Linear (-)-Zampanolide: Flexibility in Conformation-Activity Relationships.

Through an understanding of the conformational preferences of the polyketide natural product (-)-zampanolide, and the structural motifs that control these preferences, we developed a linear zampanolide analogue that exhibits potent cytotoxicity against cancer cell lines. This discovery provides a set of three structural handles for further structure-activity relationship (SAR) studies of this potent microtubule-stabilizing agent. Moreover, it provides additional evidence of the complex relationship between ligand preorganization, conformational flexibility, and biological potency. In contrast to medicinal chemistry dogma, these results demonstrate that increased overall conformational flexibility is not necessarily detrimental to protein binding affinity and biological activity.

Molecular mechanisms by which splice modulator GEX1A inhibits leukaemia development and progression.

INTRODUCTION: Splice modulators have been assessed clinically in treating haematologic malignancies exhibiting splice factor mutations and acute myeloid leukaemia. However, the mechanisms by which such modulators repress leukaemia remain to be elucidated. OBJECTIVES: The primary goal of this assessment was to assess the molecular mechanism by which the natural splice modulator GEX1A kills leukaemic cells in vitro and within in vivo mouse models. METHODS: Using human leukaemic cell lines, we assessed the overall sensitivity these cells have to GEX1A via EC50 analysis. We subsequently analysed its effects using in vivo xenograft mouse models and examined whether cell sensitivities were correlated to genetic characteristics or protein expression levels. We also utilised RT-PCR and RNAseq analyses to determine splice change and RNA expression level differences between sensitive and resistant leukaemic cell lines. RESULTS: We found that, in vitro, GEX1A induced an MCL-1 isoform shift to pro-apoptotic MCL-1S in all leukaemic cell types, though sensitivity to GEX1A-induced apoptosis was negatively associated with BCL-xL expression. In BCL-2-expressing leukaemic cells, GEX1A induced BCL-2-dependent apoptosis by converting pro-survival BCL-2 into a cell killer. Thus, GEX1A + selective BCL-xL inhibition induced synergism in killing leukaemic cells, while GEX1A + BCL-2 inhibition showed antagonism in BCL-2-expressing leukaemic cells. In addition, GEX1A sensitised FLT3-ITD+ leukaemic cells to apoptosis by inducing aberrant splicing and repressing the expression of FLT3-ITD. Consistently, in in vivo xenografts, GEX1A killed the bulk of leukaemic cells via apoptosis when combined with BCL-xL inhibition. Furthermore, GEX1A repressed leukaemia development by targeting leukaemia stem cells through inhibiting FASTK mitochondrial isoform expression across sensitive and non-sensitive leukaemia types. CONCLUSION: Our study suggests that GEX1A is a potent anti-leukaemic agent in combination with BCL-xL inhibitors, which targets leukaemic blasts and leukaemia stem cells through distinct mechanisms.

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity.

Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Brain glycogen serves as a critical glucosamine cache required for protein glycosylation.

Glycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.

Enantioselective Total Synthesis of the Putative Biosynthetic Intermediate Ambruticin†J.

The family of anti-fungal natural products known as the ambruticins are structurally distinguished by a pair of pyran rings adorning a divinylcyclopropane core. Previous characterization of their biosynthesis, including the expression of a genetically modified producing organism, revealed that the polyketide synthase pathway proceeds via a diol intermediate, known as ambruticin J. Herein, we report the first enantioselective total synthesis of the putative PKS product, ambruticin J, according to a triply convergent synthetic route featuring a Suzuki-Miyaura cross-coupling and a Julia-Kocienski olefination for fragment assembly. This synthesis takes advantage of synthetic methodology previously developed by our laboratory for the stereoselective generation of the trisubstituted cyclopropyl linchpin.

Synthesis, conformational preferences, and biological activity of conformational analogues of the microtubule-stabilizing agents, (-)-zampanolide and (-)-dactylolide.

Zampanolide and dactylolide are microtubule-stabilizing polyketides possessing potent cytotoxicity towards a variety of cancer cell lines. Using our understanding of the conformational preferences of the macrolide core in both natural products, we hypothesized that analogues lacking the C17-methyl group would maintain the necessary conformation for bioactivity while reducing the number of synthetic manipulations necessary for their synthesis. Analogues 3, 4 and 5 were prepared via total synthesis, and their conformational preferences were determined through computational and high-field NMR studies. While no observable activities were present in dactylolide analogues 3 and 4, zampanolide analogue 5 exhibited sub-micromolar cytotoxicity. Herein, we describe these efforts towards understanding the structure- and conformation-activity relationships of dactylolide and zampanolide.

GEX1A, a Polyketide from Streptomyces chromofuscus, Corrects the Cellular Defects Associated with Niemann-Pick Type C1 in Human Fibroblasts.

We report the first evidence of GEX1A, a polyketide known to modulate alternative pre-mRNA splicing, as a potential treatment for Niemann-Pick type C disease. GEX1A was isolated from its producing organism, Streptomyces chromofuscus, and screened in NPC1 mutant cells alongside several semisynthetic analogues. We found that GEX1A and analogues are capable of restoring cholesterol trafficking in NPC1 mutant fibroblasts, as well as altering the expression of NPC1 isoforms detected by Western blot. These results, along with the compound's favorable pharmacokinetic properties, highlight the potential of spliceosome-targeting scaffolds such as GEX1A for the treatment of genetic diseases.

Molecular Basis for Olefin Rearrangement in the Gephyronic Acid Polyketide Synthase.

Polyketide synthases (PKS) are a rich source of natural products of varied chemical composition and biological significance. Here, we report the characterization of an atypical dehydratase (DH) domain from the PKS pathway for gephyronic acid, an inhibitor of eukaryotic protein synthesis. Using a library of synthetic substrate mimics, the reaction course, stereospecificity, and tolerance to non-native substrates of GphF DH1 are probed via LC-MS analysis. Taken together, the studies establish GphF DH1 as a dual-function dehydratase/isomerase that installs an odd-to-even double bond and yields a product consistent with the isobutenyl terminus of gephyronic acid. The studies also reveal an unexpected C2 epimerase function in catalytic turnover with the native substrate. A 1.55-Å crystal structure of GphF DH1 guided mutagenesis experiments to elucidate the roles of key amino acids in the multistep DH1 catalysis, identifying critical functions for leucine and tyrosine side chains. The mutagenesis results were applied to add a secondary isomerase functionality to a nonisomerizing DH in the first successful gain-of-function engineering of a PKS DH. Our studies of GphF DH1 catalysis highlight the versatility of the DH active site and adaptation for a specific catalytic outcome with a specific substrate.

Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.

Conformation-guided analogue design identifies potential antimalarial compounds through inhibition of mitochondrial respiration.

The synthesis of a 2-methyl-substituted analogue of the natural product, neopeltolide, is reported in an effort to analyze the importance of molecular conformation and ligand-target interactions in relation to biological activity. The methyl substitution was incorporated via highly diastereoselective ester enolate alkylation of a late-stage intermediate. Coupling of the oxazole sidechain provided 2-methyl-neopeltolide and synthetic neopeltolide via total synthesis. The substitution was shown to maintain the conformational preferences of its biologically active parent compound through computer modeling and NMR studies. Both compounds were shown to be potential antimalarial compounds through the inhibition of mitochondrial respiration in P. falciparum parasites.

Synthesis and Biological Evaluation of 7-Deoxy-Epothilone Analogues.

The synthesis of two deoxygenated analogues of potent epothilones is reported in an effort to analyze the relative importance of molecular conformation and ligand-target interactions to biological activity. 7-deoxy-epothilone D and 7-deoxy-(S)-14-methoxy-epothilone D were prepared through total synthesis and shown to maintain the conformational preferences of their biologically active parent congeners through computer modeling and nuclear magnetic resonance (NMR) studies. The significant decrease in observed potency for each compound suggests that a hydrogen bond between the C7-hydroxyl group and the tubulin binding site plays a critical role in the energetics of binding in the epothilone class of polyketides.

α-Methylation follows condensation in the gephyronic acid modular polyketide synthase.

C-methyltransferases (MTs) from modular polyketide synthase assembly lines are relatively rare and unexplored domains that are responsible for installing α-methyl groups into nascent polyketide backbones. The stage at which these synthase-embedded enzymes operate during polyketide biosynthesis has yet to be conclusively demonstrated. In this work we establish the activity and substrate preference for six MTs from the gephyronic acid polyketide synthase and demonstrate their ability to methylate both N-acetylcysteamine- and acyl carrier protein-linked ß-ketoacylthioester substrates but not malonyl thioester equivalents. These data strongly indicate that MT-catalyzed methylation occurs immediately downstream of ketosynthase-mediated condensation during polyketide assembly. This work represents the first successful report of MT-catalyzed mono- and dimethylation of simple thioester substrates and provides the groundwork for future mechanistic and engineering studies on this important but poorly understood enzymatic domain.

Total Synthesis and Structural Reassignment of Lyngbyaloside C Highlighted by Intermolecular Ketene Esterification.

Lyngbyaloside C, a classic macrolide, isolated from Lyngbya bouilloni, has shown moderate anticancer activity against several cancer cell lines. Here, we report the first total synthesis and stereochemical configuration reassignment of lyngbyaloside C. The synthesis highlights a one-pot intermolecular ketene esterification reaction to form the crucial tertiary ester and tetrahydropyran. In addition, a novel and concise synthetic pathway towards the 1,3-syn secondary, tertiary diol fragment is described using a regio- and stereospecific electrophilic ether transfer reaction.

Conformation-activity relationships of polyketide natural products.

Polyketides represent an important class of secondary metabolites that interact with biological targets connected to a variety of disease-associated pathways. Remarkably, nature's assembly lines, polyketide synthases, manufacture these privileged structures through a combinatorial mixture of just a few structural units. This review highlights the role of these structural elements in shaping a polyketide's conformational preferences, the use of computer-based molecular modeling and solution NMR studies in the identification of low-energy conformers, and the importance of conformational analogues in probing the bound conformation. In particular, this review covers several examples wherein conformational analysis complements classic structure-activity relationships in the design of biologically active natural product analogues.

The synthesis and biological evaluation of desepoxyisotedanolide and a comparison with desepoxytedanolide.

The tedanolides are biologically active polyketides that exhibit a macrolactone constructed from a primary alcohol. Since polyketidal transformations only generate secondary alcohols, it has been hypothesized by Taylor that this unique lactone could arise from a postketidal transesterification. In order to probe this hypothesis and to investigate the biological profile of the putative precursor of all members of the tedanolide family, we embarked on the synthesis of desepoxyisotedanolide and its biological evaluation in comparison to desepoxytedanolide. The biological experiments unraveled a second target for desepoxytedanolide and provided evidence that the proposed transesterification indeed provides a survival advantage for the producing microorganism.

Draft Genome Sequence of Gephyronic Acid Producer Cystobacter violaceus Strain Cb vi76.

A draft genome sequence of Cystobacter violaceus strain Cb vi76, which produces the eukaryotic protein synthesis inhibitor gephyronic acid, has been obtained. The genome contains numerous predicted secondary metabolite clusters, including the gephyronic acid biosynthetic pathway. This genome will contribute to the investigation of secondary metabolism in other Cystobacter strains.

Elucidation of gephyronic acid biosynthetic pathway revealed unexpected SAM-dependent methylations.

Gephyronic acid, a cytostatic polyketide produced by the myxobacterium Cystobacter violaceus Cb vi76, exhibits potent and selective eukaryotic protein synthesis inhibition. Next-generation sequencing of the C. violaceus genome revealed five type I polyketide synthases and post-PKS tailoring enzymes including an O-methyltransferase and a cytochrome P450 monooxygenase. Seven methyltransferase (MT) domains embedded within the PKS subunits were found to install the methyl branches throughout the gephyronic acid skeleton. A rare loading domain from the GNAT superfamily also contains an embedded MT domain that catalyzes the in situ production of an isobutyryl starter unit. Phylogenetic analysis identified new motifs that distinguish MT domains located in PKS pathways with in cis acyltransferase (AT) domains from MT domains located in PKS pathways with trans AT enzymes. The identification of the gene cluster sets the stage for the generation of a heterologous expression system, which will allow further investigation of selective eukaryotic protein synthesis inhibitors through the generation of gephyronic acid analogues.

Conformational preferences of zampanolide and dactylolide.

The solution conformation behavior of the macrolide core of microtubule-stabilizing agents (-)-zampanolide and (-)-dactylolide has been determined through a combination of high-field NMR experiments and computational modeling. Taken together, the results demonstrate that in solution both molecules exist as a mixture of three interconverting conformational families, one of which bears strong resemblance to zampanolide's tubulin-bound conformation.

Concise enantioselective synthesis of diospongins A and B.

Ether transfer methodology is capable of stereoselectively generating 1,3-diol mono- and diethers in good yield. Surprisingly, allylic and benzylic substrates provide none of the desired products when exposed to previously optimized conditions of iodine monochloride. Herein, second-generation activation conditions for ether transfer have been developed that circumvents undesired side reactions for these substrates. The application of this chemistry to the enantioselective synthesis of diospongins A and B has now been accomplished.

Strained alkenes in natural product synthesis.

Strained molecules continue to challenge the ingenuity of chemists as their high-energy bonds serve as fuel for the promotion of complex synthetic transformations. Developments in this area have resulted in the recent emergence of strained alkenes as intermediates in natural product synthesis. This Minireview highlights these recent advances along with current developments toward understanding the unique reactivity of strained alkenes.