Discovery of rigid biphenyl Plasmodium falciparum DHFR inhibitors using a fragment linking strategy.

Plasmodium falciparum dihydrofolate reductase (PfDHFR), a historical target for antimalarials, has been considered compromised due to resistance inducing mutations caused by pyrimethamine (PYR) overexposure. The clinical candidate P218 has demonstrated that inhibitors could efficiently target both PYR-sensitive and PYR-resistant parasites through careful drug design. Yet, P218 clinical development has been limited by its pharmacokinetic profile, incompatible with single dose regimen. Herein, we report the design of new PfDHFR inhibitors using fragment-based design, aiming at improved lipophilicity and overall drug-like properties. Fragment-based screening identified hits binding in the pABA site of the enzyme. Using structure-guided design, hits were elaborated into leads by fragment linking with 2,4-diaminopyrimidine. Resulting compounds display nM range inhibition of both drug-sensitive and resistant PfDHFR, high selectivity against the human isoform, drug-like lipophilicity and metabolic stability. Compound 4 and its ester derivative 3 kill blood stage TM4/8.2 parasite at nM concentrations while showing no toxicity against Vero cells.

Anticancer Activity of (±)-Kusunokinin Derivatives towards Cholangiocarcinoma Cells.

This study aimed to investigate the cytotoxicity and anticancer activity of (±)-kusunokinin derivatives ((±)-TTPG-A and (±)-TTPG-B). The cytotoxicity effect was performed on human cancer cells, including breast cancer, cholangiocarcinoma, colon and ovarian cancer-cells, compared with normal cells, using the MTT assay. Cell-cycle arrest and apoptosis were detected using flow-cytometry analysis. We found that (±)-TTPG-B exhibited the strongest cytotoxicity on aggressive breast-cancer (MDA-MB-468 and MDA-MB-231) and cholangiocarcinoma (KKU-M213), with an IC50 value of 0.43 ± 0.01, 1.83 ± 0.04 and 0.01 ± 0.001 µM, respectively. Interestingly, (±)-TTPG-A and (±)-TTPG-B exhibited less toxicity than (±)-kusunokinin (9.75 ± 0.39 µM) on L-929 cells (normal fibroblasts). Moreover, (±)-TTPG-A predominated the ell-cycle arrest at the S phase, while (±)-TTPG-B caused cell arrest at the G0/G1 phase, in the same way as (±)-kusunokinin in KKU-M213 cells. Both (±)-TTPG-A and (±)-TTPG-B induced apoptosis and multi-caspase activity more than (±)-kusunokinin. Taken together, we conclude that (±)-TTPG-A and (±)-TTPG-B have a strong anticancer effect on cholangiocarcinoma. Moreover, (±)-TTPG-B could be a potential candidate compound for breast cancer and cholangiocarcinoma in the future.