Natural Products Discovery and Characterization Through Network Collaborations

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

The NPCS 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 2023, we have been continuing our research campaign to identify bioactive natural products that interact with a wide variety of molecular targets including the ubiquitin ligase Cbl-b, the Merkel cell carcinoma, the J-PKAc alpha kinase fusion protein, the topoisomerase-3B, the NCI60 cell line screening panel, and mesothelioma (MPM). Totally, the NPCS has completed 98 isolation projects leading to the identification of 267 natural products including 91 new compounds. Among all compounds, 151 of them including 47 new compounds exhibited bioactivities against the selected targets. (1) Cbl-b: The E3 ubiquitin-protein ligase Cbl-b represents an attractive target for immunotherapeutic intervention in cancer. In FY 2022, we reported the identification of two novel Cbl-b inhibitors, (+)-plakoramine A and (-)-plakoramine A, from a marine sponge Plakortis sp. We continued to reveal a previously undescribed, nonenzymatic route to form plakoramine A via photochemical conversion of its naturally occurring monomeric counterpart, plakinidine B, which stands for a new functional fluorophore with potential utility as both a photosensitizer and a photochemically triggered electrophilic agent. The plakoramine story has been successfully published on the ACS journal Organic Letters. (2) Merkel cell carcinoma: Merkel cell carcinoma (MCC) is a rare but highly aggressive neuroendocrine skin cancer. The treatment of advanced MCC often utilizes immune checkpoint inhibitors such as avelumab or pembrolizumab. Despite relatively high response rates to these agents, less than half of patients achieve durable benefit; thus, alternative treatments are urgently needed. Two new cytotoxic oxygenated diketopiperazines, brevianamides E1 and E2, were identified from a Penicillium brevicompactum. Both compounds showed selective cytotoxicity against the Merkel cell carcinoma cell line MCC13 cell line with IC50 values at 2.6 and 2.5 mM, respectively. (3) The J-PKAc alpha kinase fusion protein (PKADJ): Fibrolamellar Hepatocellular Carcinoma (FLHCC) is a rare liver cancer with a young patient population ( 35 years of age) and a 5-year survival rate of only 30-35%. The DNAJB1-PRKCA oncogenic gene fusion is specifically expressed in the tumor and exclusively detected in FLHCC patients. The DNAJB1-PRKCA gene fusion produces an enzymatically active chimeric protein J-PKAc alpha that is a key driver in the oncogenesis of FLHCC. A high-throughput, modified sandwich ELISA assay was developed by PCMBS to identify selective modulators of the J-PKAc alpha catalytic activity by screening the prefractionated natural product library recently created by the NCI Program for Natural Product Discovery (NPNPD). In FY 2023, the NPCS has been engaged in the bioassay-guided fractionations of 36 PKADJ-active extracts. The NPCS assisted PCMBS in the publication of the first PKADJ HTS manuscript on ACS Pharmacology & Translational Science by providing several bioactive natural products. Last year, we reported the identification of a novel PKADJ lead molecule aplithianine A (1) from a marine Aplidium sp. tunicate. Aplithianine A (1) showed potent inhibition against both J-PKAc alpha and wild-type PKAc alpha with an IC50 value 1 uM in the primary assay. Further mechanistic studies including co-crystallization and X-ray diffraction experiments revealed that 1 inhibited PKAc alpha catalytic activity by competitively binding to the ATP pocket. Human kinome profiling of 1 against a panel of 370 kinases revealed potent inhibition of select serine/threonine kinases in the CLK, DYRK, and PKG families with IC50 values ranging from 11-90 nM. An efficient, four-step total synthesis of 1 has been accomplished enabling further development of aplithianines as biologically relevant kinase inhibitors. Through the collaboration with the CCR medicinal chemistry accelerator, we have designed and synthesized more than 400 aplithianine analogs and successfully improved the biochemical potency to single digit nanomolar range. Based on data from the initial discovery and following optimization of alithianines, a manuscript and two provisional patent applications have been submitted and a following PCT application was also filed after one of the provisional applications. (4) Topoisomerase-3B: DNA Topoisomerase-3B (TOP3B) stands out among all mammalian topoisomerases due to its distinctive capability to resolve topological entanglements in both DNA and RNA. Deletion of the topoisomerase-3B (TOP3B) gene has been linked to various cancer types. This unique characteristic has made TOP3B a promising target for the development of new therapeutics for both cancer and RNA virus infections. The paired colorectal carcinoma HCT116 cell lines with (TOP3B-WT) and without TOP3B (TOP3B-KO) were developed for discovering TOP3B-specific proliferation modulators. In this high-throughput screening campaign, the NPCS has been engaged in the bioassay-guided fractionations of 38 TOP3B-active extracts leading to the identification of 76 active natural products including 20 new compounds. In FY2024, the NPCS 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. We will also incorporate new computational modeling and organic synthesis techniques/methodologies to establish our expertise in structure modification, target identification, and activity optimization. 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. 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.