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 μM.