Non-peptidic Cruzain Inhibitors with Trypanocidal Activity Discovered by Virtual Screening and in Vitro Assay

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 mu M) 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 mu M), 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 mu M, 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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Sao Carlos, Dept Quim, BR-13560 Sao Carlos, SP, BrazilUniv São Paulo, Inst Quim Sao Carlos, Grp Quim Med IQSC USP, Sao Carlos, SP, BrazilUniv Calif San Francisco, Dept Pathol, Ctr Discovery & Innovat Parasit Dis, San Francisco, CA 94140 USAUniv São Paulo, Fac Med Ribeirao Preto, Dept Bioquim & Imunol, BR-14049 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilFAPESP: 2011/01893-3,CNPq: 301614/2010-5CAPES: 5985/11-0Web of Scienc

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. the peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO42- > HPO42- > acetate(-) > HCO3- > Cl-. the activation appeared to be independent of substrate sequence and arose primarily from an increase in k(cat). A shift to higher pH was also observed for the pK(1) of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilHungarian Acad Sci, Biol Res Ctr, Inst Enzymol, H-1518 Budapest 112, HungaryUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

Cruzain inhibition by hydroxymethylnitrofurazone and nitrofurazone: investigation of a new target in Trypanosoma cruzi

Nitrofurazone (NF) and its derivative, hydroxymethylnitrofurazone (NFOH), have presented antichagasic activity. NFOH has higher activity and lower mutagenicity. The aim of this work was to assess whether NF and its derivative NFOH would also be inhibitors of cruzain, besides their trypanothione reductase inhibitory activity. In vitro cruzain inhibition tests were performed for both compounds, and the 50% inhibitory concentration (IC(50)) for NF and NFOH presented values of 22.83 +/- 1.2 mu M and 10.55 +/- 0.81 mu M, respectively. AM1 semi-empirical molecular modeling studies were performed to understand the activity of the compounds, corroborating the observed cruzain inhibitory activity.FAPESP[01/01192-3]FAPESP[04/06097-7]FAPESP[02/09518-8]CAPES-PRODOCPROBRALCNPqUniversidade de São Paulo - Pró-Reitoria de Pesquisa PRP-USP Proconte

A tellurium-based cathepsin B inhibitor: Molecular structure, modelling, molecular docking and biological evaluation

The crystallographically determined structure of biologically active 4,4-dichloro-1,3-diphenyl-4-telluraoct-2-en-1-one, 3, shows the coordination geometry for Te to be distorted psi-pentagonal bipyramidal based on a C2OCl3(lone pair) donor set. Notable is the presence of an intramolecular axial Te center dot center dot center dot O (carbonyl) interaction, a design element included to reduce hydrolysis. Raman and molecular modelling studies indicate the persistence of the Te center dot center dot center dot O(carbonyl) interaction in the solution (CHCl3) and gasphases, respectively. Docking studies of 3' (i.e. original 3 less one chloride) with Cathepsin B reveals a change in the configuration about the vinyl C = C bond. i.e. to E from Z (crystal structure). This isomerism allows the optimisation of interactions in the complex which features a covalent Te-SGCys29 bond. Crucially, the E configuration observed for 3' allows for the formation of a hypervalent Te center dot center dot center dot O interaction as well as an O center dot center dot center dot H-O hydrogen bond with the Gly27 and Glu122 residues, respectively. Additional stabilisation is afforded by a combination of interactions spanning the S1, S2, S1' and S2' sub-sites of Cathepsin B. The greater experimental inhibitory activity of 3 compared with analogues is rationalised by the additional interactions formed between 3' and the His110 and His111 residues in the occluding loop, which serve to hinder the entrance to the active site. (C) 2012 Elsevier B.V. All rights reserved.CAPES (Rede Nanobiotec-Brasil) [808/2009]CAPES (Rede NanobiotecBrasil)CNPq [308116/2010-0, 306532/2009-3]CNPqFAPESPFAPESP [06/56078-4, 07/52734-7]Ministry of Higher Education (Malaysia) [UM.C/HIR/MOHE/SC/12]Ministry of Higher Education (Malaysia

(A) Dose-response and (B) Linewaver-Burk curves for Neq30 from Table S1.

<p>Non-linear fit method was employed in the analysis.</p

Preliminary trypanocidal activity of compounds Neq42 and Neq37 evaluated against the Tulahuen lacZ strain.

<p>Preliminary trypanocidal activity of compounds Neq42 and Neq37 evaluated against the Tulahuen lacZ strain.</p

(A) Structure of the co-crystallized cruzain inhibitor K11777 and (B–D) examples of complex structures predicted by our molecular docking (Glide XP).

<p>Figure prepared using CCP4mg software <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002370#pntd.0002370-McNicholas1" target="_blank">[43]</a>.</p