Proteases regulate a broad spectrum of physiological functions by the specific processing of proteins and peptides. Elevated levels of active proteases can result in an array of physiological processes ultimately leading to disease states. Therefore, compounds designed to restore the natural equilibrium of proteases present excellent opportunities for drug candidates. Cysteine proteases, for example cathepsins B, L, and S, have been implicated in a number of diseases, including progressive cartilage and bone degradation associated with arthritis.1 Inhibitors of these cathepsins have reduced inflammation and prevented joint destruction in animal models of arthritis.2 Recently, human cathepsin 02 has been found to be highly expressed in osteoclastoma tissue.3 Cathepsins B and L have been linked to metastasis and invasion by cancer cells.4 The calcium-associated cysteine proteases calpains I and II have been associated with ischemia and hypoxia,5 Alzheimer's disease,6 and cataracts.7 Inhibition of the trypanosomal cysteine protease cruzain has proven effective in models of Chagas's disease.8Reversible inhibitors of cysteine proteases include peptide aldehydes,9 nitriles,10 and a-ketocarbonyl compounds.11 Irreversible inhibitors include peptide halomethyl ketones,12 diazomethyl ketones,13 (acyloxy)methyl ketones,14 and ketomethylsulfonium salts,15 believed to alkylate the active site thiol by formal Sn2 displacement, either directly or through a proposed hemithioketal-episulfonium ion pathway. Other irreversible inhibitors include various epoxysuccinyl compounds,16 whose oxiranes are opened through nucleophilic attack by the thiol. Previous work by Hanzlik and co-workers17 demonstrated peptide Michael acceptors as inactivators of the plant protease papain, which displayed second-order rate constants of inactivation from 0.05 to 70 M'1 s'1. However, to serve as disease modifying agents, inhibitors must inactivate target enzymes sufficiently in vivo as well as in vitro. Therefore, we designed potent inhibitors against disease-associated cysteine proteases such as the cathepsins B, L, S, and 02, calpains, and cruzain, concentrating on structures or scaffolds suitable for pharmaceutical development.

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

We previously identified a Plasmodiumfalciparum trophozoite cysteine proteinase (TCP) and hypothesized that it is required for the degradation of host hemoglobin by intraerythrocytic malaria parasites. To test this hypothesis and to evaluate TCP as a chemotherapeutic target, we examined the antimalarial effects of a panel of peptide fluoromethyl ketone proteinase inhibitors. For each inhibitor, effectiveness at inhibiting the activity of TCP correlated with effectiveness at both blocking hemoglobin degradation and killing cultured parasites. Benzyloxycarbonyl (Z)-Phe-Arg-CH2F, the most potent inhibitor, inhibited TCP at picomolar concentrations and blocked hemoglobin degradation and killed parasites at nanomolar concentrations. Micromolar concentrations of the inhibitor were nontoxic to cultured mammalian cells. These results support the hypothesis that TCP is a necessary hemoglobinase and suggest that it is a promising chemotherapeutic target. (J. Clin. Invest.

We evaluated the antimalarial effects of vinyl sulfone cysteine proteinase inhibitors. A number of vinyl sulfones strongly inhibited falcipain, a Plasmodium falciparum cysteine proteinase that is a critical hemoglobinase. In studies of cultured parasites, nanomolar concentrations of three vinyl sulfones inhibited parasite hemoglobin degradation, metabolic activity, and development. The antimalarial effects correlated with the inhibition of falcipain. Our results suggest that vinyl sulfones or related cysteine proteinase inhibitors may have promise as antimalarial agents.