Natural product discovery past, present, and future

Natural product discovery: past, present, and future

Leonard Katz 1 · Richard H. Baltz 2

Received: 13 November 2015 / Accepted: 14 December 2015 / Published online: 6 January 2016 ? Society for Industrial Microbiology and Biotechnology 2016

precipitous drop in cost for genome sequencing, it is now feasible to sequence thousands of actinomycete genomes to identify the “biosynthetic dark matter” as sources for the dis-covery of new and novel secondary metabolites. Advances in bioinformatics, mass spectrometry, proteomics, transcrip-tomics, metabolomics and gene expression are driving the new field of microbial genome mining for applications in natural product discovery and development.

Keywords Actinomycete · Antibiotic · Discovery · Genome mining · Natural product · Fungi · Secondary metabolite · Streptomyces

Introduction

Natural products (NPs) represent a large family of diverse chemical entities with a wide variety of biological activi-ties that have found multiple uses, notably in human and veterinary medicine and in agriculture [26, 36, 47, 85, 86]. They originate from bacterial, fungal, plant, and marine ani-mal sources. The bacterial and fungal NPs described in this review are products of “secondary metabolism”: molecules that are not required for survival of the host under laboratory conditions, but which undoubtedly provide some advantage to the host in its native environment. Natural macromol-ecules (DNA, RNA, and protein), their building blocks and precursors, as well as intermediates of primary metabolism, are typically excluded from the working definition of NPs.NP discovery has been driven by the findings that they are important and valuable agents: pharmaceuticals, her-bicides, insecticides, etc. Since the discovery of penicillin more than 75 years ago, >23,000 NPs have been charac-terized, the large majority of which are produced by bacte-ria, mainly by the family Actinomycetaceae [25]. After the

Abstract Microorganisms have provided abundant sources of natural products which have been developed as com-mercial products for human medicine, animal health, and plant crop protection. In the early years of natural product discovery from microorganisms (The Golden Age), new antibiotics were found with relative ease from low-through-put fermentation and whole cell screening methods. Later, molecular genetic and medicinal chemistry approaches were applied to modify and improve the activities of important chemical scaffolds, and more sophisticated screening meth-ods were directed at target disease states. In the 1990s, the pharmaceutical industry moved to high-throughput screen-ing of synthetic chemical libraries against many potential therapeutic targets, including new targets identified from the human genome sequencing project, largely to the exclusion of natural products, and discovery rates dropped dramati-cally. Nonetheless, natural products continued to provide key scaffolds for drug development. In the current millennium, it was discovered from genome sequencing that microbes with large genomes have the capacity to produce about ten times as many secondary metabolites as was previously recog-nized. Indeed, the most gifted actinomycetes have the capac-ity to produce around 30–50 secondary metabolites. With the

Special Issue: Natural Product Discovery and Development in the Genomic Era. Dedicated to Professor Satoshi ōmura for his numerous contributions to the field of natural products. * Richard H. Baltz rbaltz923@https://www.wendangku.net/doc/417127319.html,

1

Synthetic Biology Engineering Research Center, University of California-Berkeley, 5885 Hollis St. 4th Floor, Emeryville, CA 94608, USA

2

CognoGen Biotechnology Consulting, 7636 Andora Drive, Sarasota, FL 34238, USA

discovery of streptomycin by Selman Waksman and associ-

ates at Rutgers University in the 1940s, large NP discovery

efforts began in pharmaceutical companies, mainly in the

United States, Europe and Japan, and modest efforts fol-

lowed in isolated academic laboratories worldwide. Nota-

ble exceptions include the academic laboratories of Satoshi ōmura at the Kitasato Institute (now Kitasato University) in Tokyo, the late Hamao Umezawa at the Institute of Anti-

biotics in Tokyo, the late Hans Z?hner at the University of

Tübingen, and Hans Reichenbach at the Helmholtz-Zen-

trum für Infektionsforschung in Braunschweig. Working

independently, and in collaboration with others, these labo-

ratories identified more than 1000 novel NPs.

What has fascinated NP scientists is the chemical diver-

sity and complexity of NPs, examples of which are dis-

played in the compounds shown in Figs. 1, 2, 3 and 4. Not

only is there a large range of molecular weights (coumerin,

146 Da to daptomycin, 1620 Da), but many of the com-

pounds contain a significant number of stereo-specific car-

bon centers. The stereo-complexity of these molecules has

attracted synthetic chemists interested in developing routes

and reactions for total chemical synthesis, and biochem-

ists and molecular biologists interested in understanding

the enzymology, genetics, and regulation of biosyntheses.

Microbiologists interested in the discovery of microor-

ganisms that produce novel molecules and novel activities

were needed to isolate and identify the producing micro-

organisms and to test and quantify the antibacterial or anti-

fungal activities. Cell biologists tested for anti-cancer and

other pharmacological activities. The commercial require-

ment for high volume production attracted chemical engi-

neers, fermentation microbiologists and geneticists, and

isolation and analytical chemists. Organic chemists were

attracted to pharmaceutical and biotechnology companies

to develop semi-synthetic derivatives. This family of aca-

demic and industrial scientists created the field of natural

product research.

This review covers the strategies and methodologies

employed from the 1940s through the present time to dis-

cover new NPs. We describe the screening for activity

approaches employed for the first 30 years of NP discovery,

highlighting some of the important molecules discovered

during “The Golden Age”. We then discuss how the under-

standing of NP biosynthesis changed the approaches for NP

discovery. For example, sequencing efforts demonstrated

that entire NP biosynthetic gene sets were clustered, and

allowed the use of genetic probes for the search for new

NPs, enabled the mixing, matching, and substitution of NP

biosynthesis genes to generate novel “hybrid” NPs, as well

as novel derivatives that could not be produced by synthetic

chemistry. We end with a discussion of the impact of the

development of inexpensive, high-throughput Next Gen-

eration Sequencing and advanced bioinformatics tools on the genomic approaches being taken for present and future NP discovery, focusing on genome mining and silent gene activation. The history of NP discovery, as we describe it, is segmented into three overlapping periods, 1940s–1970s, 1970s–2000s, and 2000 and beyond. These periods were chosen not because there were discrete breaks in the dis-covery process but because significant advances during these eras dramatically impacted the strategies employed to discover new NPs.

This review is not intended to be comprehensive, but to provide a basic understanding of the history of NP discov-ery without a particular emphasis on molecule type or med-ical field. Because of their overall value as drugs, however, which heavily skewed NP discovery efforts in pharma-ceutical companies towards specific medical applications during the first 30 years of effort, we present a brief sum-mary of the medical uses of NPs. Finally, because we both participated in one or more aspects of NP discovery over time, in addition to citing published material, we also use unpublished, personal accounts of our experiences in this perspective. For comprehensive reviews of the impact of NPs on medicine, readers are guided to other reviews [36, 85, 86]. For insider accounts on NP discovery and develop-ment programs in the Pharmaceutical Industry, readers are referred to Feature Articles in SIM News [8, 112, 127]. Natural products as drugs

As of 2013, 1453 new chemical entities (NCEs) have been approved by the US Food and Drug Administration, of which ca. 40 % are NPs or NP-inspired (semi-synthetic NP derivatives, synthetic compounds based on NP pharmaco-phores, or NP mimics) [36, 68]. In the past 30 years, the percentage of NP or NP-inspired NCEs has risen to approx-imately 50 % of the total, and to ca. 74 % in the anti-tumor arena [26, 47]. During the period 2008–2013, 25 NP or NP-derived drugs were approved for use. Here we present a brief summary of the many pharmaceutical applications of NPs or NP-inspired compounds and a short overview of their mechanisms of action or molecular targets. The struc-tures of the NPs highlighted in bold are shown in Figs. 1, 2, 3 and 4.

The most prominent medicinal use of NPs has been in the area of anti-infectives, notably anti-bacterial therapy (Fig. 1). Dozens of NPs, and their second- and third-gener-ation semisynthetic derivatives have been, and continue to be used to treat Gram-positive or Gram-negative bacterial infections in humans and animals. Briefly, azithromycin or clarithromycin, both semi-synthetic derivatives of the macrolide erythromycin, doxycycline, a second-genera-tion tetracycline, amoxicillin, a semi-synthetic penicillin derivative, and cephalexin, a derivative of cephalosporin

C, are all commonly used as outpatient treatments of bac-terial skin or respiratory infections. The macrolide tylosin and its semi-synthetic derivative tilmicosin are used to treat bacterial infections in animals. Macrolide antibacterial agents related to erythromycin or tylosin act as translation inhibitors by binding to the 50S ribosomal subunit, block-ing translation of the nascent polyptide chain. They are generally bacteriostatic for susceptible pathogens, but are bactericidal for Streptococcus pneumonia.Tetracycline

also blocks translation by binding to the 50S ribosomal Fig. 1 Structures of antibacterial natural products

subunit at a site distinct from the macrolides. Chloram?phenicol, a third ribosome binding agent is very active against Gram-negative bacteria, but is no longer used clini-cally, due to rare side effects. It binds to the A site of the 50S ribosome preventing the binding of aminoacyl-tRNA. The bactericidal penicillins and cephalosporins covalently bind to penicillin binding proteins in the bacterial cell wall and block cell wall synthesis, leading to exposure of the cell membrane to its osmotically unfavorable environment, resulting in cell lysis. Clavulanic acid, an inhibitor of sev-eral β-lactamases (which inactivate penicillins) is combined with the semi-synthetic penicillin analog amoxicillin in the compound Augmentin for use against penicillin-resist-ant pathogens. Glycopeptides, such as vancomycin and teicoplanin or their recently approved second-generation structural relatives (e.g., telavancin, dalbavancin, and orita-vancin) are used for Gram-positive infections that are not readily treated by macrolides and tetracyclines. The mem-brane active cyclic peptide daptomycin is used for hospi-tal-based difficult to treat Gram-positive infections, includ-ing methicillin-resistant Staphylococcus aureus (MRSA), and the penems (e.g., meripenem, derived from thienamy?cin) are used to treat infections caused by Gram-negative microorganisms. Natural penems are cephalosporin ana-logs. Aminoglycosides, such as tobramycin, are used in hospitalized patients carrying Gram-negative infections. They interact with mRNA in the 30S–50S ribosome junc-tion and cause mistranslation. Lipiarmycin (independently identified from three different actinomycetes, and also named clostomicin, tiacumicin and fidaxomycin), recently approved by the FDA, is effective against Clostridium difficile, the agent associated with severe colitis and diar-rhea. This macrolide does not bind ribosomes, but inhibits the β-subunit of RNA polymerase blocking transcription. Rifampicin, a semi-synthetic analog of rifamycin, also inhibits RNA polymerase and is used in combination with other agents, including streptomycin, for the treatment of tuberculosis.

Two different families of NPs are used to treat fungal infections in humans. Polyene macrolides such as ampho?tericin B (Fig. 2) and nystatin bind to ergosterol creat-ing holes in the fungal membrane and the echinocandins, such as caspofungin, inhibit the fungal cell wall form-ing enzyme glucan synthase. A large family of polyethers including monensin, narasin, salinomycin, and lasalocid are used in cattle feed as growth promoting agents or in chicken feed to prevent coccidiosis. The antimicrobial and antiparasidic effects of the polyethers are through their activities as ionophores, chelating mono- or divalent cati-ons. Ivermectin, a semi-synthetic derivative of avermectin, is active against parasitic worms in humans and animals. It is used to treat onchocerciasis (river blindness) and lym-phatic filariasis (elephantiais) in humans. Artemisinin is used to treat the malaria causing parasite Plasmodium fal-ciparum. Artemisinin is derived from a plant, but is now produced by the yeast Saccharomyces cerevisiae, based on a successful synthetic biology approach to heterologously

express the NP pathway. Spinosad, an antiparasitic and Fig. 2 Structures of antifungal and antiparasitic natural products

Fig. 3 Structures of anti-tumor natural products

insecticidal agent comprised of a mixture of the polyke-tides spinosyn A and spinosyn D, is approved for the topi-cal treatment of head lice in humans, and as an important insecticide active against a wide range of lepidopterous and dipterous insect pests in agriculture.

A large number of NPs or NP-inspired agents have been found to exhibit anti-tumor activity [26, 47]. Approved drugs from bacterial sources include the anthracycline mitomycin C (Fig. 3) and the mixed polyketide-nonribo-somal peptide (PK-NRP) bleomycin, both of which act by causing breaks in DNA, though via different chemical mechanisms; the di-cyclic peptide actinomycin D, a tran-scription inhibitor; doxorubicin, a semi-synthetic derivi-ative of the anthracycline polyketide daunorubicin, that acts by intercalating into DNA and blocking the enzyme topisomerase II; and romidepsin, a histone deacetylase inhibitor. Anti-tumor terpenes from plant sources include vincristine, vinblastine and paclitaxel. These agents block mitosis by binding to microtubules. Other anti-tumor agents from plants are the topoisomerase II inhibitor etopo-side, a semi-synthetic agent related to podophyllotoxin; the topoisomerase I inhibitor camptothecin, a quinoline alkyloid, as well as its water-soluble semi-synthetic deriva-tive topotecan; and the recently approved translation inhibi-tor omacetaxine mepesuccinate, a natural ester of the alka-loid cephalotaxine. Other recently approved anti-cancer drugs include the tetrapeptide epoxyketone carfilzomib, a semi-synthetic derivative of epoxomicin, and eribulin, a macrocylic ketone analog of the NP halichondrin B. Salinosporamide A is a proteosome inhibitor not yet approved as an anti-cancer agent. Many NPs that exhibit anti-tumor activities are too cytotoxic to be administered as drugs by themselves. A number has been conjugated to monoclonal antibodies for the purpose of target-specific delivery. NPs present in approved antibody–drug-conju-gates (ADCs) include the ansamycin polyketide maytan?sine and the acylpeptide dolastatin. The formerly approved ADC containing calicheamicin, an enediyne polyketide was recently withdrawn due to severe toxic side effects. However, many more enediynes have been discovered by focused genome mining [101], thus providing a wide range of chemical warheads for potential drug development.

An important advance in the treatment of hypercholes-terolemia was the development of the class of NPs known as statins, e.g., compactin (Fig. 4), lovastatin, and semi-synthetic derivatives simvastatin, atorvastatin, pravasta-tin. These compounds inhibit the mammalian and fungal enzyme HMG-CoA reductase in the cholesterol biosynthe-sis pathway. They continue to be among the highest rev-enue generating pharmaceuticals worldwide. The bacterial polyketides rapamycin and FK506 (tacrolimus), and the fungal cyclic peptide cyclosporine A are immunosupres-sants that are widely used after organ transplantation to sup-press rejection. Fingolimod, a drug used to treat multiple sclerosis, was developed from the fungal compound myri?ocin

, an inhibitor of serine palmitoyltransferase, by using Fig. 4 Structures of natural products with various bioactivities

structure–activity studies to determine the pharmacophore. Canagliflozin, a semi-synthetic derivative of the flavanoid phlorizin, found in many plants, is used to treat type 2 dia-betes, and capsaicin, derived from chili peppers, is used topically in over-the-counter creams to reduce minor aches and pains. In addition, many plant-derived NPs, e.g., cou?marin, are using in food as flavorings or as neutraceuticals.

Many of the examples described above represent FDA approved NPs or NP-inspired compounds. More than 100 other NPs or NP-inspired synthetic or semi-synthetic deriva-tives originally obtained from bacterial, fungal, plant, and marine sources are currently in clinical trials as anti-infective, anti-cancer, or other pharmaceutical agents. For a comprehen-sive list of NP-based compounds currently in clinical trials, the reader is directed to the excellent review by Butler et al. [30].

Each of the NPs described exhibits a distinct mechanism of action on their respective targets, although compounds within a given structural class with the same pharmacological activ-ity share binding sites in a common target. Erythromycin and tylosin both bind to the 23S rRNA in domain V of the bacterial ribosome, but the third sugar on tylosin also binds to domain II of 23S rRNA. The study of drug mechanism of action has revealed that similar pharmacological activities can be obtained from compounds of dissimilar structure. For example, rapa-mycin and cyclosporine A are both immunosuppressants, but have different targets and mechanisms of action. Other com-pounds, such as tetracycline and daunorubicin, have high struc-tural similarity but exhibit different pharmacological effects: antibacterial via binding to ribosome and anti-tumor via DNA intercalation, respectively. Finally some compounds have more than one pharmacological effect. For example, in addition to its antibacterial activity, erythromycin exhibits binding to motilin receptor in the human gut and is used clinically for treatment of diabetic gastroparesis, as well as to stimulate gastrointesti-nal motor activity when needed in patients following surgery. Because erythromycin is a large compound, the pharmacoph-ores responsible for the different properties are composed of different sub-sets of atoms of the molecule. These examples illustrate the variety of binding targets and pharmacological effects NPs have and underscore not only the values they have brought as medicines, but also as agents that have been used to deepen our understanding of fundamental biology.

Table 1 presents a list of 100 important NPs from diverse microbial sources and with diverse biosynthetic mecha-nisms. Most have use in human or animal medicine, or in agriculture. Several are important research tools, which will be further discussed below.

Strategies and methodologies for NP discovery Over the past seven decades, natural product discovery has been in a process of evolution; strategies were relatively simple during the first 30 years, then grew more diverse and complex, accelerated by advancements in science and technology in the next two decades. Overall efforts in NP discovery have decelerated with the waning interest from the pharmaceutical industry over the past twenty-years. In more recent times, inexpensive microbial genome sequenc-ing has opened totally new strategies for secondary metab-olite drug discovery. We recount some of the strategies and approaches in the following sections.

The first 30 years (1940s–1970s): phenotypic screening

Although the development of penicillin for wide use dur-ing WWII demonstrated success at bringing a NP to mar-ket, it was the systematic screening of actinomycetes obtained from soil by the Waksman group at Rutgers in the 1940s leading to the discoveries of actinomycin, strep-tothricin, and, most notably, streptomycin, that prompted pharmaceutical companies to begin screening extracts of actinomycetes and fungi for activities primarily against pathogenic bacteria. The process required acquisition of microorganisms, fermentation, product isolation, and test-ing of fermentation broths and purified compounds against test organisms. For testing, companies used bioassays for phenotypic screening. In general, a phenotypic screen employs a cell line (bacteria, yeast, eukaryotic cells or tis-sues in culture, etc.) and a method to determine a response to an applied test compound, without prior knowledge of its mechanism of action. The easiest read-out is cell inhibition or death, and, in the first 30 years this was used to iden-tify more than 1000 NPs that had antibacterial or antifungal activity, including dozens that were ultimately approved as drugs. Early cancer drugs were also detected in a pheno-typic screen using stable cancer cell lines cultivated first in experimental animal models and later, in vitro. Acquisition of microorganisms

Acquisition of actinomycetes and fungi was facilitated by the abundant numbers of these microbes in soil. At Eli Lilly and Company, Abbott Laboratories, and other phar-maceutical companies, employees were encouraged to col-lect soil samples when they traveled around the world on business or vacation. At Abbott, a large map of the world was kept on the wall of the soil screening laboratory with more than 2000 pins stuck at the locations from which soils were collected. Every continent, and almost every country had at least one pin. In many cases, new microbes produc-ing important compounds could be tracked back to the originating soils. The most famous example involved Pro-fessor Satoshi ōmura of the Kitasato Institute collecting a

Table 1 Diverse natural products produced as secondary metabolites by bacteria or fungi

Secondary metabolite Biosynthetic origin Major use Producing organism Type of organism Pubmed citations a

Acarbose Glycoside Antidiabetic (HM)Actinoplanes sp.Actinomycete1878 Actinomycin NRPS Antitumor (HM)Streptomyces anulatus Actinomycete24,705 Actinorhodin PKS II Antibacterial (RT)Streptomyces coelicolor Actinomycete448 Adriamycin PKS II Antitumor (HM)Streptomyces peucetius Actinomycete59,850 Albomycin Peptidyl-nucleoside Antibacterial Streptomyces sp. ATCC

700974

Actinomycete130 Amphomycin NRPS Antibacterial Streptomyces canus Actinomycete97 Amphotericin B PKS I Antifungal (HM)Streptomyces nodosus Actinomycete20,386 Antimycin NRPS-PKS I Research tool; Piscicide Streptomyces (multiple

sp.)

Actinomycete4558

Apramycin Aminoglycoside Antibacterial (AH, RT)Streptoalloteicus hindu-

stanus

Actinomycete303

Ascomycin NRPS-PKS I Immunomodulator

(HM)Streptomyces hygro-

scopicus

Actinomycete255

Avermectin (Ivermectin)b PKS I Anthelmintic (HM, AH)Streptomyces avermitilis Actinomycete741 (6208)b Bialphos NRPS Herbicide (CP)Streptomyces viridochro-

mogenes

Actinomycete182

Bleomycin NRPS-PKS I Antitumor (HM)Streptomyces verticillus Actinomycete17,239 Calcimycin (A23187)PKS I Calcium ionophore (RT)Streptomyces chartru-

ensis

Actinomycete11,796 (16,702)

Candicidin PKS I Antifungal (HM)Streptomyces (multiple

sp.)

Actinomycete291 Capreomycin NRPS Antitubercular (HM)Saccharothrix mutabilis Actinomycete545 Carbomycin PKS I Antibacterial (HM)Streptomyces halstedii Actinomycete166 Cephalosporin NRPS Antibacterial (HM)Cephalosporium acre-

monium

Fungus45,337

Cephamycin NRPS Antibacterial (S)Streptomyces cla-

vuligerus

Actinomycete3462 Chloramphenicol Shikimate modification Antibacterial (HM)Streptomyces venezuelae Actinomycete38,609 Chlorobiocin Aminocoumarine Antibacterial (S)Strteptomyces roseo-

chromogenes

Actinomycete96

Chloroeremomycin

(oritavancin)

NRPS Antibacterial (HM)Amycolatopsis orientalis Actinomycete39 (247)

Clavulanic acid (aug-mentin)Other Antibacterial (HM)Streptomyces cla-

vuligerus

Actinomycete6348 (12,346)

Compactin PKS Cardiovascular (HM)Penicillium brevicom-

pactum

Fungus662 Coumermycin Aminocoumarin Antibacterial (RT)Streptomyces rishiriensis Actinomycete353 Cyclosporin A NRPS Immunomodulator

(HM)

Tolypocladium inflatum Fungus50,544

d-Cycloserine Other Antitubercular (HM)Streptomyces lavendulae Actinomycete3045 Daptomycin NRPS Antibacterial (HM)Streptomyces roseospo-

rus

Actinomycete2183 Dalbavancin NRPS Antibacterial (HM)Nonomuraea sp.Actinomycete235 Daunorubicin PKS II Antitumor (HM)Streptomyces peucetius Actinomycete53,337 Echinocandin NRPS Antifungal (HM)Aspergillus nidulans Fungus3079 Epothilone NRPS-PKS I Anti-tumor (HM)Sorangium cellulosum Myxobacteria971 Ergometrine Ergolin alkaloid Cardiovascular (HM)Claviceps purpurea Fungus2180 Erythromycin PKS I Antibacterial (HM)Saccharopolyspora

erythraea

Actinomycete31,179 Filipin PKS I Antifungal (RT)Streptomyces filipinensis Actinomycete1303 Fusidic acid Terpine Antibacterial (HM)Fusidium coccineus Fungus2167

Geldanamycin PKS I Antitumor (S)Streptomyces hygro-

scopicus

Actinomycete1627

Gentamicin Aminoglycoside Antibacterial (HM)Micromonospora pur-

purea

Actinomycete27,233 Gramicidin S NRPS Antibacterial (RT)Bacillus subtilis Bacillales3841 Hygromycin Aminoglycoside Antibacterial (AH)Streptomyces hygro-

scopicus

Actinomycete2092

Josamycin PKS I Antibacterial (HM)Streptomyces narbon-

ensis

Actinomycete676

Kanamycin (amikacin)Aminoglycoside Antibacterial (HM)Streptomyces kanamy-

ceticus

Actinomycete18,934 (8055) Kirromycin NRPS-PKS I Antibacterial (RT)Streptomyces collinus Actinomycete219 Lasalocid PKS I Coccidiostat (AH)Streptomyces lasaliensis Actinomycete875

Lincomycin (clindamy-cin)Other Antibacterial (HM)Streptomyces lincoln-

ensis

Actinomycete7702 (9958)

Lipiaramycin (fidax-omicin)PKS I Antibacterial (HM)Dactosporangium

aurantiacum

Actinomycete155 (264)

Lipstatin (Xenical)Fatty acyl-lactone Antiobesity (HM)Streptomyces toxitricini Actinomycete25 (1559) Lovastatin PKS Cardiovascular (HM)Asperigillus terreus Fungus10,266 Milbemycin PKS I Anti-parasidic (AH)Streptomyces hygro-

scopicus

Actinomycete736 Mithramycin PKS II Antitumor (HM)Streptomyces plicatus Actinomycete1342 Mitomycin C Quinone Antitumor (HM)Streptomyces lavendulae Actinomycete18,112 Moenomycin Phosphoglycolipid Antibacterial (AH)Streptomyces ghanaensis Actinomycete228 Monensin PKS I Coccidiostat (AH)Streptomyces cinnamon-

ensis

Actinomycete4675

Mycophenolic acid PKS Immunomodulator

(HM)Penicillium brevicom-

pactum

Fungus7109

Narasin PKS I Coccidiostat (AH)Streptomyces aureofa-

ciens

Actinomycete221

Natamycin (pimaracin)PKS I Antifungal (HM)Streptomyces (multiple

sp.)

Actinomycete817 (894) Neomycin Aminoglycoside Antibacterial (HM)Streptomyces fradiae Actinomycete13,737 Netropsin NRPS Antitumor (RT)Streptomyces ambofa-

ciens

Actinomycete719 Nikkomycin Peptidyl-nucleoside Antifungal Streptomyces tendae Actinomycete246

Nisin RiPP Food preservative Lactococcus lactis Lactobacillales1720 Nystatin PKS I Antifungal (HM)Streptomyces noursei Actinomycete4686 Oligomycin PKS I Toxin (RT)Streptomyces avermitilis Actinomycete4784 Oxytetracycline PKS II Antibacterial (HM)Streptomyces rimosus Actinomycete7379 Paclitaxel Isoprenoid Antitumor (HM)Several endophytic fungi Fungus27,268 Penicillin NRPS Antibacterial (HM)Penicillium crysogenum Fungus96,006 Phosphomycin Phosphone Antibacterial (HM)Streptomyces wedmo-

rensis

Actinomycete2410 Pleuromutalin Diterpine Antibacterial (HM)Clitopilus scyphoides Fungus161 Pneumocandin NRPS Antifungal (HM)Glarea lozoyensis Fungus77 Polymyxin (colistin)NRPS Antibacterial (HM)Paenibacillus polymyxa Bacillales9611 (4084) Polyoxin (D)Nucleoside Antifungal (CP)Streptomyces cacaoi Actinomycete120 Pristinamycin IA NRPS Antibacterial (HM)Streptomyces pristi-

naespiralis

Actinomycete521

Pristinamycin IIA NRPS-PKS I Antibacterial (HM)Streptomyces pristi-

naespiralis

Actinomycete131

soil sample from a golf course in Tokyo in the early 1970s from which his research group discovered avermectin from a new species of Streptomyces that he named S. avermiti-lis. Other examples include the erythromycin producer Saccharopolyspora erythraea, discovered in 1949 in the garden of a local physician in the Phillipines employed by Eli Lilly and Company [https://www.wendangku.net/doc/417127319.html,/1994/11/ medicine-philippines-who-really-discovered-erythromycin-1-an-inter-press-service-feature], the daptomycin producer Streptomyces roseosporus, tracked to a soil sample from

Ramoplanin NRPS Antibacterial (HM)Actinoplanes sp. ATCC

33076

Actinomycete155

Rapamycin NRPS-PKS I Immunomodulator

(HM)Streptomyces hygro-

scopicus

Actinomycete26,695

Rebeccamycin Alkaloid Antitumor Lechevalieria aerocolo-

negenes

Actinomycete128

Rifamycin (rifampicin)PKS I Antibacterial (HM)Amycolatopsis mediter-

ranei

Actinomycete19,004 (24,279) Ristocetin NRPS Antibacterial (RT)Amycolatopsis lurida Actinomycete2953 Salinomycin PKS I Coccidiostat (AH)Streptomyces albus Actinomycete624 Sinefungin Nucleoside Antifungal (RT)Streptomyces griseolus Actinomycete255 Spectinomycin Aminoglycoside Antibacterial (HM)Streptomyces spectabilis Actinomycete2408 Spinosyn (spinosad)PKS I Insecticidal (CP)Saccharopolyspora

spinosa

Actinomycete92 (548)

Spiramycin PKS I Antibacterial (HM)Streptomyces ambofa-

ciens

Actinomycete1367

Staurosporin (aglycone)Alkaloid Antitumor (S)Streptomyces stauro-

sporeus

Actinomycete10,116 Streptomycin Aminoglycoside Anti-tubercular (HM)Streptomyces griseus Actinomycete29,437 Streptothricin Aminoglycoside Antibacterial Streptomyces (multiple

species)

Actinomycete337

Streptozotocin Glucosamine-nitros-

ourea Antitumor (HM)Streptomyces acromo-

genes

Actinomycete24,436

Surfactin NRPS Surfactant Bacillus subtilis Bacillales595

Tacrolimus (FK-506)NRPS-PKS I Immunomodulator

(HM)Streptomyces tsukubae-

nsis

Actinomycete19,514 (19,841)

Teicoplanin NRPS Antibacterial (HM)Actinoplanes teichomy-

ceticus

Actinomycete3358

Tetracenomycin PKS II Antitumor Streptomyces glauce-

scens

Actinomycete101 Tetracycline PKS II Antibacterial (HM)Streptomyces rimosus Actinomycete35,863 Thienamycin Other Antibacterial (HM)Streptomyces cattleya Actinomycete324 Thiostrepton RiPP Antibacterial (AH, RT)Streptomyces azureus Actinomycete486 Tobramycin Aminoglycoside Antibacterial (HM)Streptoalloteicus hindu-

stanus

Actinomycete6636 Tunicamycin Nucleoside Antibacterial (RT)Streptomyces chartreusis Actinomycete4971

Tylosin PKS I Antibacterial (AH)Streptomyces fradiae Actinomycete1557 Undecylprodigiosin Other Antibacterial (RT)Streptomyces coelicolor Actinomycete161 Validamycin Glycoside Antifungal (CP)Streptomyces hygro-

scopicus

Actinomycete99

Viomycin NRPS Antibacterial (HM)Streptomyces sp. 11861Actinomycete831 Virginiamycin NRPS, NRPS-PKS I Antibacterial (AH)Streptomyces virginiae Actinomycete1372

AH animal health, CP crop protection, HM human medicine, PKS polyketide synthase, NRPS nonribosomal peptide synthetase, S scaffold for chemical semi-synthesis, RiPP ribosomally synthesized and post-translationally modified peptide, RT research tool

a PubMed citations on 8/31/2015

b Compounds of commercial products in parentheses correspond to citations in parentheses

Mount Ararat in Turkey [41], and the spinosad producer Saccharopolyspora spinosa, tracked to a defunct sugar mill rum still in the Virgin Islands [125]. The practice of collect-ing soil from foreign sources and bringing samples back to the home country went on unabated until countries signed the treaty known as the Convention on Biological Diversity (CBD) in the early 1990s. Until then, the countries from which the soil samples originated did not share in the reve-nues brought by the subsequent drugs that were developed. CBD ensured that revenues would return to these countries.

During the first three decades of antibiotic discovery in the pharmaceutical industry, many important NPs were dis-covered from microbial sources, many of which, or their second, third, or fourth generation semi-synthetic deriva-tives, are still in use today. Many of the important NPs listed in Table 1 were discovered in this timeframe. Thou-sands of new chemical entities (NCEs) were discovered from soil screening, but only a small number were devel-oped as drugs. Nevertheless, the success achieved from the commercial launch of a single compound was sufficient to keep the process going unchanged for more than 30 years, at least in the companies with which we are familiar. In general, soil screening was a low-throughput process which, as described below, was limited by the slow growth of the actinomycetes and fungi examined, and importantly by the amount of shaker space available to companies.

In the early years of screening, most companies focused on isolating and identifying Streptomyces as the major bac-terial genus to search for NCEs. As can be seen in Table 1, Streptomyces species yielded many important compounds in several antibiotic classes: macrolides (tylosin, spiramy-cin); aminoglycosides (neomycin, kanamycin,); β-lactams (cephamycin, carbapenems); tetracyclines (tetracycline, chlortetracycline, oxytetracycline); polyenes (candicidin, amphotericin B, nystatin); peptides (actinomycin); and chloramphenicol. However, three important antibiotics discovered by Eli Lilly and Company, erythromycin, van-comycin and tobramycin [8], were originally thought to be produced by Streptomyces species, but the producing actinomycetes were later reclassified as Saccharopolyspora erythraea, Amycolatopsis orientalis, and Streptoalloteicus hindustanus, respectively (Table 1). In the late 1950s, the US-based Schering Corp. collected a large group of soil actinomycetes classified as Micromonospora and began screening them for antibacterial activity [124, 127]. This genus yielded the aminoglycosides gentamicin (a fam-ily consisting of more than 5 congeners), sisomicin, the fortimicins, and sagamicin, the macrolides rosaramicin, juvenimicin, and the megalomicins, as well as the oligo-saccharide everninomicin. Only gentamicin was developed as an antibacterial drug. Lepetit (later Dow Lepetit then Biosearch Italia) began to screen rare actinomycetes in the 1960s. The company discovered the macrolide lipiarmycin from Dactosporangium (later developed as fidoxamicin by Optimer and Cubist), and the glycopeptides teichopla-nin and ramoplanin from different Actinoplanes species. It is now apparent from the early discoveries in the pharma-ceutical industry that in addition to the highly productive Streptomyces, other less abundant actinomycetes have con-tributed substantially to the wealth of important NPs dis-covered in the first 30 years.

Detection, fermentation and isolation of secondary metabolites

To determine if they produced active compounds, indi-vidual colonies isolated from agar plates were grown in small shake flask fermentations employing several media compositions, and in some cases under a range of tempera-tures and agitation speeds. Initial detection for antibacterial or antifungal activity involved assay of solvent extracted culture broths. Small amounts of extracted material were deposited on filter paper discs which were then placed on agar plates seeded with a susceptible test organism. Detec-tion was as simple as visualization of growth inhibition of test organisms after overnight incubation. Activities were quantified by standard MIC analyses employing panels of various pathogens. For more polar compounds that were not solvent extractable (e.g., peptidic or glycosidic agents), Schering used an agar plug assay. Small (2–3 cm diam-eter) plugs were removed from agar plates seeded with a test organism, allowing the administration of up to 5 mL of whole culture broths of soil isolates (Micromonospora strains). Detection of small zones of inhibition after over-night incubation was used to identify antibiotic producers. After the activities were identified, the active components were often separated by paper chromatography and assayed by bioautography: the chromatogram was placed on an agar plate seeded with a susceptible test strain; after overnight incubation, the plate was examined for zones of inhibition corresponding to the migration of the active ingredient. Bioautography was used to determine if there was more than a single active compound in the culture. After initial discovery, product titers were improved initially by fer-mentation optimization to produce enough material for iso-lation and evaluation. In some cases, the producing strain was subjected to mutagenesis and selection for product titer improvement at an early stage using chemical or physical mutagenic agents. Promising strains were then scaled in pilot plant fermenters to obtain greater quantities of mate-rial for isolation and structural characterization, and further activity evaluation. Because of the ease of collecting sam-ples and obtaining different colonies, each colony (strain) was typically given a single chance, as described above, to produce an active product, although many companies kept the isolated colonies as lyophilized spore suspensions in

their culture collections. Initial shake flask cultures were generally discarded. Because of the sheer volume of shake flasks and shaker capacity, companies could test only a limited number of different microbes per year. At Eli Lilly and Company, that number was about 35,000, translating to about 1,000,000 strains screened over three decades. [8, 11]. By the mid-1990s, when NP screening was halted, Abbott Laboratories had screened about 400,000 isolates (J. McAlpine, personal communication).

We estimate, for all companies, institutes, and research laboratories worldwide engaged in screening during the period from 1950 to 2000, that about 10–20 million isolates were screened. Undoubtedly, many of the more common actinomycete species were screened many times by many companies. From these, thousands of NCEs were identified and hundreds, either the NPs themselves or their deriva-tives, were developed as drugs. Although, as we describe briefly below, the methods to screen for pharmacological activities have changed dramatically over the last 50 years, the processes employed to isolate organisms from envi-ronmental samples, to grow them in liquid culture, and to extract and purify natural products have undergone only minor evolution. High-speed shakers capable of providing sufficient aeration to allow actinomycetes to grow in micro-titer plates [80] replaced the need for shake flask cultivation so that more isolates could be screened in less space, and major improvements in NMR [29] and MS [49] analyses have made structure determination of complex NPs easier and quicker. Large-scale fermentation has remained basi-cally unchanged.

The first 30 years of NP discovery followed the para-digm: (1) phenotypic screening, (2) compound isola-tion and structural characterization, (3) mode of action studies in some cases, (4) preclinical development, and if successful, (5) clinical development and commerciali-zation. Efforts were directed primarily to discover anti-bacterial and antifungal compounds. All of the NPs with antibacterial or antifungal activities discovered during this period, with a single exception, employed pheno-typic screens that used cell killing (or growth inhibition) as the read-out. Because Merck was interested in discov-ering agents specifically targeted to the biosynthesis of the cell, they developed a screen in which the read out was the conversion of bacteria to spheroplasts. This led to the discovery of fosfomycin [53, 110]. Moreover, with a single exception, all the antibacterials and antifun-gals discovered during this era that went on to be devel-oped as drugs were all NPs. Naladixic acid is based on the structure of the NP quinine, and was identified in a search for synthetic derivatives of anti-malarial drugs, but was found to have serendipitous antibacterial proper-ties. In the 1980s, a number of natural quinolones with antibacterial properties were discovered from the bacte-rium Pseudonocardia spp. CL3849, as well as various plant sources [50].

As early as the 1950s, companies became interested in expanding their screening efforts to find compounds for treatment of cancer. Anti-cancer agents were screened by observing the reduction of transplanted or experimentally induced tumors in rodents or by observing cytotoxic effects of tumor cell lines grown in culture. Whole animal tests were also used to distinguish antibacterial or antifungal activity from cytotoxic activity and were used to inform companies that antibacterial agents such as kirromycin or oligomycin, or the antifungal agent rapamycin, for exam-ple, were too toxic to be considered for drug development. On the other hand, the same assays were used to initially identify the anti-tumor activities of daunorubicin, mito-mycin C, bleomycin, and camptothecin (Fig. 1) [39, 46, 63, 122]. In the 1960s and 1970s, companies also started to become interested in finding NPs for treatment of heart disease, neurologic disorders, and metabolic diseases, but facile assays to screen for effective compounds in these therapeutic areas were not readily available. Sandoz (now Novartis) discovered cyclosporine A (Fig. 4) using an assay developed to test both immunosuppressive and anti-tumor activities in a single mouse. [55, 109]. Blood drawn from mice 7 days after they were immunized with sheep erythrocytes (antigen) agglutinated when the antigen was added to the test tube. To test for immunosuppressive activ-ity, the agent was given by intravenous administration to the mice at the time of inoculation with the antigen, and for a number of days thereafter. If the blood drawn at 7 days did not agglutinate in the presence of the antigen, the com-pound was said to have blocked the immune response–immunosuppressive. Because the same mice carried trans-planted tumors, antitumor activity could be measured by suppression of tumor growth after the test compound was administered. This expensive, low-throughput method was replaced by the mixed lymphocyte reaction (MLR) test in the 1980s, which is described below.

The second 30 years (1970s–2000s):

knowledge?based approaches

As mentioned above, other than some miniaturization of organism cultivation and improvements in NMR and MS analyses for structure determination, used extensively for dereplication purposes (to eliminate rediscovery of known compounds), the processes used to produce NPs for discovery during the second 30 years remained largely unchanged. New sources for NPs were sought during this period. The hallmark of the second 30 year period was the

great expansion in screening methodologies and strategies. Advancements in recombinant DNA and other technolo-gies enabled researchers to rapidly determine mechanism of action (MOA) of NPs, their semi-synthetic analogues, and synthetic compounds, and rapidly led to the develop-ment of biochemical or whole cell assays, collectively known as target-based screening. In addition, the growing understanding of cell cycle regulation, the role of recep-tors in biologic responses, the genetic bases of cancer, etc., along with improvements in cell culture methodology, led to the development of a large number of selective pheno-typic screens for agents active against cancers, various neu-rologic, metabolic, or cardiac disorders or abnormalities. Both target-based and phenotypic screening are too vast to be described comprehensively here, but are covered in a number of reviews [74, 81, 102, 118]. We present here a brief overview of some of the approaches taken, including a number of examples that led to successful development of NP-based drugs.

New sources of NPs

Phenotypic screening of soil samples for NPs continued in the 1970s and 1980s, but was expanded, as described below, beyond the search for antibacterial or antifungal drugs. In addition, new sources beyond soils were sought for NP discovery. This included marine environments, with a heavy focus on the discovery of novel anti-cancer agents. Samples collected by certified divers or sampling devices included bacteria, algae, and marine invertebrates (tuni-cates, corals, bryzoans, sea slugs, sponges, etc.), and the NPs extracted from the samples were tested in new anti-cancer screens described below.

Phenotypic screening

As described above, the early cancer drugs, mitomycin C, daunoribucin, and actinomycin (Fig. 3) were discovered using cancer cell lines transplanted into animals. Starting in the 1960s, companies and academic laboratories interested in anti-cancer agents began to produce stable cell lines that were differentiated for specific types of cancer (e.g., lung, breast, etc.) to screen fermentation broths, as well as their compound collections consisting of purified NPs, semi-synthetic and synthetic chemicals. In the 1980s, the US National Cancer Institute began to develop their own col-lection of stable, differentiated specific cancer cell lines, culminating in the collection known as NCI-60, a panel of 60 cell lines differentiated for breast, lung, colon, prostate, renal, ovarian, or CNS cancers, as well as those for leuke-mia, and melanoma, and set up a service to test investiga-tional compounds (including NPs) for efficacy (providing dose response curves) and specificity (IC50 determinations using the entire NCI-60 panel). For compounds discov-ered before the panel was in use, for example paclitaxel (Fig. 3), the results provided by NCI led to the develop-ment of the drug for use in breast, lung and ovarian cancer. The primary use of the NCI-60 panel today, however, is for screening (efficacy, specificity, and MOA) of new NPs, and other putative anti-cancer agents [104].

As of 2014, three compounds derived from NPs from marine invertebrates have been approved for treatment of various cancers. These include eribulin, a synthetic derivative of halichondrin B (Fig. 3), trabectidin, a semi-synthetic derivative of the NP safricin B, and PM-10450, a synthetic compound derived from jorumycin from a sea slug, and renieramycin J from a marine invertebrate. It is not yet known if these compounds are produced from the marine invertebrates themselves, of from yet to be identi-fied bacterial symbionts. A number of other semi-synthetic or synthetic derivatives, or antibody-conjugates of marine invertebrate NPs are in various stages of clinical trials. The compound salinosporamide A (Fig. 3), isolated from the marine bacterium Salinispora tropica is the single example to date of a potential anti-cancer NP from a marine bacte-rium that made it into clinical trials [30].

A second example of a phenotypic screen developed in the second period of NP discovery is the mixed lympho-cyte reaction (MLR), used to screen for immunosuppres-sive agents. In general when lymphocytes of two different allotypes are mixed, an immunologic response causes them to grow and divide. If one of the set of lymphocytes is uv-inactivated, it can serve as a stimulator for growth and divi-sion of the un-irradiated responder lymphocytes. Growth can be measured by following 3H-thymidine incorporation (into DNA) or by following OD570 in cells treated with the tetrazolium dye MTT (3-[4,5-dimethylthiozol-2-yl]-2,5-di-phenyltetrazoium bromide) [37, 82]. Immunosuppressive activity is detected by inhibition of growth of the responder cells. Separate cell-based assays are used to distinguish the immunosuppression from cytoxicity. Many versions of the MLR have been developed, from employing whole blood from different donors, to using splenocytes drawn from dif-ferent lines of mice [60]. Validation of the MLR employed the use of cyclosporin A (Fig. 4) to demonstrate immu-nosuppression. The MLR assay played a prominent role in the discovery of two new NPs currently approved as immunosuppressive agents, FK506 (tacrolimus) and rapa?mycin (Fig. 4). The immunosuppressive activity of FK506, produced by Streptomyces tsukubaensis, was detected at Fujisawa (now Astellas) in a primary screen using the MLR assay. Rapamycin, isolated from a strain of Streptomyces hygroscopicus collected on Easter Island, was discovered in the early 1970s by microbiologists at Ayerst Research Lab-oratories in an antifungal screen, but the compound proved to be too toxic to develop as an antifungal drug [123]. Two

years after the initial report of the antifungal activity, phar-macologists at Ayerst reported immunosuppressive activity demonstrating the complete prevention of experimentally induced immunogenic disorders in rats [75]. Work on the mode of action of rapamycin, including demonstration that at least one binding target of the drug differed from that of FK506 continued during the 1980s, but serious develop-ment of rapamycin as an immunosuppressive drug did not begin until the late 1990s, after direct comparisons of activ-ity between rapamycin and FK506 or cyclosporin A using the MLR assay were published [52].

Target?based approaches

In phenotype screens, the mode of action (i.e., molecular target) of the compound tested is not known or assumed. In contrast, target-based screening is based on the implica-tion or the validated understanding that interaction of tested compound with the designated target (usually an enzyme, enzyme complex, or a receptor) will result in the desired pharmacological effect. The screen is an assay that pro-vides a measurable read-out if the tested compound inter-acts specifically with the target. Target-based assays can be as simple as in vitro enzyme assays. Target-based assays can also employ genetically engineered yeast or bacteria in whole-cell survival read outs, similar to that used in pheno-typic screening.

Only a very small number of NPs were identified by using a specific enzyme assay and later developed as drugs. Two are described here: lovastatin and clavulanic acid. As early as the middle 1960s, the biochemical pathway of cho-lesterol biosynthesis was fairly well understood and the enzyme hydroxymethylglutaryl-CoA reductase (HMGR) was known to convert HMG-CoA to the intermediate mevalonate. Akira Endo at Sankyo developed a three step assay (two phenotypic screens and a target-based assay) using liver tissues extracted from rats to screen fermenta-tion broths to find NPs that specifically inhibited HMGR [43]. In the first screen, the broths were screened examin-ing the conversion of 14C-acetate to 14C-non-saponifiable lipids (e.g., cholesterol). Broths that exhibited inhibition were then screened in an assay that measured conversion of 3H-mevalonate to 3H-lipids. Broths that did not inhibit this conversion were then assayed for the conversion of 14C-HMG-CoA to 14C-mevalonate. Using this approach, Endo discovered the compound ML-236B, later named compactin (Fig. 4), from the broth of a strain of Penicil-lium citrinum that was isolated from a rice sample collected from a vendor in Kyoto. Compactin was not developed as a drug, however. Using screening strategies analogous to that used to discover compactin, Endo and a group at Merck independently discovered lovastatin, a methylated analog of compactin, from two different fungi, Monascus ruber and Aspergillus terreus, respectively [2, 42]. Merck developed lovastatin to become the first in class of the statins successfully used to treat hypercholesteremia. Sec-ond-generation synthetic derivatives of compactin and lov-astatin, known under the trade names Pravachol?, Zocor?, and Lipitor? are among the world’s largest selling drugs. In retrospect, the contributions that Endo brought to human health with the discovery of compactin may not have been adequately recognized.

Clavulanic acid was discovered by Beecham [98] in a culture broth of a cephamycin producing strain of Strep-tomyces clavuligerus first described by Lilly scientists in 1971 [8, 83]. Although clavulanic acid was not active enough to be considered for development as an antibacte-rial agent, it was later shown to be an effective competi-tive inhibitor of a number of β-lactamases which, by as early as the 1950s, were known to be mediators of clinical resistance of Gram positive and Gram negative pathogens to penicillin and other β-lactam antibiotics [95]. Clavu-lanic acid has been combined with amoxicillin, a semi-syn-thetic derivative of penicillin, in the drug Augmentin? to widen the spectrum of pathogens susceptible to antibiotic treatment.

During the second 30 year period, discovery of new NPs from screening continued. We cannot accurately state the number of NCEs discovered during this period, but the number of patents filed world-wide exceeded 1300 [70]. Yet less than two dozen novel NPs, their-semi-synthetic or completely synthetic derivatives that were discovered dur-ing this period were developed as drugs [114]. In contrast to the first 30 year period, which gave rise to the introduc-tion of dozens of antibacterials NPs into clinical practice, the only novel anti-bacterial NP discovered during the sec-ond 30 year period that went through clinical development was daptomycin; the echinocandins represent the only class of novel antifungal NPs discovered during this period that were later approved as drugs. On the other hand, numerous 2nd, 3rd, and in the case of the cephalosporins, 4th genera-tion semi- or completely synthetic derivatives of antibac-terial NPs, representing many chemical classes, were suc-cessfully developed during this period.

During the 1990s, combinatorial chemistry was devel-oped that could give rise to thousands of unique com-pounds from hundreds of chemical scaffolds. Most phar-maceutical companies either purchased or developed their own ‘combi-chem’ libraries that numbered in the 5 × 105 to 4 × 106 range. In addition, advances in robotics together with the development of receptor-binding or enzyme-based assays with facile read-outs enabled companies to run these libraries through multiple high-throughput screens (HTS) simultaneously. To handle the large numbers, mixtures were made containing known compounds and screened; mixtures yielding “hits” were deconvoluted in subsequent

screening rounds. In contrast, NP preparation of broths could not be scaled up practically and, hence, could not keep pace with the generation of known synthetic chemi-cals. The prospect of screening endless numbers of known compounds rapidly versus the low-throughput phenotypic screens of small numbers of broths that may or may not have contained NPs was too enticing for management in pharmaceutical companies to pass up. By the mid-1990s, most pharmaceutical companies in the US and Europe stopped screening natural products. The exception was Novartis, which continues NP screening [102].

During the period between the mid-1990s to the mid-2000s, millions of compounds were screened against hun-dreds of targets in areas relating to metabolic diseases, cancer, neurobiology, cardiovascular, immunology, and infectious disease. More compounds were screened in this period than in the preceding 60 years of drug discovery, yet this effort returned very few quality compounds, and to date has produced no approved drugs. GSK ran a library of upwards of 500,000 synthetic compounds through 70 antibacterial screens on targets from Streptococcus pneu-monia. All the targets were validated as essential proteins involved in macromolecular biosynthesis, including targets for which there were known NP antibiotics: MurA, fosfo-mycin; FabI/FabH, cerulenin, thiolactomycin; PBP2, peni-cillins, cepahlosporins. All of the screens either returned no hits, or hits that were not turned into leads. The same 500,000 compound library was also run through whole cell antibacterial screens using E. coli and Staphyloccocus aureus, again resulting in no useful leads [96]. Mutasynthesis and precursor?directed biosynthesis Mutational biosynthesis, commonly referred to as muta-synthesis, is the production of novel NPs through the incorporation of analogs of NP intermediates into biosyn-thetic pathways. This approach, coined by Kenneth Rine-hart, was started in the late 1960s in his laboratory, and used extensively throughout the 1970s and 1980s. Feed-ing streptamine or epistreptamine, analogs of the normal precursor 2-deoxystreptamine, to mutants of Strepomyces fradiae blocked in neomycin biosynthesis, caused produc-tion of neomycin analogs [103]. Similar mutasynthesis programs were used to produce analogs of novobiocin, the nucleoside antibiotic nikkomycin, as well as other NPs [129]. The early studies were undertaken before full knowledge of the biochemical pathways became available. Understanding the steps in secondary metabolite biosynthe-sis, coupled with mutagenesis to block specific steps has enabled a related approach termed product-directed bio-conversion [10]. An example is the approach was taken at Eli Lilly and Company in the 1980s to generate macrolide antibiotics related to tylosin. Macrocin, the immediate precursor to tylosin, was produced by a mutant blocked in terminal O-methylation generated by N-methyl-N’-nitro-N-nitrosoguinadine (MNNG) mutagenesis, affording a fer-mentation process to overproduce macrocin [22]. Purified macrocin was then fed to Streptomyces thermotolerans, a strain that expresses 4″-mycarosyl isovaleryl-CoA trans-ferase, leading to the biosynthesis of 4″-isovalerylmacro-sin, a highly active antibiotic that was evaluated as an ani-mal health product by Lilly Elanco [121]. Although Lilly Elanco chose not to develop this compound, other compa-nies currently manufacture a related compound, 3-acetyl-4″-isovaleryltylosin (AIV or Tylvalosin), for treatment of Mycoplasma gallisepticum air sac infections in chickens, by feeding tylosin to Streptomyces thermotolerans for bio-conversion [135].

Other mutants blocked in tylosin biosynthesis produced high levels of other precursors or shunt metabolites which served as starting materials for semi-synthesis. One mutant produced desmycosin, which lacked a single sugar resi-due. Desmycosin was used as a scaffold for semi-synthesis culminating in the discovery of tilmicosin [35], marketed by Lilly Elanco as Micotil?. Semi-synthesis has also been used to generate a second generation derivative of spino-sad (Fig. 2), a glycosylated type I polyketide insecticide marketed by Dow AgroSciences for plant crop protec-tion. MNNG-induced mutants of Saccharopolyspora spi-nosa blocked in spinosad biosynthesis were isolated and evaluated for insecticidal activities [69, 125]. One of the mutants was defective in the O-methylation of the 3′-OH of the ramnosyl moiety and accumulated high levels of fac-tors J and L, the starting materials for the semi-synthesis of spinetoram, which is marketed as Radiant? by Dow Agro-Sciences [45, 108].

Precursor-directed biosynthesis also employs the addi-tion of specific compounds to the growth medium that ulti-mately get incorporated into the pathway resulting in the biosynthesis of a novel NP. Unlike mutasynthesis, however, the compound supplied does not replace an intermediate in the pathway, but a precursor, hence mutants employed are not in the biochemical pathway per se. The best example of precursor-directed biosynthesis is the production of the anthelminthic compound doramectin. By the early 1980s, it was well understood that the avemectins employed the precursors isobutyryl-CoA (e.g., avemectin 1B; Fig. 2) or 2-methylbutyryl-CoA to initiate their biosyntheses in S. avermitilis. In the 1990s, the Pfizer group identified the set of bkd genes in S. avermitilis that were respon-sible for the breakdown of branched-chain amino acids valine, isoleucine, and leucine that resulted in the produc-tion of isobutyryl-CoA and 2-methylbutyryl-CoA, as well as isovaleryl-CoA. After disrupting this pathway, they found that numerous carboxylic acids could be introduced into the “starting” position of avermectin, giving rise to a

variety of novel analogs [38]. Among them was the com-pound named doramectin, which carries cyclohexane in place of the isobutyate or 2-methylbutyrate side chain. Ser-endipitously, the S. avermitilis avermectin producing strain contains one or more acyl-CoA synthetases that have broad substrate specificities which, upon direct feeding of free acids, enable them to generate a variety of acyl-CoAs that can initiate avermectin biosynthesis. A similar approach to changing the structure of the starter employed in rapamy-cin biosynthesis was attempted. The dihydroxycyclohexyl starter unit of rapamycin was replaced with a hydroxycy-clohexyl- or hydroxycyclohepatanyl-unit when the corre-sponding carboxylic acids were fed to a rapK mutant of the rapamycin producer, Streptomyces hygroscopicus, blocked in the production of the natural starter [48]. Similarly, feed-ing of sulfur-containing analogs of the precursor pipecolic acid to a rapL mutant blocked in pipecolic acid biosynthe-sis generated a number of sulfide- or sulfoxide-containing rapamycin analogs [65]. None of these naturally produced rapalogs were advanced to clinical trials, however.

Two important scientific and technological develop-ments, briefly reviewed here, took place during this 30 year period that had significant impact on NP discovery: the development of genetic tools for streptomycetes, and the detailed understanding of NP biosynthesis.

Genetic tools for streptomycetes

The development of recombinant DNA (rDNA) technol-ogy in Escherichia coli took place in the early 1970s; NPs played an important role in its development as all vectors used for cloning depended on the use of genes that con-ferred resistance to antibiotics (penicillin, kanamycin, chloramphenicol, etc.) [33]. Development of rDNA tech-nologies in Streptomyces species lagged for about a dec-ade. Some of the initial advancements included the devel-opment of plasmid vectors and protoplast transformation and regeneration techniques for a variety of both academic and industrial strains [e.g., see 27, 76]. Later, bifunctional vectors were constructed that facilitated engineering of secondary metabolite genes in E. coli followed by conju-gation into streptomycetes and site-specific integration into bacteriophage attachment (attB) sites for stable main-tenance of the engineered genes or gene clusters [18, 28]. These advancements were followed by improved genetic engineering methods facilitated by the use of the λ RED recombination system of cloned genes and gene clusters in bacterial artificial chromosomes (BACs) in E. coli, fol-lowed by conjugation and site-specific integration into any of a number of attB sites to facilitate combinatorial bio-synthesis [3, 20, 87, 88]. In addition, all analytical meth-ods developed for determining gene expression (transcrip-tomics, RNA-Seq), enzyme levels (proteomics), precursor supply (metabolomics) in E. coli were directly applicable to streptomycetes.

Biochemistry and genetics of NP biosynthesis Biochemical pathways for the production of penicillins and cephalosporins were determined by the middle 1960s, employing feeding experiments and biochemical assays on isolated enzymes [97]. When DNA cloning in actinomy-cetes and DNA sequencing became available, biochemical pathways of NPs were inferred largely from the sequences of the genes that determined the corresponding enzymes, although enzymology was always prominent in academic labs interested in NP biosynthesis. Involvement of genes in biochemical pathways was often determined by gene disruption, followed in some instances by complementa-tion analysis. As more biosynthetic genes were sequenced, informatics was used to construct the biochemical steps determined by the predicted enzymes. This paradigm has been used for more than 100 NPs where pathways have been verified genetically, as well as for hundreds more of their structural relatives for which genetic verifica-tion is lacking. All of these data established that, overall, biochemical pathways of NPs appear to contain discrete, single function enzymes that act on and release diffusible compounds, and large multifunctional enzyme complexes that carry out many processive enzymatic steps in a linear order where all compounds are tethered to the complex in the form of thioester linkages. A brief overview is pro-vided here. Detailed descriptions of biochemical pathways, genetics of NP biosynthesis, and structural information of multifunctional enzymes can be found in other reviews [54, 56, 71, 111, 128]. Aminoglycosides and other NPs consist-ing exclusively of sugars, deoxysugars, or acylated sugars, are produced by discrete single-function enzymes. The tripeptide backbones of penicillins and cephalosporins, the tetrapeptide subtilisin, and the cyclic tridecapeptide dap-tomycin are each produced by enzyme complexes collec-tively named non-ribosomal peptide synthetases (NRPS). NRPSs consist of modules, which themselves consist of domains that are responsible for the incorporation of one selected amino acid into the nacent polypeptide chain. The peptide backbone of the glycopeptide vancomycin consist-ing of seven amino acids is biosynthesized by three NRPS proteins containing a total of seven modules. The sugar, vancosamine, is generated from glucose by seven discrete enzymes. Interestingly, the NRPS that produces the cyclic decapeptide cyclosporin A consists of a single protein con-taining ten modules. Similarly, the large macrocyclic back-bones of the polyketides erythromycin, tylosin, spinosyn A, avermectin, amphotericin B, etc., are produced by specific multifunctional enzymes designated polyketide synthases (PKSs) which incorporate, in a processive fashion, specific

acyl-CoA precursors, into a growing acyl (polyketide) chain which is tethered to the multienzyme as a thioester until released at the termination of synthesis. These type I PKSs are also composed of modules which themselves consist of enzymatic domains responsible for a single step in the biosynthesis of the polyketide backbone. The sugar components of erythromycin, tylosin, and others are pro-duced by discrete enzymes.

Early cloning experiments, followed by sequencing of biosynthesis genes initially, and entire genomes afterward consistently revealed that the genes for entire pathways of NP biosynthesis were clustered, even in eukaryotes (e.g., fungi) whose genomes are composed of multiple chromo-somes. The erythromycin biosynthesis cluster consists of three large PKS genes (35 kb) flanked by 17 genes involved in the biosynthesis of sugars, hydroxylation of the pol-yketide backbone, methylatation of one of the sugars, and a thioesterase, as well as a gene conferring resistance to erythromycin [113]. The entire cluster is greater than 50 kb. Some NP biosynthesis clusters are much larger. Some clus-ters (e.g., tylosin [34]), contain a number of positive and negative regulatory genes. Clustering facilitates the clon-ing of the entire NP biosynthesis pathway into vectors, as described above, for production of NPs in heterologous hosts, or for combinatorial biosynthesis.

Combinatorial biosynthesis

At Eli Lilly and Company, advances in genetic engineering methodologies facilitated the generation of novel polyke-tides and glycopeptides. Building on the precursor-directed biosynthesis studies described earlier [7], scientists at Lilly cloned the 4″-mycarosyl isovaleryl-CoA transferase gene (carE) from Streptomyces thermotolerans and expressed it in Streptomyces ambofaciens, the spiramycin producer [44]. Expression of carE in S. ambofaciens caused the pro-duction of isovalerylspiramycin. This served as an early validation of what would later be termed combinatorial biosynthesis [10]. A second example was the reprogram-ming of the polyketide synthase involved in spiramycin biosynthesis by exchanging a module with a different acyl-transferase specificity to produce a novel hybrid polyketide [72]. A novel glycopeptide was generated by cloning the glucosyltransferase gene (gtfE) from the vancomycin pro-ducer, A. orientalis, into the A47934 producer, Streptomy-ces toyocaensis [107]. This was the first novel glycopeptide produced by genetic engineering, and it further validated the concept of combinatorial biosynthesis in Streptomyces species.

At Cubist Pharmaceuticals, the use of λ RED recom-bineering of lipopeptides cloned in BAC vectors facilitated combinatorial biosynthesis around the core cyclic peptide structures of daptomycin and A54145 to generate about 120 novel derivatives for evaluation as potent antibacterial agents active against Gram-positive pathogens [3, 4, 20, 87, 88]. The work at Cubist helped define the “rules” for suc-cessful engineering of NRPS pathways, and demonstrated that complex pharmacological properties can be modulated by changing the primary amino acid sequences of the tride-capeptides of daptomycin and A54145 in ways not address-able by synthetic chemistry.

Combinatorial biosynthesis of modular PKSs has had limited successes at Lilly, Abbott, Kosan, and Biotica. Changes to the structure of erythromycin, tylosin, pikromy-cin, epothilone and other polyketides were initially accom-plished by domain swaps employing cumbersome and time consuming double reciprocal, two step recombination in the producing organism [93, 100, 116, 117]. At Kosan, the erythromycin PKS was re-synthesized employing E. coli codon usage and unique restriction sites so that domain swaps could be done combinatorially using restriction site cloning leading to multiple changes in the structure of the erythromycin polyketide backbone [77]. In addition, scien-tists at Kosan showed that the terminal two modules of the erythromycin PKS, carried on a single PKS polypeptide, could be replaced by the last two modules of the pikro-mycin or oleandomycin PKSs to yield hybrid polyketides. This work was carried out by combining plasmids carrying separate PKS modules from the ery, pik, or ole PKSs in the host Streptomyces lividans [116]. At Kosan, hybrid PKS composed from the tylosin and chalcomycin PKSs or the tylosin and spiramycin PKSs produced the expected hybrid macrolactone backbones. When these hybrid PKSs were placed in a strain of Streptomyces fradiae deleted for the native PKS genes, the hybrid macrolactones produced were then acted upon by the tylosin tailoring enzymes (glycosyl transferases, hydroxylases, methytransferases) to produce novel, fully elaborated NPs [99]. More progress in PKS engineering has been made in recent years as better under-standing of PKS structure has emerged, but the promise of new polyketide-based drugs has not yet been realized. Genomics?based approaches (2000s and beyond)

In the early 2000s, the first two Streptomyces genome sequences revealed the striking observation that many more secondary metabolite gene clusters (SMGCs) are encoded in these large genomes than predicted from their expressed secondary metabolomes [24, 31, 58, 59]. These observations have since been generalized to other actino-mycetes with large genomes [6, 14, 17, 23, 57, 62, 84, 90]. It is now estimated that <10 % of SMGCs are expressed in sufficient quantities to be observed under routine fermenta-tion analyses, and the others require special conditions or genetic manipulations to reveal their products [21, 89, 133,

134]. Among the actinomycetes, the number of SMGCs varies widely [14, 84], and actinomycetes with very large genomes tend to be the most “gifted” [17, 19]. An extreme example is Streptomyces rapamycinicus (a rapamycin pro-ducer) which has a 12.7 Mb genome with 3.0 Mb (24 %) devoted to the biosynthesis of 48 SMGCs [23]. Sequence information also revealed the high frequencies of biosyn-thetic clusters of the NPs discovered in the early discovery period, as well as the presence of antibiotic “resistomes” (multiple genes for antibiotic resistance) among soil iso-lates [131]. Indeed, current knowledge on mechanisms of antibiotic resistance can be exploited to select directly for producers of vancomycin and related glycopeptides from soil actinomycetes [119]. This concept can be extended to other antibiotic classes where the mechanism of antibacte-rial resistance is known and easily selectable.

With inexpensive genome sequencing, advances in understanding secondary metabolite biosynthesis, and advances in analytical and bioinformatics methods, it is now possible to predict at least partial structures of new SMs related to known SMs, and truly novel SMs by scan-ning DNA sequences [78, 120, 126]. Although gene clus-ter sequences are not yet available for all known SMs, this process continues to improve as more SMGC sequences become available. Predicting new and novel pathways from genome sequence data bypasses the tedious task of derep-lication of known compounds, which has been the bane of natural products discovery in recent years [9, 11, 13]. Fur-thermore, improved technology for DNA synthesis, clon-ing, and heterologous expression allows the rapid trans-plantation of novel NP clusters from the sequenced host into well characterized, genetically tractable engineered hosts that have high levels of needed precursors, and con-stitutively expressed strong promoters, together with new high-throughput MS detection methodology [105, 132], to enable high probability production and detection of novel NPs [21].

Genome mining and activation of cryptic pathways

As described above, it is now apparent that actinomy-cetes with large genomes encode multiple (mostly cryptic) SMGCs [1, 6, 23, 24, 31, 58, 59, 90] ranging from ~20 to 50 per genome. It is now feasible to sequence 10,000 [62] to over 100,000 actinomycete genomes in pools (Warp Drive Bio, unpublished data) to identify new and novel SMGCs. There are a number of genetic and fermentation methods to activate the expression of cryptic or poorly expressed SMGCs. Genetic methods include: (1) chemical and transposon mutagenesis; (2) gene cluster duplication/ amplification; (3) overexpression of positive regulators;

(4) disruption of negative regulators; (5) modulation of the transcription apparatus; (6) modulation of the translation apparatus; (7) modulation of post translation of carrier proteins; (8) heterologous expression; (9) synthetic biol-ogy refactoring of promoters and ribosome binding sites; and (10) combinations of (1) through (9). These and other methods to activate or improve the production of SMs are reviewed in detail elsewhere [15, 16, 21].

Aside from exploring multiple fermentation media, there are a number of elicitors or stimulators of SM production, including certain types of stress or chemicals that can be added to fermentations (e.g., heat, ethanol, rare earth ele-ments, glucosamine, and others) [89, 133, 134] or antibiot-ics added at sub-inhibitory concentrations [5, 115]. This is an area ripe for future investigations.

Advances in combinatorial biosynthesis

Early attempts at combinatorial biosynthesis as a means to produce novel NP structures were limited by a lack of understanding of the fundamental structural requirements of these megasynthases for activity. Much progress has been made in understanding the important protein–protein interactions critical to the functioning of PKS and NRPS megaenzymes [12, 20, 40, 66, 73, 128, 130]. In addition, much is now known about effective approaches for com-binatorial biosynthesis of post-PKS and other modifica-tions of SMs [12, 51, 91, 92]. Combinatorial biosynthesis can also be coupled with medicinal chemistry to further expand the numbers and variety of compounds available for evaluation [67]. With the rapid accumulation of new DNA sequences for novel PKS, NRPS and other SMGCs afforded by inexpensive microbial genome sequencing, which can serve as important new sources of “parts and devices” for combinatorial biosynthesis, it is time to rein-vigorate this important approach as an adjunct to microbial genome mining for drug discovery.

The future of natural product discovery

We believe that three key elements will drive natural prod-ucts discovery from microbes in the coming years. 1. We have already learned that microbes (particularly actino-mycetes) with large genomes encode multiple SMGCs, most of which are cryptic, and many of which appear to encode novel SMs. Many more cryptic pathways will be uncovered by inexpensive genome sequencing. Though structural details of truly novel SMs cannot be predicted accurately by bioinformatics alone, the current programs are rapidly improving their predictive power and can read-ily identify truly novel SMGCs [61, 78, 105, 120, 126]. These novel gene clusters can be expressed by genetic and physiological manipulations of the producing microbes or by expression in a heterologous hosts. 2. Scientists have

sampled a miniscule fraction of soil environments [9, 13] and metagenomic and other studies indicate that there exists a wealth of novel SMGCs yet to be discovered [32, 64, 79, 94, 106]. Vastly expanded sampling of soil will provide an inexhaustible source of new microbes (cultiva-ble or not) for genome mining. 3. Advances in structural biology of complex PKS megaenzymes [40, 128, 130] and in functional analysis of engineered NRPS megaenzymes [20] bode well for future applications of combinatorial biosynthesis to exploit novel PKS and NRPS modules and domains identified by genome mining. These three cor-nerstones will be supported by continuing advancements in enzymology, structural biology, bioinformatics, tran-scriptomics, proteomics, metabolomics, and advances in mass spectral analysis. In addition, as understanding of cellular function and the molecular basis of human disease increases, more “smart-screens” will be developed to find NPs with desired activities. Indeed, we predict a second “Golden Age for Antibiotics” and natural product discov-ery. Stay tuned!

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史上最全的外语类科研项目申报干货汇总.

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一、数据表

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地震勘探技术新进展_杨勤勇

第25卷第1期2002年2月 勘探地球物理进展 Progress in Exploration Geophysics Vol.25,No.1 Feb.2002地震勘探技术新进展 杨勤勇1徐丽萍2 (1.中国石化石油勘探开发研究院南京石油物探研究所,江苏南京210014; 2.西北石油局规划设计研究院,乌鲁木齐830011) 摘要:近几年来,地震勘探技术得到了很大的发展。超万道地震仪的投入使用,以及优化采集设计技术的发展,有效地提高了采集效率和资料质量;叠前深度偏移技术使复杂构造的成像更为清晰;3D可视化技术和虚拟现实技术大大提高了地震解释的能力、精度和速度;地震属性技术的发展把地震解释向定量化解释推进了一步;井中地震技术、多波多分量地震技术以及时延地震技术的发展,有力地增强了油气静态描述和动态监测的能力;复杂介质中地震波传播规律的研究向传统的层状介质理论发起了冲击。 关键词:可视化;虚拟现实;地震属性;成像;井中地震;VSP;多分量;时延地震 中图分类号:TE132.1+1文献标识码:A 地震勘探是利用地层岩石的弹性特性来研究地下地质结构,推断岩体物性,预测油气的一种勘查方法。几十年来,地震勘探以其高信噪比、高分辨率、高保真度、高精确度、高清晰度和高可信度等赢得了广大用户的信任,成为找油找气的关键技术。在油气勘探开发中,应用地震勘探已有效地解决了一系列复杂的地质问题,在各种复杂构造油气藏和隐蔽油气藏的勘查方面取得了重大成果,给油气公司带来了可观的经济效益。 近几年来,以PC计算机群大规模投入使用,可视化、虚拟现实、网络技术飞速发展为标志,以高分辨率地震、3D地震为代表,以4D地震、井中地震、多波多分量地震为发展前沿的地震勘探技术正跃上新的台阶,高密度采集和3D空间成像归位技术以其精确、灵活显示等优点,在国内外已卓有成效地用于查明各种复杂构造油气藏和隐蔽油气藏。 1主要进展 1.13D可视化技术[1~4] 可视化技术是把描述物理现象的数据转化为图形、图像,并运用颜色、透视、动画和观察视点的实时改变等视觉表现形式,使人们能够观察到不可见的对象,洞察事物内部结构。方法包括以图形为基础(或称为面可视化)和以体素为基础(体可视化)的可视化。在以体素为基础的体可视化中,每一个数据采样点被转换成一个体素(一个3D象素的大小近似于面元间隔和采样间隔)。每一个体素有一个对应于源3D数据体的值,一个RGB(红色、绿色、蓝色)色彩值以及可被用来标定数据透明度的暗度变量。 多年来,许多公司致力于地学可视化应用软件的开发,取得了可喜的成果。在3D图形工作站环境支持下,各种基于数据体操作、图素提取与曲面造型、体绘制技术的应用软件相继出现,它们基本上代表了当今综合解释工作站3D可视化软件功能的发展水平(见表1)。 表1有代表性的可视化解释处理软件 公司软件 Landmark 3DVI(3D体积解释) Voxcube(3D立体动画) Geoquest GeoViz(交互3D解释) Paradigm Voxel Geo(真3D地震解释系统) DGI Earth Vision (基于3D空间地质建模) Photo3DViz(3D体可视化) 体可视化允许解释人员直接进行地层解释,识别地震相,改进油藏特征描述。它通过数据的3D 立体显示,使解释人员能够作构造、断层、地层沉积、岩性、储集参数和油气等的交互解释。解释结果在三度空间内立体显示,可以激发资料处理解释人员的科学灵感,赋予他们无限的想像空间与创造力,极大地提高了工作效率和工作质量。 1.2虚拟现实技术 虚拟现实(Virtual Reality,简写为VR)是一种 收稿日期:2001-12-31 作者简介:杨勤勇(1964-),高级工程师,1985年毕业于中国地质大学物探系,现从事情报研究。