Natural Products Discovery and Characterization Through Network Collaborations

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We utilized high throughput screening technologies to help identify compounds and extracts that can specifically interact with or modulate the function of selected biochemical targets or processes. Bioassay-guided chemical fractionation of natural products extracts is employed to isolate and purify the individual bioactive compounds. Identification and structural characterization of these compounds provides new structural classes or molecular scaffolds for the development of potential drug leads or biological probes that can interact with the desired molecular target. In addition to extensive NMR and mass spectroscopic analyses, our efforts include rigorous evaluation of a new compound's potency, molecular target specificity, and mode of action. In FY 2021, we have been continuing our research campaign to identify bioactive natural products that interact with a wide variety of molecular targets including the oncogenic transcription factor PAX3-FOXO1, the ubiquitin ligase Cbl-b, the hymocyte maturation regulator Tbata, the NADPH oxidase isoforms DUOX, and PKA kinase fusion protein. Neopetrothiazide, isolated from a Neopetrosia sp. sponge, represents a novel isoquinoline quinone that showed potent inhibition against PAX3-FOXO1-driven luciferase expression with an IC50 value of 1.4 micromolar . The unprecedented thiazide-fused structural scaffold of neopetrothiazide comprised of 14 contiguous nonprotonated centers (C, N, and S) was assigned by comprehensive analysis of sophisticated NMR experiments with newly optimized pulse sequences. Three new aryl alkaloids named suberitamides A-C were isolated from an extract of the marine sponge Pseudosuberites sp. collected along the coast of North Carolina. Suberitamides A and B inhibited Cbl-b, an E3 ubiquitin ligase that is an important modulator of immune cell function, with IC50 values of approximately 11 micromolar . Structure elucidation of suberitamide A was aided by the application of new NMR methodologies including the PIP HSQMBC IPAP experiment for measuring key long-range heteronuclear (C, H) coupling constants. Bioassay-guided separation of the extract of a Sinularia sp. soft coral led to the identification of a series of terpenoid-derived spermidine and spermine amides that were named sinularamides A-G. All of the isolated compounds showed Cbl-b inhibitory activities with IC50 values that ranged from approximately 6.5 to 33 micromolar . New methodologies for rapid isolation and identification of biologically active natural products from prefractionated libraries in HTS have been developed and applied in recent isolation projects against the PKA kinase fusion protein. Twelve PKA-fusion inhibitors including a novel purine-thiazine-imidazole-conjugated alkaloid have been rapidly isolated from six plant and marine extracts within a short period. We will continue to improve the efficiency of our HTS-based bioassay-guided fractionation platform by optimizing the analytical-fingerprint-based dereplication and project selection pipelines. The long-term focus of this project is to exploit the vast spectrum of chemical diversity within the NPR for potential anticancer and anti-HIV applications. It relies on close integration with the MTP Assay Development and Screening Section, Chemical Diversity Development Section, and the Protein Chemistry and Molecular Biology Section for extract screening, data analysis, bioassay support, and functional analysis of isolated compounds. Our CCR collaborators who study aspects of cancer biology, genetics, and immunology provide expertise for target selection and subsequent compound evaluation. We have assembled a broad consortium of intramural and extramural partners with expertise in organic synthesis, chemical biology, molecular pharmacology, computational sciences, and spectroscopic analysis to help characterize and advance our natural product discoveries. With 25 years of experience studying the chemistry of extracts from the NPR, a proven capability to develop and apply targeted screening assays, and access to CCR investigators with broad expertise in cancer and HIV biology, the Natural Products Chemistry Section is uniquely positioned within the NCI to combine molecular target-based discovery with natural products chemistry. Natural products are a source of structural complexity and biological activity that can provide insight on the function of new targets, pathways, or cellular processes. They play an important role in dissecting and understanding the intricacies of cancer development and progression, so continued natural products discovery efforts can complement the goals of the CCR and NCI.