Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.

Discovery, Synthesis, and Biological Evaluation of Anaenamides C and D from a New Marine Cyanobacterium, Hormoscilla sp.

Our ongoing efforts to explore the chemical space associated with marine cyanobacteria from coral reefs of Guam have yielded two new members of the anaenamide family of natural products, anaenamides C (3) and D (4). These compounds were isolated from a novel Hormoscilla sp. (VPG16-58). Our phylogenetic profiling (16S rDNA) of this cyanobacterium indicated that VPG16-58 is taxonomically distinct from the previously reported producer of the anaephenes, VPG16-59 (Hormoscilla sp.), and other previously documented species of the genus Hormoscilla. The planar structures of 3 and 4 were determined via spectroscopic methods, and absolute configurations of the α-hydroxy acids were assigned by enantioselective HPLC analysis. To address the requirement for sufficient material for testing, we first adapted our published linear synthetic approach for 1 and 2 to generate anaenoic acid (7), which served as a point for diversification, providing the primary amides 3 and 4 from synthetic intermediates 5 and 6, respectively. The compounds were then tested for effects on HCT116 colon cancer cell viability and in an ARE-luciferase reporter gene assay for Nrf2 modulation using HEK293 human embryonic kidney cells. Our findings indicate that, in contrast to cytotoxic methyl esters 1 and 2, the primary amides 3 and 4 activate the Nrf2 pathway at noncytotoxic concentrations. Overall, our data suggest that the anaenamide scaffold is tunable to produce differential biological outcomes.

Bifunctional Doscadenamides Activate Quorum Sensing in Gram-Negative Bacteria and Synergize with TRAIL to Induce Apoptosis in Cancer Cells.

New cyanobacteria-derived bifunctional analogues of doscadenamide A, a LasR-dependent quorum sensing (QS) activator in Pseudomonas aeruginosa, characterized by dual acylation of the pyrrolinone core structure and the pendant side chain primary amine to form an imide/amide hybrid are reported. The identities of doscadenamides B-J were confirmed through total synthesis and a strategic focused library with different acylation and unsaturation patterns was created. Key molecular interactions for binding with LasR and a functional response through mutation studies coupled with molecular docking were identified. The structure-activity relationships (SARs) were probed in various Gram-negative bacteria, including P. aeruginosa and Vibrio harveyi, indicating that the pyrrolinone-N acyl chain is critical for full agonist activity, while the other acyl chain is dispensable or can result in antagonist activity, depending on the bacterial system. Since homoserine lactone (HSL) quorum sensing activators have been shown to act in synergy with TRAIL to induce apoptosis in cancer cells, selected doscadenamides were tested in orthogonal eukaryotic screening systems. The most potent QS agonists, doscadenamides S10-S12, along with doscadenamides F and S4 with partial or complete saturation of the acyl side chains, exhibited the most pronounced synergistic effects with TRAIL in triple negative MDA-MB-231 breast cancer cells. The overall correlation of the SAR with respect to prokaryotic and eukaryotic targets may hint at coevolutionary processes and intriguing host-bacteria relationships. The doscadenamide scaffold represents a non-HSL template for combination therapy with TRAIL pathway stimulators.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

Ahp-Cyclodepsipeptides as tunable inhibitors of human neutrophil elastase and kallikrein 7: Total synthesis of tutuilamide A, serine protease selectivity profile and comparison with lyngbyastatin 7.

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors.

Upregulation of lncRNA snoRNA host gene 6 regulates NUAK family SnF1-like kinase-1 expression by competitively binding microRNA-125b and interacting with Snail1/2 in bladder cancer.

BACKGROUNDS/AIMS: Numerous studies have reported that long noncoding RNAs (lncRNAs) play critical roles in the development and progression of bladder cancer (BC). LncRNA snoRNA host gene 6 (SNHG6) is ectopically expressed in tumor tissues of patients with BC and BC cell lines. However, little is known about the molecular mechanism of SNHG6-mediated bladder urothelial carcinoma cell migration and invasion. METHODS: We detected the SNHG6 levels in human BC specimens and cell lines by quantitative real-time polymerase chain reaction and Western blot, and investigated its role in BC using in vitro assays. RESULTS: We showed that overexpression of SNHG6 induced epithelial-mesenchymal transition (EMT) and promoted the migration and invasion capabilities of BC cells. Mechanistically, SNHG6 induced EMT of BC cells by upregulating the expression levels of Snail1/2 and regulated BC cell migration and invasion by tumor suppressive hsa-miR-125b and its target gene NUAK Family Kinase 1 (NUAK1). Furthermore, we found that SNHG6 was positively correlated with Snail1/2 expression, and negatively correlated with hsa-miR-125b expression in BC specimens. Further study showed that SNHG6 repressed hsa-miR-125b expression to upregulate Snail1/2. Conversely, hsa-miR-125b knockdown augmented SNHG6 expression in BC cells. CONCLUSION: Overall, our study demonstrated that SNHG6 promotes BC cell migration and invasion partly via the hsa-miR-125b/Snail1/2/NUAK1 pathway. Therefore, SNHG6 may be a potential prognostic biomarker in BC, and targeting hsa-miR-125b/Snail1/2/NUAK1 axis may be a promising therapeutic approach for BC patients.

Development of apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent and its preliminary evaluation in an orthotopic patient-derived xenograft (PDX) model.

Despite the significant progress in the field of cancer therapeutics, the incidence of pancreatic cancer (PC) has continuously increased. One possible mechanism for this increasing burden is impaired drug delivery and drug resistance resulting from a unique tumor microenvironment and genetic mutations. Apratoxins are potent anticancer agents and cotranslational translocation inhibitors with potential therapeutic applications to treat cancers with active secretory pathways. Here, we developed apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent which potently inhibited the growth of both established and patient-derived primary pancreatic cancer cells. We validated its mechanism of action on pancreatic cancer cells by demonstrating the downregulation of multiple receptor tyrosine kinases and inhibition of growth factor and cytokine secretion. Apra S10 also inhibited a number of cytokines secreted by stromal cells, suggesting that Apra S10 not only inhibited pancreatic cancer cell secretion, but also reduced the level of factors secreted by other cell types active within the tumor microenvironment. As Apra S10 tissue distribution indicated its high enrichment in pancreas tissue, an orthotopic pancreatic patient-derived xenograft mouse model that closely mimics the human pancreatic tumor microenvironment was for the first time used in apratoxin studies. Apra S10 showed promising antitumor effect in this pancreatic cancer model and this effect was mediated through anti-proliferation properties.

Discovery of new A- and B-type laxaphycins with synergistic anticancer activity.

Two new cyclic lipopeptides termed laxaphycins B4 (1) and A2 (2) were discovered from a collection of the marine cyanobacterium Hormothamnion enteromorphoides, along with the known compound laxaphycin A. The planar structures were solved based on a combined interpretation of 1D and 2D NMR data and mass spectral data. The absolute configurations of the subunits were determined by chiral LC-MS analysis of the hydrolysates, advanced Marfey's analysis and 1D and 2D ROESY experiments. Consistent with similar findings on other laxaphycin A- and B-type peptides, laxaphycin B4 (1) showed antiproliferative effects against human colon cancer HCT116 cells with IC50 of 1.7 µM, while laxaphycins A and A2 (2) exhibited weak activities. The two major compounds isolated from the sample, laxaphycins A and B4, were shown to act synergistically to inhibit the growth of HCT116 colorectal cancer cells.

Bladder cancer cells induce immunosuppression of T cells by supporting PD-L1 expression in tumour macrophages partially through interleukin 10.

Immunotherapy based on BCG vaccination is an effective treatment in bladder cancer, but a positive response is restricted to a subset of patients and for a limited period of time only. This suggests that T cells antitumour responses are effective but can become compromised in bladder cancer. To investigate the underlying mechanisms, we first identified peripheral blood monocytes and tumour macrophages using the pan-monocyte/macrophage marker CD14, and found that the PD-L1 expression on the monocytes/macrophages in bladder cancer patients was significantly higher than that in controls. The monocytes from bladder cancer patients were also more capable at inducing apoptosis and inhibiting proliferation in activated autologous T cells than monocytes from controls, which was directly associated with the level of PD-L1 expression. We next investigated the tumour cells' participation in upregulating PD-L1 in monocytes/macrophages. Significant elevation of PD-L1 was observed in monocytes after culturing with autologous tumour cells, which did not require direct contact but required soluble factors. The STAT phosphorylation pattern in monocytes after tumour cell co-culture was consistent with effects of the interleukin (IL)-10 signalling pathway. We then found that removal of IL-10 in monocyte-tumour cell co-culture reduced the PD-L1 upregulation in monocytes, but IL-10 by itself was unable to directly upregulate PD-L1. Primary bladder tumour cells secreted significant levels of IL-10, indicating that they could serve as the source of IL-10. Together, these results demonstrated a novel pathway that bladder cancer cells induced immunosuppression of T cells by supporting PD-L1 expression in tumour macrophages partially through IL-10.

Total Synthesis of the Potent Marine-Derived Elastase Inhibitor Lyngbyastatin 7 and in Vitro Biological Evaluation in Model Systems for Pulmonary Diseases.

Lyngbyastatin 7 (1) is a marine cyanobacteria-derived lariat-type cyclic depsipeptide of which the macrocyclic core possesses modified amino acids, including a featured 3-amino-6-hydroxy-2-piperidone (Ahp) moiety and a (Z)-2-amino-2-butenoic acid (Abu) moiety. The first total synthesis of 1 was successfully established via 31 steps, and the conditions of several crucial steps were optimized to ensure smooth operations. The previously reported structural assignment and elastase inhibitory activity of the isolated natural product were confirmed. According to the extensive in vitro biological evaluation, compound 1 displayed low nanomolar IC50 in blocking elastase activity and strong ability in protecting bronchial epithelial cells against elastase-induced antiproliferation and abrogating the elastase-triggered induction of pro-inflammatory cytokine expression. Its overall performance was superior over sivelestat, the only approved small molecule drug targeting elastase, which indicated its potential in developing as a pharmacotherapeutic against elastase-mediated pathologies. The success in total synthesis, designed with a novel convergent strategy, not only overcame the supply issue for thorough preclinical studies but also paved the way for convenient synthesis of analogues with improved potency and druglike properties.

Tumor-suppressive microRNA-145 induces growth arrest by targeting SENP1 in human prostate cancer cells.

Prostate cancer (PCa) prevails as the most commonly diagnosed malignancy in men and the third leading cause of cancer-related deaths in developed countries. One of the distinct characteristics of prostate cancer is overexpression of the small ubiquitin-like modifier (SUMO)-specific protease 1 (SENP1), and the upregulation of SENP1 contributes to the malignant progression and cell proliferation of PCa. Previous studies have shown that the expression of microRNA-145 (miRNA-145) was extensively deregulated in PCa cell lines and primary clinical prostate cancer samples. Independent target prediction methods have indicated that the 3'-untranslated region of SENP1 mRNA is a potential target of miR-145. Here we found that low expression of miR-145 was correlated with high expression of SENP1 in PCa cell line PC-3. The transient introduction of miR-145 caused cell cycle arrest in PC-3 cells, and the opposite effect was observed when miR-145 inhibitor was transfected. Further studies revealed that the SENP1 3'-untranslated region was a regulative target of miR-145 in vitro. MicroRNA-145 also suppressed tumor formation in vivo in nude mice. Taken together, miR-145 plays an important role in tumorigenesis of PCa through interfering SENP1.

Evaluation of class I HDAC isoform selectivity of largazole analogues.

Largazole is a potent class I selective histone deacetylase (HDAC) inhibitor. The majority of largazole analogues to date have modified the thiazole-thiazoline and the warhead moiety. In order to elucidate class I-specific structure-activity relationships, a series of analogues with modifications in the valine or the linker region were prepared and evaluated for their class I isoform selectivity. The inhibition profile showed that the C2 position of largazole has an optimal steric requirement for efficient HDAC inhibition and that substitution of the trans-alkene in the linker with an aromatic group results in complete loss of activity. This data will aid the design of class I isoform selective HDAC inhibitors.

Largazole pharmacokinetics in rats by LC-MS/MS.

A highly sensitive and specific LC-MS/MS method for the quantitation of largazole thiol, the active species of the marine-derived preclinical histone deacetylase inhibitor, largazole (prodrug), was developed and validated. Largazole thiol was extracted with ethyl acetate from human or rat plasma along with the internal standard, harmine. Samples were separated on an Onyx Monolithic C18 column by a stepwise gradient elution with 0.1% formic acid in methanol and 0.1% aqueous formic acid employing multiple reaction monitoring (MRM) detection. Linear calibration curves were obtained in the range of 12.5-400 ng/mL with 200 µL of human plasma. The overall intra-day precision was from 3.87% to 12.6%, and the inter-day precision was from 7.12% to 9.8%. The accuracy at low, medium and high concentrations ranged from 101.55% to 105.84%. Plasma protein bindings of largazole thiol in human and rat plasma as determined by an ultrafiltration method were 90.13% and 77.14%, respectively. Plasma drug concentrations were measured by this LC-MS/MS method. The pharmacokinetics of largazole thiol in rats was studied following i.v. administration at 10 mg/kg and found to follow a two-compartment model. Largazole thiol was rapidly eliminated from systemic circulation within 2 h. The established LC-MS/MS method is suitable for the analysis of largazole thiol in human plasma, as well.

VCP/p97 mediates nuclear targeting of non-ER-imported prion protein to maintain proteostasis.

Mistargeting of secretory proteins in the cytosol can trigger their aggregation and subsequent proteostasis decline. We have identified a VCP/p97-dependent pathway that directs non-ER-imported prion protein (PrP) into the nucleus to prevent the formation of toxic aggregates in the cytosol. Upon impaired translocation into the ER, PrP interacts with VCP/p97, which facilitates nuclear import mediated by importin-ß. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import are independent of ubiquitination. In vitro experiments revealed that VCP/p97 binds non-ubiquitinated PrP and prevents its aggregation. Inhibiting binding of PrP to VCP/p97, or transient proteotoxic stress, promotes the formation of self-perpetuating and partially proteinase resistant PrP aggregates in the cytosol, which compromised cellular proteostasis and disrupted further nuclear targeting of PrP. In the nucleus, RNAs keep PrP in a soluble and non-toxic conformation. Our study revealed a novel ubiquitin-independent role of VCP/p97 in the nuclear targeting of non-imported secretory proteins and highlights the impact of the chemical milieu in triggering protein misfolding.

Phenethylammonium bromide interlayer for high-performance red quantum-dot light emitting diodes.

Interfacial modification is vital to boost the performance of colloidal quantum-dot light-emitting diodes (QLEDs). We introduce phenethylammonium bromide (PEABr) as an interlayer to reduce the trap states and exciton quenching at the interface between the emitting layer (EML) with CdSe/ZnS quantum-dots and the electron transport layer (ETL) with ZnMgO. The presence of PEABr separates the EML and the ETL and thus passivates the surface traps of ZnMgO. Moreover, the interfacial modification also alleviates electron injection, leading to more improved carrier injection balance. Consequently, the external quantum efficiency of the PEABr-based red QLED reached 27.6%, which outperformed those of the previously reported devices. Our results indicate that the halide ion salts are promising to balance charge carrier injection and reduce exciton quenching in the QLEDs.

Epigenetic small-molecule screen for inhibition and reversal of acinar ductal metaplasia in mouse pancreatic organoids.

Background: Acinar ductal metaplasia (ADM) is among the earliest initiating events in pancreatic ductal adenocarcinoma (PDAC) development. Methods: We developed a novel morphology-based screen using organoids from wildtype and p48Cre/+ (Cre) mice to discover epigenetic modulators that inhibit or reverse pancreatic ADM more effectively than the broad-spectrum HDAC inhibitor trichostatin A (TSA). Results: Of the 144 compounds screened, nine hits and two additional natural product HDAC inhibitors were validated by dose-response analysis. The class I HDAC inhibitors apicidin and FK228, and the histone methyltransferase inhibitor chaetocin demonstrated pronounced ADM inhibition and reversal without inducing significant cytotoxicity at 1 µM. Thioester prodrug class I HDAC inhibitor largazole attenuated ADM while its disulfide homodimer was effective in both ADM inhibition and reversal. Prioritized compounds were validated for ADM reversal in p48Cre/+; LSL-KrasG12D/+ (KC) mouse organoids using both morphological and molecular endpoints. Molecular index analysis of ADM reversal in KC mouse organoids demonstrated improved activity compared to TSA. Improved prodrug stability translated into a stronger phenotypic and molecular response. RNA-sequencing indicated that angiotensinogen was the top inhibited pathway during ADM reversal. Conclusion: Our findings demonstrate a unique epigenetic mechanism and suggest that the phenotypic screen developed here may be applied to discover potential treatments for PDAC.

Discovery of small molecule guanylyl cyclase B receptor positive allosteric modulators.

Myocardial fibrosis is a pathological hallmark of cardiovascular disease (CVD), and excessive fibrosis can lead to new-onset heart failure and increased mortality. Currently, pharmacological therapies for myocardial fibrosis are limited, highlighting the need for novel therapeutic approaches. The particulate guanylyl cyclase B (GC-B) receptor possesses beneficial antifibrotic actions through the binding of its natural ligand C-type natriuretic peptide (CNP) and the generation of the intracellular second messenger, cyclic guanosine 3',5'-monophosphate (cGMP). These actions include the suppression of fibroblast proliferation and reduction in collagen synthesis. With its abundant expression on fibroblasts, the GC-B receptor has emerged as a key molecular target for innovative CVD therapeutics. However, small molecules that can bind and potentiate the GC-B/cGMP pathway have yet to be discovered. From a cell-based high-throughput screening initiative of the NIH Molecular Libraries Small Molecule Repository and hit-to-lead evolution based on a series of structure-activity relationships, we report the successful discovery of MCUF-42, a GC-B-targeted small molecule that acts as a positive allosteric modulator (PAM). Studies herein support MCUF-42's ability to enhance the binding affinity between GC-B and CNP. Moreover, MCUF-42 potentiated cGMP levels induced by CNP in human cardiac fibroblasts (HCFs) and notably also enhanced the inhibitory effect of CNP on HCF proliferation. Together, our findings highlight that MCUF-42 is a small molecule that can modulate the GC-B/cGMP signaling pathway, potentially enhancing the antifibrotic actions of CNP. Thus, these data underscore the continued development of GC-B small molecule PAMs as a novel therapeutic strategy for targeting cardiac fibrosis and CVD.

Chemical Genetics in C. elegans Identifies Anticancer Mycotoxins Chaetocin and Chetomin as Potent Inducers of a Nuclear Metal Homeostasis Response.

C. elegans numr-1/2 (nuclear-localized metal-responsive) is an identical gene pair encoding a nuclear protein previously shown to be activated by cadmium and disruption of the integrator RNA metabolism complex. We took a chemical genetic approach to further characterize regulation of this novel metal response by screening 41,716 compounds and extracts for numr-1p::GFP activation. The most potent activator was chaetocin, a fungal 3,6-epidithiodiketopiperazine (ETP) with promising anticancer activity. Chaetocin activates numr-1/2 strongly in the alimentary canal but is distinct from metal exposure, because it represses canonical cadmium-responsive metallothionine genes. Chaetocin has diverse targets in cancer cells including thioredoxin reductase, histone lysine methyltransferase, and acetyltransferase p300/CBP; further work is needed to identify the mechanism in C. elegans as genetic disruption and RNAi screening of homologues did not induce numr-1/2 in the alimentary canal and chaetocin did not affect markers of integrator dysfunction. We demonstrate that disulfides in chaetocin and chetomin, a dimeric ETP analog, are required to induce numr-1/2. ETP monomer gliotoxin, despite possessing a disulfide linkage, had almost no effect on numr-1/2, suggesting a dimer requirement. Chetomin inhibits C. elegans growth at low micromolar levels, and loss of numr-1/2 increases sensitivity; C. elegans and Chaetomiaceae fungi inhabit similar environments raising the possibility that numr-1/2 functions as a defense mechanism. There is no direct orthologue of numr-1/2 in humans, but RNaseq suggests that chaetocin affects expression of cellular processes linked to stress response and metal homeostasis in colorectal cancer cells. Our results reveal interactions between metal response gene regulation and ETPs and identify a potential mechanism of resistance to this versatile class of preclinical compounds.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.

Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.

Heterologous Production of the C33-C45 Polyketide Fragment of Anticancer Apratoxins in a Cyanobacterial Host.

A polyketide synthase subcluster of cytotoxic apratoxin A was isolated from a Moorena bouillonii environmental DNA library and engineered with a thioesterase II domain for heterologous expression in the filamentous cyanobacterium Anabaena sp. PCC7120. Further engineering with a rhamnose-inducible promoter led to the production of (2R,3R,5R,7R)-3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid, a stereogenically rich chiral building block that is important to the efficient synthesis of apratoxin analogues, representing the first synthetic biology attempt for this type of polyketide fragment.