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

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.

Identification of Anti-Inflammatory and Anti-Hypertensive Drugs as Inhibitors of Bacterial Diguanylate Cyclases

“…The degree of structural similarity was calculated using ComboScore, the sum of the Shape Tanimoto and the Scaled Color values. 33 EON 34,35 is an electrostatics comparison program, it compares electrostatic potential maps of pre-aligned molecules and determines the Tanimoto measures for the comparison. The similarity between the compounds and GTP-α-S was also accessed in terms of electrostatic potential with the program EON.…”

“…The combination of ROCS and EON potentially yields compounds with similar shape and electrostatics. 34,35 Target based virtual screening methods FRED (Fast Exhaustive Docking, version 2.2.5), was used for docking purposes. The 3D structure of diguanylate cyclase PLED (PDB ID 2V0N) used for docking was prepared with the MAKE RECEPTOR program.…”

Planejamento, síntese e avaliação de inibidores da enzima cruzaína e de agentes tripanossomicidas derivados de imidazopiridina

“…These set of information guided us to propose novel cruzain inhibitors to be synthesized. Then, the best HQSAR model obtained was used to predict the pIC 50 values of these new compounds. Some compounds identified using this method has shown calculated potencies higher than those which have originated them.…”

“…Assim como em outras áreas do conhecimento humano, o uso do termo "Química As amostras do grupo teste foram escolhidas cuidadosamente para compor a validação interna, de forma que os compostos com valores extremos de pIC 50 fossem evitados, mas que uma distribuição regular fosse mantida ao longo de todo o conjunto. Assim como, uma análise detalhada das similaridades estruturais, para que aquelas moléculas com características únicas fossem mantidas no conjunto de treinamento.…”

“…No capítulo 3, estratégias de química medicinal foram empregadas nas sínteses de 23 novos análogos, contendo o esqueleto básico de imidazopiridina. Do total de compostos sintetizados, sete e doze compostos exibiram EC 50 ≤ 1µM in vitro contra os parasitos Tripanosoma cruzi (T. cruzi) e brucei (T. brucei), respectivamente. Com os resultados promissores de atividade biológica in vitro, citotoxicidade, estabilidade metabólica, ligação proteica e propriedades farmacocinéticas, o composto 41 foi selecionado como candidato para os estudos de eficácia in vivo.…”