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.

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A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.

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Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis.

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Natural polymer-based films, due to their favorable biological and mechanical properties, have demonstrated great potential as coatings for biomedical applications. Among them, chitosan films have been widely studied both as coating materials and as controlled drug release systems. Crosslinkers are often used to tune chitosan’s crosslinking degree and thus to control the drug release kinetics. For this purpose, quercetin, a plant-derived natural polyphenol, has gained attention as a crosslinker, mainly for its intrinsic anti-inflammatory, antioxidant, and antibacterial features. In this study, chitosan films crosslinked with three different concentrations of quercetin (10, 20, and 30% w/w) have been used as controlled release systems for the delivery of the antibacterial drug trimethoprim (TMP, 10% w/w). Physicochemical and antimicrobial properties were investigated. Surface wettability and composition of the films were assessed by contact angle measurements, X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectroscopy (FTIR), respectively. The release kinetic of TMP in phosphate-buffered saline (PBS) and 2-(N-morpholino) ethanesulfonic acid (MES) was studied over time. Finally, antibacterial properties were assessed on E. coli and S. aureus through Kirby–Bauer disc diffusion and micro-dilution broth assays. Results show that quercetin, at the tested concentrations, clearly increases the crosslinking degree in a dose-dependent manner, thus influencing the release kinetic of the loaded TMP while maintaining its bactericidal effects. In conclusion, this work demonstrates that quercetin-crosslinked chitosan films represent a promising strategy for the design of antibiotic-releasing coatings for biomedical applications.