Antipain and its analogues, natural product inhibitors of cathepsin K isolated from streptomyces

In human bone, 90% of organic bone matrix is composed of type 1 collagen. Cathepsin K is a cysteine protease involved in osteoclast mediated bone absorption and has been identified as a major drug target for the treatment of osteoporosis. Numerous potent inhibitors of cathepsin K have already been identified from natural sources including epoxide inhibitors such as E-64 isolated from the fungi Aspergillus japonicus as well as various peptide aldehydes such as Leupeptin and alpha-MAPI purified from Streptomyces. 350 soil and lichen-associated bacterial strains collected in the rain forests of British Columbia were screened and 22 samples were identified containing significant cathepsin K inhibitory activity. From those active samples, L-91-3 was selected as one of the most potent samples for further characterization of their cathepsin inhibitor content. Three Antipain-related peptide inhibitors were identified from L-91-3 strain of Streptomyces. Antipain (Ki 41nM +/- 37nM) and Vince 2 (cyclized P1 derivative Ki 295nM +/- 123nM) were isolated by traditional purification and subsequent NMR and Mass Spectrometry analysis. The cyclized compound, Vince 2 (phenylalanyl-ureido-arginyl-valinyl- cycloarginal), lacked the aldehyde function and resulted in a lower binding affinity towards cathepsin K. Using Cathepsin K as bait for active site directed inhibitors a third compound, named Lichostatinal was identified by x-ray crystallography where recombinant human cathepsin K was co-crystallized with the semi-crude fermentation broth resulting in 2.0 Å resolution crystal structure. Lichostatinal is a peptide-based aldehyde with the amino acid composition (agmatinyl-ureido-serine-valinyl-arginal). The P1-P4 substrate residues of Lichostatinal interact with the non primed S1-S4 subsites of cathepsin K.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat