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 product extracts is employed to isolate and purify the individual bioactive compounds. Identification and structural characterization of these compounds provide 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. We also incorporated new computational modeling and organic synthesis techniques/methodologies to extend the translational potential of natural product leads through structure modification, target identification, and SAR optimization. In FY 2025, we have been continuing our research campaign to identify bioactive natural products that interact with a wide variety of molecular targets including the topoisomerase-3B, the inositol-tetrakisphosphate 1-kinase (ITPK1), and diffuse pleural mesothelioma (DPM). Totally, the NPCS has completed 56 isolation projects leading to the identification of >60 bioactive natural products. Based on the structures of the bioactive natural products, NPCS also generated 57 synthetic/semi-synthetic analogs including 43 new compounds. During this FY2025, the NPCS has contributed to 15 peer-reviewed publications, including 4 papers with NPCS staff as lead authors (e.g. first and/or corresponding authors). Based on the discoveries of NPCS research, the MTP filed an EIR and 6 new patent applications. (1) Topoisomerase-3B: Topoisomerases are essential enzymes that solve topological constraints of nucleic acids.101 Among all six known human topoisomerases, topoisomerase-3B (TOP3B) is the only dual DNA and RNA topoisomerase. Recent studies suggest TOP3B as a promising therapeutic target for viral infections and it is reported to be essential for the efficient replication of positive-sense RNA viruses in hosts. The exact roles of TOP3B in cancer are still an area of active discovery and may be cancer type dependent. While the study by Zhang, X. et al. demonstrates that TOP3B is a functional target of the anticancer molecule cinobufagin, and it is crucial for the survival of colon cancer cells under cellular stress, TOP3B genetic inactivation has also been associated with breast and renal cancer. To identify small-molecule modulators of TOP3B-associated colon cancer cell viability and investigate the role of TOP3B in colon cancer cells, the MTP developed a high-throughput differential cell viability assay using paired human colon carcinoma HCT116 cell lines with (TOP3B-WT) and without TOP3B (TOP3B-KO). Seven new furanoheliangolide class of sesquiterpene lactones, centratherolides A-G, along with goyazensolide and (2,3-epoxybutyryloxy)-goyazensolanolide were isolated from the organic solvent extract of the terrestrial plant Centratherum punctatum through bioassay-guided fractionation. We reported the development of a multicomponent analytical strategy that enables accurate stereochemical assignment of the oxidized, chiral side chains in sesquiterpene lactone structures. This general approach can be widely applied to a broad spectrum of natural product classes bearing similar chiral ester functionalities. Several compounds showed cytotoxicity against the HCT116 TOP3B-KO cell line (1.4 µM < IC50s < 10.4 µM) with relatively better potency than that against the HCT116 TOP3B-WT cell line (3.3 µM < IC50s less than 23.8 µM), affording similar selectivity index (SI) values at ~2.3. The results of this project led to a recent publication in the Journal of Natural Products. (2) Diffuse pleural mesothelioma (DPM): Diffuse pleural mesothelioma (DPM) is a type of rare yet aggressive cancer that kills approximately 40,000 people worldwide per year. With a long latency period of 30-50 years, DPM affects mostly older individuals who have been occupationally exposed to asbestos fibers, with a median survival of around 18 months. There are three distinct histologic subtypes of DPM phenotypes: epithelioid, sarcomatoid, and biphasic. Epithelioid mesothelioma accounts for more than half of all DPM cases with a 5-year survival rate of around 10% - 15% after surgery. DPMs are largely untreatable, due to the limited efficacy of the current standard of care, the lack of routine predictive biomarkers (associated with extensive interpatient heterogeneity), and the high incidence of recurrence. Thus, more effective therapeutic options are a significant need for the treatment of malignant pleural mesothelioma. To identify selective small-molecule modulators of DPM cell viability, the MTP developed a high-throughput cell-based screen composed of three cell lines, including the epithelioid DPM cell line MB52, the sarcomatoid DPM cell line MB24, and the normal pleural cell line NP1. The NPCS has identified two known terthiophene esters 5-hydroxymethyl-(2,2:5,2)-terthienyl angelate and tiglate from the plant Eclipta prostrata (Asteraceae) exhibiting selective anti-proliferative and cytotoxic activities against the epithelioid DPM cell line MB52 (IC50 values 623 and 1287 nM, respectively) with the Selectivity Index [SI, IC50(MB24 or NP1)/IC50(MB52)] of 76 and 38 for NP1 and 127 and 63 for MB24, respectively. To investigate the SAR, a series of terthiophene analogs were synthesized. All analogs retained the cell line selectivity with IC50 values greater than 10000 nM for MB24 and NP1. Many analogs showed improved potency against the MB52 cell viability including the propionate diester analog showed the most potent cytotoxic activity with an IC50 value of 9 nM. The mechanisms underlying the selective cytotoxicity against the epithelioid DPM cell lines are currently ongoing in Hoang's lab. Current findings indicated terthiophene analogs induced apoptosis and immunogenic cell death exclusively in epithelioid DPM cell lines, at least partially due to the selective cellular uptake by the epithelioid cell lines. In FY2026, 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. 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 product chemistry. Natural products are a source of structural complexity and biological activity that can provide insight into 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 product discovery efforts can complement the goals of the CCR and NCI.