Yanwen Duan

Strain prioritization for natural product discovery by a high-throughput real-time PCR method.

Natural products offer unmatched chemical and structural diversity compared to other small-molecule libraries, but traditional natural product discovery programs are not sustainable, demanding too much time, effort, and resources. Here we report a strain prioritization method for natural product discovery. Central to the method is the application of real-time PCR, targeting genes characteristic to the biosynthetic machinery of natural products with distinct scaffolds in a high-throughput format. The practicality and effectiveness of the method were showcased by prioritizing 1911 actinomycete strains for diterpenoid discovery. A total of 488 potential diterpenoid producers were identified, among which six were confirmed as platensimycin and platencin dual producers and one as a viguiepinol and oxaloterpin producer. While the method as described is most appropriate to prioritize strains for discovering specific natural products, variations of this method should be applicable to the discovery of other classes of natural products. Applications of genome sequencing and genome mining to the high-priority strains could essentially eliminate the chance elements from traditional discovery programs and fundamentally change how natural products are discovered.

Biosynthetic potential-based strain prioritization for natural product discovery: a showcase for diterpenoid-producing actinomycetes.

Natural products remain the best sources of drugs and drug leads and serve as outstanding small-molecule probes to dissect fundamental biological processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here we report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to our approach is the innovative preparation, by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from our actinomycete collection, for their biosynthetic potential of four classes of natural products, aromatic polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products.

Actinopolysporins A-C and tubercidin as a Pdcd4 stabilizer from the halophilic actinomycete Actinopolyspora erythraea YIM 90600.

Our current natural product program utilizes new actinomycetes originating from unexplored and underexplored ecological niches, employing cytotoxicity against a selected panel of cancer cell lines as the preliminary screen to identify hit strains for natural product dereplication, followed by mechanism-based assays of the purified natural products to discover potential anticancer drug leads. Three new linear polyketides, actinopolysporins A (1), B (2), and C (3), along with the known antineoplastic antibiotic tubercidin (4), were isolated from the halophilic actinomycete Actinopolyspora erythraea YIM 90600, and the structures of the new compounds were elucidated on the basis of spectroscopic data interpretation. All four compounds were assayed for their ability to stabilize the tumor suppressor programmed cell death protein 4 (Pdcd4), which is known to antagonize critical events in oncogenic pathways. Only 4 significantly inhibited proteasomal degradation of a model Pdcd4-luciferase fusion protein, with an IC50 of 0.88±0.09 μM, unveiling a novel biological activity for this well-studied natural product.

Bafilomycins produced by an endophytic actinomycete Streptomyces sp. YIM56209

Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA; Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery, Changsha, Hunan 410329, China; Yunnan Institute of Microbiology, Yunnan University, Kunming, Yunnan 650091, China; Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA; University of Wisconsin National Cooperative Drug Discovery Group, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA; and Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA

Erythronolides H and I, new erythromycin congeners from a new halophilic actinomycete Actinopolyspora sp. YIM90600.

Erythronolides H and I, novel congeners of the clinically important antibacterial drug erythromycin A, have been isolated from the new halophilic actinomycete Actinopolyspora sp. YIM90600. In addition to producing the new erythromycin congeners, A. sp. YIM90600 produces erythromycin C in a high titer. The presence of the C-14 hydroxyl moiety and the C-6/C-18-epoxide in erythronolide H and the spiroketal moiety of erythronolide I sheds new insights into structural diversity of erythromycin analog libraries potentially accessible by combinatorial biosynthesis.

Discovery and Total Synthesis of a New Estrogen Receptor Heterodimerizing Actinopolymorphol A from Actinopolymorpha rutilus

Estrogen receptor ERalpha and ERbeta heterodimerization has been implicated in cancer chemoprevention. The discovery, structural elucidation, and total synthesis of a new natural product, actinopolymorphol A (1), from Actinopolymorpha rutilus (YIM45725) that preferentially induces ERalpha/beta heterodimerization is reported. Total synthesis of 1 has allowed us to determine its absolute stereochemistry and that of a previously known deacetylated congener, and 1 represents the first member of a new class of natural products not previously recognized to modulate ER function.

Angucyclines and Angucyclinones from Streptomyces sp. CB01913 Featuring C-Ring Cleavage and Expansion

Angucyclines and angucyclinones are aromatic polyketides with a tetracyclic benz[a]anthracene skeleton. The benz[a]anthracene scaffold is biosynthesized by type II polyketide synthases that catalyze the decarboxylative condensation of a short acyl-CoA starter and nine extender units. Angucyclines and angucyclinones, the largest group of polycyclic aromatic polyketides, achieve structural diversity via subsequent oxidation, ring cleavage, amino acid incorporation, and glycosylation. We here report the discovery of 14 angucyclinones and two angucyclines (1-16) from Streptomyces sp. CB01913, identifying 12 new compounds featuring various oxidations on rings A and C (1, 2, and 4), different sugar moieties attached to rings A and B (3 and 6), and C-ring cleavage (5 and 10-14) and expansion (8). These new structural features, highlighted by C-ring cleavage and expansion, enrich the structural diversity of angucyclines and angucyclinones. All compounds were tested for cytotoxicity and antibacterial activities, with 1, 5, 15, and 16 showing moderate activities against selected cancer cell lines or bacterial strains.

Cycloheximide and congeners as inhibitors of eukaryotic protein synthesis from endophytic actinomycetes Streptomyces sps. YIM56132 and YIM56141

Cycloheximide and congeners as inhibitors of eukaryotic protein synthesis from endophytic actinomycetes Streptomyces sps. YIM56132 and YIM56141

Strain Prioritization and Genome Mining for Enediyne Natural Products

ABSTRACT The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family. Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family.

Recent advances in inkjet dispensing technologies: applications in drug discovery

Introduction: Inkjet dispensing technology is a promising fabrication methodology widely applied in drug discovery. The automated programmable characteristics and high-throughput efficiency makes this approach potentially very useful in miniaturizing the design patterns for assays and drug screening. Various custom-made inkjet dispensing systems as well as specialized bio-ink and substrates have been developed and applied to fulfill the increasing demands of basic drug discovery studies. The incorporation of other modern technologies has further exploited the potential of inkjet dispensing technology in drug discovery and development. Areas covered: This paper reviews and discusses the recent developments and practical applications of inkjet dispensing technology in several areas of drug discovery and development including fundamental assays of cells and proteins, microarrays, biosensors, tissue engineering, basic biological and pharmaceutical studies. Expert opinion: Progression in a number of areas of research including biomaterials, inkjet mechanical systems and modern analytical techniques as well as the exploration and accumulation of profound biological knowledge has enabled different inkjet dispensing technologies to be developed and adapted for high-throughput pattern fabrication and miniaturization. This in turn presents a great opportunity to propel inkjet dispensing technology into drug discovery.