CPB expression is regulated so that CPB1 and CPB2, the first two genes of the tandem array, are expressed in the infectious metacyclic stage and the remaining genes in the intracellular amastigote stage that causes the disease. Due to their high sequence identity, the multiple isoforms present in amastigotes are expected to have similar inhibitor susceptibilities. Inhibitors of cysteine proteases typically rely on the presence of a warhead, an electrophilic functionality that is attacked by the catalytic cysteine thiolate in the active site of the target enzyme. Inhibitors containing a reversible reactive warhead-type might be expected to possess better safety profiles with regards to their potential application as drugs for treating parasitic infections, examples of such reactive inhibitors of L. mexicana CPB are compounds of the class of ��- ketoheterocycles. In order to identify new warhead-types that are reversibly reactive and have some specificity for cysteine proteases of trypanosomatid parasites, high throughput screening of a compound library against L. mexicana CPB2.8 and bovine cathepsin B as a counter assay was performed. Homology modeling and covalent docking studies to rationalize the experimental findings were also carried out. In this study, 74,339 structurally diverse compounds coming from a general screening library have been Axitinib VEGFR/PDGFR inhibitor tested as inhibitors of a recombinant form of the cathepsin L-like cysteine protease CPB present in the parasite L. mexicana. In a separate assay, the compounds were evaluated for their ability to inhibit cathepsin B from bovine spleen. BtCatB was chosen over the higly similar HsCatB because of easy accessibility. Two unexpected results emerge from this study. First, molecules with novel cysteine protease warheads were not identified in this study. This is startling, given the effort devoted to identify inhibitors bearing covalent reversible warheads in screening libraries. Because the structurally diverse screening set was filtered from a general purpose screening library of 2,000,000 compounds, we assumed to find more suitable warheads. Even taking into account the lack of target bias in the chemotypes represented in it, the result (+)-JQ1 side effects suggests that potent covalent reversible inhibition of CPB2.8��CTE is limited to only a few warheads. Second, only compounds from the thiosemicarbazone and semicarbazone warhead-type were identified as specific, reversible inhibitors of CPB2.8��CTE but with no activity against BtCatB ; the selectivity ratio was for the most active inhibitor CP229988. These two findings will be considered in turn. The lack of novel covalent reversible acting warheads identified in the CPB2.8��CTE HTS highlights the limitations of screening a relatively small and unbiased library within a large chemical possibility space.