Solid-Phase Library Synthesis, Screening, and Selection of Tight-Binding Reduced Peptide Bond Inhibitors of a Recombinant Leishmania mexicana Cysteine Protease B

A one-bead−two-compound inhibitor library was synthesized by the split−mix method for the identification of inhibitors of a recombinant cysteine protease from Leishmania mexicana, CPB2.8ΔCTE. The inhibitor library was composed of octapeptides with a centrally located reduced bond introduced by reductive amination of the resin-bound amines with Fmoc amino aldehydes. The library was screened on solid phase, and less than 1% of the library contained active compounds. The inhibitors displayed great specificity in the subsites flanking the enzyme catalytic triad with Cha and Ile/Leu preferred in P₂, Phe in P₁, Cha and Ile/Leu in P₁‘, and Ile/Leu in P₂‘. Some of the inhibitors were resynthesized, and the kinetics of inhibition were determined in solution-phase assays. Most of the inhibitors had micromolar K_i values, and a few inhibited the enzyme at nanomolar concentrations. One inhibitor, DKHF(CH₂NH)LLVK (K_i = 1 μM), was tested for antiparasite efficacy and shown to affect parasite survival with an IC₅₀ of approximately 50 μΜ.