The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 appear to be required for hemoglobin hydrolysis by intraerythrocytic malaria parasites. Previous studies showed that peptidyl vinyl sulfone inhibitors of falcipain-2 blocked the development of P. falciparum in culture and exerted antimalarial effects in vivo. We now report the structure-activity relationships for inhibition of falcipain-2, falcipain-3, and parasite development by 39 new vinyl sulfone, vinyl sulfonate ester, and vinyl sulfonamide cysteine protease inhibitors. Levels of inhibition of falcipain-2 and falcipain-3 were generally similar, and many potent compounds were identified. Optimal antimalarial compounds, which inhibited P. falciparum development at low nanomolar concentrations, were phenyl vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides with P 2 leucine moieties. Our results identify independent structural correlates of falcipain inhibition and antiparasitic activity and suggest that peptidyl vinyl sulfones have promise as antimalarial agents.Malaria is one of the most important infectious diseases in the world. Plasmodium falciparum, the most virulent human malaria parasite, is estimated to cause over 300 million new cases and 1 million deaths annually (33). Further complicating this grim scenario is the emergence of the widespread resistance of P. falciparum to available antimalarial drugs (25). New drugs to combat malaria are urgently needed.Among potential new targets for antimalarial chemotherapy are enzymes that mediate hemoglobin hydrolysis. Intraerythrocytic P. falciparum trophozoites derive amino acids for protein synthesis from the hydrolysis of host cell hemoglobin in an acidic food vacuole (12,20,27). Proteases that hydrolyze hemoglobin in the food vacuole include members of the aspartic protease (1), cysteine protease (38, 39), and metalloprotease (9) families. Cysteine protease inhibitors arrested the erythrocytic life cycle of P. falciparum (26). Examination of inhibitortreated parasites revealed abnormally swollen food vacuoles filled with undigested hemoglobin, indicating that the block in parasite development was due to the inhibition of hemoglobin hydrolysis (26).P. falciparum contains three fairly typical papain family cysteine proteases, known as falcipains (28,38,39). Falcipain-2 and falcipain-3 appear to be the principal cysteine protease hemoglobinases (38, 39). Both of these proteases localize to vacuolar parasite fractions and readily hydrolyze hemoglobin under physiological reducing conditions at acidic pHs (37). Falcipain-2 is considerably more active against small peptide substrates, but the specificities of the two proteases are similar; both enzymes display a strong preference for leucine at the P 2 position (38, 39). The role of falcipain-1 in hemoglobin hydrolysis is unknown.In earlier studies, peptidyl vinyl sulfones inhibited falcipain-2 activity and parasite development at nanomolar concentrations and were active in vivo against murine malaria (22,29,30). We have now init...

The rate of base-mediated ester hydrolysis in monolayers of 11-mercaptoundecyl isonicotinate on gold is monitored by infrared spectroscopy and cyclic voltammetry. The hydrolysis product, a surface-confined alcohol, can be converted to a trifluoroacetate, increasing the sensitivity of infrared monitoring. Pentaammineruthenium(II) can be attached to the pendant isonicotinate either prior to or after monolayer assembly, leading to monolayers with a highly-charged monolayer/solution interface and a built-in electrochemical probe. The rate of ester hydrolysis within the monolayer is controlled by steric factors associated with monolayer packing and by the amount of charge at the monolayer/solution interface. The stability of the pendant Ru(III) complex is very sensitive to pH and interfacial charge. These results are correlated with Gouy-Chapman double-layer theory.

A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.