[Show abstract][Hide abstract] ABSTRACT: A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/α-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an L-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound >40-fold, going from the starting ~60mg/L to 2.5g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy.
Preview · Article · Nov 2015 · Metabolic Engineering
[Show abstract][Hide abstract] ABSTRACT: Borrelidin (1) is a nitrile-containing bacterially derived polyketide that is a potent inhibitor of bacterial and eukaryotic threonyl-tRNA synthetases. We now report the discovery of borrelidin B (2), a tetrahydro-borrelidin derivative containing an aminomethyl group in place of the nitrile functionality in borrelidin. The discovery of this new metabolite has implications for both the biosynthesis of the nitrile group and the bioactivity of the borrelidin compound class. Screening in the SToPS assay for tRNA synthetase inhibition revealed that the nitrile moiety is essential for activity, while profiling using our in-house image-based cytological profiling assay demonstrated that 2 retains biological activity by causing a mitotic stall, even in the absence of the nitrile motif.
No preview · Article · Nov 2014 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: The spliceostatin class of natural products was reported to be potent cytotoxic agents via inhibition of the spliceosome, a key protein complex in the biosynthesis of mature mRNA. As part of an effort to discover novel leads for cancer chemotherapy, we re-examined this class of compounds from several angles, including fermentation of the producing strains, isolation and structure determination of new analogues, and semisynthetic modification. Accordingly, a group of spliceostatins were isolated from a culture broth of Burkholderia sp. FERM BP-3421, and their structures identified by analysis of spectroscopic data. Semisynthesis was performed on the major components 4 and 5 to generate ester and amide derivatives with improved in vitro potency. With their potent activity against tumor cells and unique mode of action, spliceostatins can be considered potential leads for development of cancer drugs.
Full-text · Article · Aug 2014 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer-Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate-dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer-Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain-the dioxygenase fr9P(-) mutant-that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable.
Preview · Article · Aug 2014 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.
[Show abstract][Hide abstract] ABSTRACT: The conversion of the C40 secondary hydroxyl group of rapamycin into the azido group was followed by copper catalyzed cycloaddition of the resulting azido-rapamcin with various unprotected propargyl O- and S-glycosides and a C-ethynyl derivative. This approach furnished a collection of triazole-bridged rapamycin glycoconjugates (14 examples) in 44–83% isolated yield.
No preview · Article · Dec 2013 · Tetrahedron Letters
[Show abstract][Hide abstract] ABSTRACT: Covering: up to the end of 2012The Antibody Drug Conjugate (ADC) is a therapeutic modality consisting of a monoclonal antibody attached to a cytotoxic, small-molecule payload. The antibody portion of the ADC serves as a transport vehicle that recognizes and binds to a protein antigen expressed in tumor tissues. The localized delivery and release of the payload within or near malignant cells allows for targeted delivery of a potent cytotoxic agent to diseased tissue, while reducing damage to antigen-negative, normal tissues. Recent years have witnessed an explosive increase in ADC-based therapies, due mainly to clinical reports of activity in both hematologic and epithelial cancers. Accompanying this upsurge in ADC development is a renewed interest in natural product cytotoxins, which are typically highly potent cell-killing agents, but suffer from poor drug-like properties and narrow safety margins when systemically administered as conventional chemotherapeutics. In this review, we discuss recent advances related to the construction of ADCs, the optimization of ADC safety and efficacy, and the increasingly pivotal roles of natural product payloads in the current and future landscape of ADC therapy.
No preview · Article · Mar 2013 · Natural Product Reports
[Show abstract][Hide abstract] ABSTRACT: The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of S. rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 & BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
Full-text · Article · Nov 2012 · Metabolic Engineering
[Show abstract][Hide abstract] ABSTRACT: Natural products are an unsurpassed source of lead structures for drug discovery. However, these molecules, many of which fall into the beyond-rule-of-5 chemical space, are often difficult to optimize by chemical means because of their complex structures. Biosynthetic engineering of the producing host organism offers an important tool for the modification of complex natural products, leading to analogues which are unattainable by chemical semisynthesis. This review describes the current role of natural products in lead generation and the principles behind biosynthetic medicinal chemistry. It then goes on to describe five distinct drugs – salinosporamide, geldanamycin, FK506, rapamycin and epothilone – to exemplify how biosynthetic engineering approaches have contributed to the advancement of natural product clinical candidates.
No preview · Article · Aug 2012 · Medicinal Chemistry Communication
[Show abstract][Hide abstract] ABSTRACT: The pyrroloquinoline alkaloid family of natural products, which includes the immunosuppressant lymphostin, has long been postulated to arise from tryptophan. We now report the molecular basis of lymphostin biosynthesis in three marine Salinispora species that maintain conserved biosynthetic gene clusters harboring a hybrid nonribosomal peptide synthetase-polyketide synthase that is central to lymphostin assembly. Through a series of experiments involving gene mutations, stable isotope profiling, and natural product discovery, we report the assembly-line biosynthesis of lymphostin and nine new analogues that exhibit potent mTOR inhibitory activity.
Full-text · Article · Aug 2011 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
Full-text · Article · Mar 2011 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: In the course of processing extracts from fermentations with Nodulisporium hinnuleum Smith (ATCC 36102) to obtain demethoxyvirdin, we noticed that this fungus produced several other secondary metabolites as well. In an effort to identify these components, four new, related natural products, designated hinnulin A-D (1-4)
were isolated and characterized. Structure elucidation of the hinnulins, primarily by NMR spectroscopy, revealed
that these belong to the rare class of 1,2’-binaphthyl natural products.
Preview · Article · Jan 2011 · Records of Natural Products
[Show abstract][Hide abstract] ABSTRACT: Microbial sources of natural products are increasingly valued for their chemical diversity and access to biosynthetic pathways. It appears that an incredible repository of untapped microbial life and associated chemical diversity remain to be exploited. Molecular techniques such as “metagenomics” are beginning to provide access to cryptic biosynthetic pathways, which are also revealing new chemistry. Refined libraries of natural products with well-characterized components provide enhanced value as screening sources. These libraries continue to provide new chemical entities that inform biological processes and provide leads for therapeutic agents. There is a renaissance of phenotypic screening that promises to uncover numerous new links between secondary metabolites and their roles in biology. Genomic methods are growing in value as our understanding of biosynthetic processes at the molecular level expands. Screening of DNA sequences for pathways that yield particular chemistries is now a reality for certain types of biosynthetic processes, especially type I polyketide synthases and nonribosomally produced peptides.Keywords:biodiversity;marine actinomycetes;microbial genomics;natural products lead generation;natural products libraries;screening;secondary metabolites
[Show abstract][Hide abstract] ABSTRACT: J. Neurochem. (2010) 113, 1331–1342.
One of the major barriers to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that originate from the myelin sheath and glial scar. So far, only a small number of pharmacological compounds have exhibited functional activity against CNS inhibitors in promoting axon regeneration after injury. To search for novel compounds that enhance neurite outgrowth in vitro, we initiated a screen of a collection of natural products. We identified four compounds with the potential to promote growth over a myelin substrate. Of these, Amphotericin B (AmB) was shown to enhance neurite outgrowth and antagonize activities of major myelin associated inhibitors and glial-scar-derived chondroitin sulfate proteoglycans. AmB was found to activate Akt and thereby suppress the activity of glycogen synthase kinase 3 beta. Also, a cell permeable peptide that inhibits Akt activity was shown to block the effect of AmB in promoting axonal growth, while another peptide that increases Akt activity stimulated axonal growth in the presence of the myelin associated inhibitors. Our results suggest that AmB can promote neurite outgrowth over a wide range of inhibitory substrates via a mechanism that involves activation of Akt.
No preview · Article · Mar 2010 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation, and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.
No preview · Article · Mar 2009 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: Bioassay-directed fractionation of a fermentation of Pochonia bulbinosa, culture 38G272, led to the isolation of a series of structurally novel, prospective cell wall-active lipopeptides. The main component of this suite is 1, a linear hexapeptide with a delta-hydroxymyristic acid amide substituted N-terminus. The structure was deduced using high-field microsample NMR, Fourier transform mass spectrometry, and microscale chemical degradation. The potent cell wall activity and synthetically accessible structure of 1 make it a potential lead for further investigation.
No preview · Article · Dec 2008 · Journal of Natural Products
[Show abstract][Hide abstract] ABSTRACT: Two natural products, diazepinomicin (1) and dioxapyrrolomycin (2), containing stable isotopic labels of (15)N or deuterium, were used to demonstrate the utility of Fourier transform ion cyclotron resonance mass spectrometry for probing natural product biosynthetic pathways. The isotopic fine structures of significant ions were resolved and subsequently assigned elemental compositions on the basis of highly accurate mass measurements. In most instances the mass measurement accuracy is less than one part per million (ppm), which typically makes the identification of stable-isotope labeling unambiguous. In the case of the mono-(15)N-labeled diazepinomicin (1) derived from labeled tryptophan, tandem mass spectrometry located this (15)N label at the non-amide nitrogen. Through the use of exceptionally high mass resolving power of over 125,000, the isotopic fine structure of the molecular ion cluster of 1 was revealed. Separation of the (15)N(2) peak from the isobaric (13)C(15)N peak, both having similar abundances, demonstrated the presence of a minor amount of doubly (15)N-labeled diazepinomicin (1). Tandem mass spectrometry amplified this isotopic fine structure (Deltam=6.32 mDa) from mDa to 1 Da scale thereby allowing more detailed scrutiny of labeling content and location. Tandem mass spectrometry was also used to assign the location of deuterium labeling in two deuterium-labeled diazepinomicin (1) samples. In one case three deuterium atoms were incorporated into the dibenzodiazepine core; while in the other a mono-D label was mainly incorporated into the farnesyl side chain. The specificity of (15)N-labeling in dioxapyrrolomycin (2) and the proportion of the (15)N-label contained in the nitro group were determined from the measurement of the relative abundance of the (14)NO(2)(1-) and (15)NO(2)(1-) fragment ions.
No preview · Article · Dec 2008 · Bioorganic & medicinal chemistry