation; however, some of these approaches have emerged to be overly costly and lacking in flexibility; it is predicted that future development in HTS will focus more on quality of assays and their physiological relevance, rather than further miniaturization (Mayr and Bojanic, 2009). Thus, the newest approaches accentuate flexibility and cost effectiveness matched with quality test compounds being evaluated in new and intriguing areas of disease biology. Final analytical HPLC scale purifications on small samples can occur very rapidly to yield sufficient compound for structure elucidation by very sensitive mass spectrometry (MS) and nuclear magnetic resonance (NMR) instrumentation (dubbed ‘‘nanoscale structure elucidation’’) (Molinski, 2010), such that leads are generated from materials available on only the microgram scale. These data, coupled with informative databases of natural products that contain analytical information (e.g., SciFinder, AntiMarine, MarinLit, ChemSpider) allows for the rapid identification of compounds as being previously known, the so-called process of ‘‘dereplication,’’ or representing new chemical entities. However, an obvious consequence and issue that has emerged from finding and determining active molecules on an ever smaller scale is that there is not sufficient compound produced to adequately explore their biological properties. Oftentimes, samples are obtained in this small scale because the producing organisms are present in small natural abundance and are difficult or impossible to recollect. Thus, supply problems emerge at an even earlier stage in the drug discovery process, and underscore the critical importance of synthetic and biosynthetic methods to resupply compounds and analogs. Moreover, structures determined at the nanoscale, just as for compounds elucidated when more plentiful, should be confirmed for structural and stereochemical accuracy by independent methodologies, such as total chemical synthesis (Nicolaou and Snyder, 2005). Finally, while chemoinformatics and in silico screening of natural products to protein targets has occurred to some extent, this is a relatively little developed aspect of the field and thus represents an exciting frontier, as described further below. Origins and Development of Marine Chemical Biology The molecular revolution has forever changed the face of marine natural products chemistry (Lane and Moore, 2011). With the explosion of numerous ‘‘omic’’ approaches—genomics, proteomics, metabolomics, transcriptomics—now employed by marine natural product practitioners, new areas of science and inquiry have emerged over the past decade that begin to answer long-term questions in the field and provide fresh ideas for new research directions. The bridging of natural products chemistry with modern molecular biology has empowered researchers with the ability to address fundamental questions about the biosynthetic capacity of marine organisms, the synthetic role of microbes in marine invertebrate natural product chemistry, and the bioengineering potential of marine drugs. In this section, we highlight recent trends in marine natural product research that have been impacted by modern omic approaches with a focus on small molecule biosynthesis. Without a doubt, marine organisms synthesize a plethora of small molecules with fascinating chemical structures and potent biological properties. Early biosynthetic knowledge developed primarily from isotope tracer experiments involving a wide range of marine microbes, algae, and invertebrates (Moore, 2005, 2006). These studies often revealed biosynthetic strategies in marine organisms distinct from their terrestrial counterparts. At the turn of the 21st century, the molecular basis of marine microbial natural product biosynthesis was first established for the streptomycete antibiotic enterocin (Piel et al., 2000) and the cyanobacterial agent barbamide (Chang et al., 2002) based on the targeted cloning and sequencing of their respective biosynthetic gene clusters (Figure 1). This targeted gene sequencing approach to the identification of secondary metabolic pathways relied upon the ability to generate specific gene probes that in some instances were time consuming to develop and at other times misleading to implement. Nonetheless, over the past 10 years numerous discoveries were established of natural product biosynthetic gene sets from laboratory-cultured marine isolates to field-collected samples (Lane and Moore, 2011). With the emergence of faster and cheaper DNA sequencing protocols, it is now much more efficient to sequence entire genomes rather than specific gene sets, especially when searching for biosynthetic pathways with little biochemical precedence or exploring organisms with multiple pathways of interest. Not only does genome sequencing provide key molecular 86 Chemistry & Biology 19, January 27, 2012 ª2012 Elsevier Ltd All rights reserved Chemistry & Biology Review

information about how, when and sometimes why a natural product is assembled, it also bestows additional information about precursor supply networks, competing or complementary metabolic pathways, and basic genetic traits of the producing organism, inter alia. The first completed marine actinomycete genome sequence was for the 5,183,331 bp Salinispora tropica in 2007 and revealed the molecular basis of at least 17 natural product biosynthetic pathways spanning approximately 10% of the genome (Udwary et al., 2007). Over the past few years, knowledge of the benthic S. tropica genome facilitated the molecular cloning of the salinosporamide (Eusta´ quio et al., 2009), sporolide (McGlinchey et al., 2008), and lymphostin (Miyanaga et al., 2011) pathways, the discovery of the novel macrolactam salinilactam A (Udwary et al., 2007), a new salinosporamide analog (Eusta´ quio et al., 2011) and numerous biosynthetic enzymes, and the linking of natural products to functional adaptation concepts in the Salinispora genus (Penn et al., 2009). This example highlights the far-reaching translational opportunities of having access to a completed genome in being able to speed up the rate of scientific discovery. More recently, the draft genome of the benthic filamentous cyanobacterium Moorea producta 3L (formally Lyngbya majuscula; Engene et al., 2011), a well-known producer of complex natural products such as barbamide (Chang et al., 2002) and curacin A (Chang et al., 2004), was found to harbor several surprises, including a relatively limited number of natural product pathways (Jones et al., 2011). Its 8.5 Mbp draft genome supports approximately 293 kbp of DNA sequence in secondary metabolism, far too low to encode the full suite of reported M. producta metabolites of approximately 200 different compounds. This maiden genome sequencing project suggests that the rich complement of natural products in M. producta will ultimately be strain specific. Genome sequencing of organisms not normally associated for their biosynthetic prowess, such as the pelagic cyanobacteria Trichodesmium erythraeum ISM101 and Prochlorococcus MIT9313, has enabled through sequence gazing, or ‘‘genome mining,’’ the hypothesis-driven discovery of the postranslationally modified ribosomal peptides trichamide (Sudek et al., 2006) and the prochlorosins (Li et al., 2010), respectively. This genomics-driven discovery of new chemical entities is a key, first step in understanding the specialized chemical biology of these important planktonic organisms. The prochlorosins represent a fascinating finding from a biosynthetic perspective in which N S N O CCl3 OCH3 H barbamide NH O O O Cl OH salinosporamide A O O H3CO O OH O O OH OH HO H enterocin N S O N NH O S N O N HN O H N N H O O patellamide A onnamide A NH O H2N NH NH COOH O O O OCH3 H N OH O CH3O O HO O O O HO O H3COOC OAc OH COOCH3 O O OH O OH bryostatin 1 N N O NH2 H O N OCH3 lymphostin H3CO H N O N O N O N O O O N S OH apratoxin A A B H H H H Figure 1. Examples of Marine Natural Products with Characterized Biosynthetic Pathways (A) Laboratory cultured and (B) environmental uncultured marine microbes whose biosynthetic pathways have been established by a variety of omic approaches (includes ecteinascidin-743 shown in Figure 4). Chemistry & Biology 19, January 27, 2012 ª2012 Elsevier Ltd All rights reserved 87 Chemistry & Biology Review

Table 1. Six Marine Natural Products and Fourteen Marine Natural Products Inspired Compounds that Are FDA-Approved Agents or in Clinical Trial with Details of Their Collected Source, Predicted Biosynthetic Source, Molecular Target, and Disease Treated Clinical Status Compound Name Natural Product or Derivative Collected Source Organism Predicted Biosynthetic Source Biosynthetic Class of Agent Molecular Target Disease Area FDA approved Cytarabine (Ara-C) Derivative Sponge Bacterium Nucleoside DNA polymerase Cancer FDA approved Vidarabine (Ara-A) Derivative Sponge Bacterium Nucleoside Viral DNA polymerase I Antiviral FDA approved Ziconotide Natural Product Cone Snail Mollusk Cysteine Knot Peptide N-type Ca channel Pain FDA approved Eribulin Mesylate (E7389) Derivative Sponge Bacterium Complex Polyketide Microtubules Cancer FDA approved Omega-3-acid ethyl esters Derivative Fish Microalgae Omega-3 fatty acids Triglyceridesynthesizing enzymes Hypertriglyceridemia FDA approved Trabectedin (ET-743) (EU registered only) Natural Product Tunicate Bacterium NRPS-derived Alkaloid Minor groove of DNA Cancer FDA approved Brentuximab vedotin (SGN-35) Derivative Mollusk Cyanobacterium Antibody drug conjugate (MM auristatin E) – Linear NRPS/PKS CD30 and microtubules Cancer Phase III Plitidepsin (Aplidine) Natural Product Tunicate Bacterium Cyclic Depsipeptide Rac1 and JNK activation Cancer Phase II DMXBA (GTS-21) Derivative Worm Worm? Alkaloid a7 nicotinic acetylcholine receptor Cognition, Schizophrenia Phase II Plinabulin (NPI 2358) Derivative Fungus Fungus Diketopiperazine Microtubules and JNK stress protein Cancer Phase II Elisidepsin Derivative Mollusk Bacterium Cyclic Depsipeptide Plasma membrane fluidity Cancer Phase II Zalypsis (PM00104) Derivative Nudibranch Bacterium Alkaloid DNA binding Cancer Phase II Glembatumumab vedotin (CDX-011) Derivative Mollusk Cyanobacterium Antibody drug conjugate (MM auristatin E) – Linear NRPS/PKS Glycoprotein NMB & microtubules Cancer Phase I Marizomib (Salinosporamide A; NPI-0052) Natural Product Bacterium Bacterium NRPS with b-lactone & g-lactam 20S proteasome Cancer Phase I Trabectedin analog (PM01183) Derivative Tunicate Bacterium NRPS Alkaloid Minor groove of DNA, Nucleotide Excision Repair Cancer Phase I SGN-75 Derivative Mollusk Cyanobacterium Antibody drug conjugate (MM auristatin F) – Linear NRPS/PKS CD70 and microtubules Cancer Phase I ASG-5ME Derivative Mollusk Cyanobacterium Antibody drug conjugate (MM auristatin E) - linear NRPS/PKS ASG-5 and microtubules Cancer Phase I Hemiasterlin derivative (E7974) Derivative Sponge Bacterium Modified linear tripeptide (NRPS-PKS) Microtubules Cancer 88 Chemistry & Biology 19, January 27, 2012 ª2012 Elsevier Ltd All rights reserved Chemistry & Biology Review

a diverse collection of polycyclic, conformationally constrained lantipeptides are efficiently synthesized from up to 29 different ribosomally synthesized peptide precursors by the action of a single promiscuous processing enzyme (Li et al., 2010). In this way, although the genome size of P. MIT9313 is a modest 2,410,873 bp (Rocap et al., 2003), it still has the capacity to synthesize a collection of specialized chemicals more often associated with antibiotic-producing streptomycetes whose genomes are several times larger (Nett et al., 2009a). Marine macroorganisms, such as sponges, tunicates and bryozoans, have long been known to harbor natural products of biomedical significance that structurally resemble compounds produced by terrestrial microorganisms (Piel, 2004). This structural resemblance for decades begged the question of the biosynthetic source of invertebrate natural product chemistry, yet went unanswered due to difficulties in the isolation and culturing of community-based, natural product-producing microbes. Early experimental work involving cell sorting and in situ hybridization studies helped establish the symbiosis hypothesis (Haygood et al., 1999), yet unequivocal proof was not ultimately achieved until metagenomic approaches confirmed the central importance of bacteria in marine natural product biosynthesis (Piel, 2009). The success of the metagenomic method is based on the relatively unbiased sequence analysis of total environmental DNA, which in the examples discussed here involves the marine organism and its associated microflora. Large clonal libraries often numbering in the millions are prepared and screened by phenotype or genotype to identify biosynthetic genes or pathways of interest (Piel, 2011). However, due to the large size of natural product biosynthetic gene clusters that make them diffi- cult to heterologously express, genotype screens are often preferred over phenotype screens. In this way, the bacterial origin and molecular basis were established for the pederin family of polyketide antitumor agents (onnamide [Piel et al., 2004] and psymberin [Fisch et al., 2009]) from sponges, the bryostatin macrolide anticancer agents from bryozoans (Sudek et al., 2007), and the cyanobactin family of posttranslationally modified ribosomal peptides from ascidians (Schmidt et al., 2005). Onnamide, psymberin, and bryostatin all share a common biosynthetic strategy involving large, trans-AT PKSs that to date have precluded their heterologous expression; this is in contrast to the patellamide and other cyanobactin ribosomal peptides whose biosynthetic gene clusters are considerably smaller allowing not only for functional expression but also for their genetic remodeling (Donia et al., 2008). These seminal studies confirm the power of metagenomics in capturing the genetic blueprint of natural product biosynthesis in marine invertebrates and point toward the future biotechnological approaches in helping solve the supply problem that often limits marine natural products from clinical development. Some Marine Drug Discovery Success Stories To date, there are seven therapeutic agents (four anticancer, one antiviral, one pain control, and one for hypertriglyceridemia) that derive from the marine environment in some sense (Mayer et al., 2010). Of these, two are the actual chemical structure as isolated, and five others are synthetic agents that capture their chemical idea from a marine product. In addition, a further 13 agents are in phase I, II, or III clinical trials (Table 1). Figure 2A summarizes the collected sources of organisms that have yielded these agents and reveals that mollusks, sponges, and tunicates are the richest collected source of these most valuable to substances. However, as noted above, the collected source has oftentimes been shown or is strongly suspected of harboring or feeding upon microorganisms that are the actual producers of the bioactive agent. Figure 2B displays the actual or suspected metabolic source of the most important agents and reveals that heterotrophic bacteria and cyanobacteria are the real metabolic jewels of the world’s oceans, accounting for fully 80% of these clinical trial and approved pharmaceutical agents. An early finding of the marine drug discovery field was that the Caribbean sponge Cryptotethia crypta possessed metabolites with a very interesting but relatively simple modification of a nucleoside; the normal 2-deoxyribose ring of deoxythymine and uracil are replaced by b-D-arabinofuranose (Bergmann et al., 1957). Ensuing biological and medicinal chemistry exploration of the impacts of such a subtle alteration revealed that cytosine arabinoside was a potent disrupter of DNA replication and led to cellular toxicity (Brunton et al., 2011), while the arabinoside derivative of adenosine had antiviral effects (Sagar et al., 2010). Metabolic activation of Ara-C to the corresponding triphosphate yields a substrate mimic of deoxycytidine 50 -triphosphate, and following incorporation into the DNA backbone, inhibits the DNA polymerase as well as DNA repair enzymes. While Ara-C has found greatest utility in inducing remissions of acute myelocytic leukemia, more significantly these discoveries from nature helped illuminate nucleoside chemistry as a viable therapeutic strategy that later gained favor in antiviral chemotherapy. Soon after the discovery of unusual nucleoside sponge-based chemistry, potent anticancer activity was detected in extracts of the tunicate Ecteinascidia turbinata. However, it would be nearly 30 years until the structure of the active compound, ecteinascidin 743 (ET-743, = trabectedin), was finally elucidated (Rinehart Table 1. Continued Clinical Status Compound Name Natural Product or Derivative Collected Source Organism Predicted Biosynthetic Source Biosynthetic Class of Agent Molecular Target Disease Area Phase I Bryostatin 1 Natural Product Bryozoan Bacterium Polyketide Protein kinase C Cancer, Alzheimer’s Phase I Pseudopterosins Natural Product Soft Coral Bacterium? Diterpene glycoside Eicosanoid metabolism Wound healing Additional perspectives on approved FDA drugs and clinical trial agents that were derived or inspired by marine natural products can be found in Mayer et al. (2010) and Newman and Cragg (2010). Chemistry & Biology 19, January 27, 2012 ª2012 Elsevier Ltd All rights reserved 89 Chemistry & Biology Review