Esterase mutation is a mechanism of resistance to antimalarial compounds

Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance

Development of Potent and Selective Trypanocides by Targeting of the Trypanosomal Protease Tbcat B

Human African trypanosomiasis (HAT) is a major health concern in sub-Saharan Africa and is caused by the subspecies of Trypanosoma brucei. This protozoan parasite relies on a limited repertoire of cysteine cathepsins for growth and replication in the mammalian host. The essentiality and lack of redundancy within this protease family in T.brucei makes it a promising point for therapeutic intervention. Recent genetic studies have validated the trypanosomal cathepsin TbcatB as essential for survival in vivo, and we have developed a series of over 100 inhibitors targeted to TbcatB based on the thiosemicarbazone and purine nitrile scaffolds. This approach has yielded several potent trypanocides with excellent selectivity relative to a panel of mammalian cell lines. In addition, we have developed inhibitors with good selectivity for TbcatB relative to both trypanosomal and mammalian homologues by utilizing structure based approaches. These biochemical and cellular inhibitor studies confirm that TbcatB is an essential target in T.brucei, and that targeting this protease is a promising therapeutic strategy

Whole-Body Physiologically Based Pharmacokinetic Model for Nutlin-3a in Mice after Intravenous and Oral AdministrationS⃞

Nutlin-3a is an MDM2 inhibitor that is under investigation in preclinical models for a variety of pediatric malignancies, including retinoblastoma, rhabdomyosarcoma, neuroblastoma, and leukemia. We used physiologically based pharmacokinetic (PBPK) modeling to characterize the disposition of nutlin-3a in the mouse. Plasma protein binding and blood partitioning were assessed by in vitro studies. After intravenous (10 and 20 mg/kg) and oral (50, 100, and 200 mg/kg) dosing, tissue concentrations of nutlin-3a were determined in plasma, liver, spleen, intestine, muscle, lung, adipose, bone marrow, adrenal gland, brain, retina, and vitreous fluid. The PBPK model was simultaneously fit to all pharmacokinetic data using NONMEM. Nutlin-3a exhibited nonlinear binding to murine plasma proteins, with the unbound fraction ranging from 0.7 to 11.8%. Nutlin-3a disposition was characterized by rapid absorption with peak plasma concentrations at approximately 2 h and biphasic elimination consistent with a saturable clearance process. The final PBPK model successfully described the plasma and tissue disposition of nutlin-3a. Simulations suggested high bioavailability, rapid attainment of steady state, and little accumulation when administered once or twice daily at dosages up to 400 mg/kg. The final model was used to perform simulations of unbound tissue concentrations to determine which dosing regimens are appropriate for preclinical models of several pediatric malignancies