Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [15N]Cys, [15N]His, and [13C]Met labeling was performed to quickly assess cruzain–ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme–inhibitor interactions to facilitate lead compound identification and subsequent structural studies.
"Crystal structure of cruzain co - complexed with compound Neq176 . Recombinant cruzain was expressed and purified in a modified version as described recently by Lee et al . 2012 [ 32 ] . The 0 . 5 mg mL 21 solution of procruzain ( in 100 mM sodium acetate , 10 mM EDTA and 300 mM NaCl , at pH 5 . 2 ) was activated at 37uC for 3 . 5 hours with 5 mM DTT . After activation , cruzain was immediately inhibited with the covalent reversible inhibitor methyl methanethiosulfonate ( MMTS ) to a final concentration of 1 mM"
[Show abstract][Hide abstract] ABSTRACT: A multi-step cascade strategy using integrated ligand- and target-based virtual screening methods was developed to select a small number of compounds from the ZINC database to be evaluated for trypanocidal activity. Winnowing the database to 23 selected compounds, 12 non-covalent binding cruzain inhibitors with affinity values (K i) in the low micromolar range (3-60 µM) acting through a competitive inhibition mechanism were identified. This mechanism has been confirmed by determining the binding mode of the cruzain inhibitor Nequimed176 through X-ray crystallographic studies. Cruzain, a validated therapeutic target for new chemotherapy for Chagas disease, also shares high similarity with the mammalian homolog cathepsin L. Because increased activity of cathepsin L is related to invasive properties and has been linked to metastatic cancer cells, cruzain inhibitors from the same library were assayed against it. Affinity values were in a similar range (4-80 µM), yielding poor selectivity towards cruzain but raising the possibility of investigating such inhibitors for their effect on cell proliferation. In order to select the most promising enzyme inhibitors retaining trypanocidal activity for structure-activity relationship (SAR) studies, the most potent cruzain inhibitors were assayed against T. cruzi-infected cells. Two compounds were found to have trypanocidal activity. Using compound Nequimed42 as precursor, an SAR was established in which the 2-acetamidothiophene-3-carboxamide group was identified as essential for enzyme and parasite inhibition activities. The IC50 value for compound Nequimed42 acting against the trypomastigote form of the Tulahuen lacZ strain was found to be 10.6±0.1 µM, tenfold lower than that obtained for benznidazole, which was taken as positive control. In addition, by employing the strategy of molecular simplification, a smaller compound derived from Nequimed42 with a ligand efficiency (LE) of 0.33 kcal mol(-1) atom(-1) (compound Nequimed176) is highlighted as a novel non-peptidic, non-covalent cruzain inhibitor as a trypanocidal agent candidate for optimization.
[Show abstract][Hide abstract] ABSTRACT: An improved, Weinreb amide-based, synthesis of anti-trypanosomal lysine-containing vinyl sulfones is described incorporating, as a feature, diversity at the ε-lysine amino group. Members of this family demonstrated moderate to good efficacy as anti-trypanosomal agents and a fluorescent dansyl () derivative was used to investigate subcellular localisation of the compound class.
[Show abstract][Hide abstract] ABSTRACT: A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A Ki value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.
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