Computational Strategies Targeting Inhibition of Helicobacter pylori and Cryptococcus neoformans Ureases.

Helicobacter pylori and Cryptococcus ssp. are pathogenic ureolytic microorganisms that cause several disorders in the host organism and, in severe cases, lead to death. Both infections have the urease enzyme as a key virulence factor since they use its ability to produce ammonia to soften the inhospitable pH to which they are subjected. In this review, we describe two ureases as possible molecular targets for drug discovery and provide insights for developing potent inhibitors against ureases from these pathogenic microorganisms through computer-aided drug discovery approaches, such as structure-based drug design (SBDD) and structure-activity relationship (SAR). The SAR studies have indicated several essential subunits and groups to be present in urease inhibitors that are critical for inhibitory activity against H. pylori or Cryptococcus spp. Since the threedimensional structure of C. neoformans urease has yet to be determined experimentally, the plant urease of Canavalia ensiformis was used in this study due to its structural similarity. Therefore, in the SBDD context, FTMap and FTSite analyses were performed to reveal characteristics of the urease active sites in two protein data bank files (4H9M, Canavalia ensiformis, and 6ZJA, H. pylori). Finally, a docking-based analysis was performed to explore the best inhibitors described in the literature to understand the role of the ligand interactions with the key residues in complex ligand-urease stabilization, which can be applied in the design of novel bioactive compounds.

Proteasome as a Drug Target in Trypanosomatid Diseases.

Some diseases caused by trypanosomatid parasites, like Leishmaniasis, Chagas Disease, and Human African Trypanosomiasis (HTA), are challenging to manage, mainly concerning pharmacological therapy because they are associated with vulnerable populations. Unfortunately, there is a lack of significant investments in the search for new drugs. Therefore, one of the strategies to aid the discovery of new drugs is to identify and inhibit molecular targets essential to the parasite's survival, such as the proteasome, which degrades most proteins in the parasite cells. Our study has presented several proteasome inhibitors with various pharmacophoric cores, and two of them, 5, and 13, have stood out in the clinical phase of treatment for leishmaniasis.

CYP1B1: A Promising Target in Cancer Drug Discovery.

CYP1B1 plays an essential role in cancer's pathogenesis since it activates procarcinogens. Significantly, this enzyme catalyzes the hydroxylation of 17ß-estradiol, leading to carcinogenic metabolites involved in carcinogenesis and cancer progression. Therefore, the inhibition of CYP1B1 activity is considered a therapeutic target for chemotherapy. In addition, CYP1B1 is overexpressed in hormone-dependent cancer cells and could be related to resistance to anticancer drugs. However, the activity of CYP1B1 in the tumor microenvironment can metabolize and activate prodrugs in cancer cells, providing more selectivity and being useful for chemoprevention or chemotherapy strategies. Furthermore, due to its importance in anticancer drug design, recent studies have reported using computational methods to understand the intermolecular interactions between possible ligands and CYP1B1. Therefore, in this perspective, we highlight recent findings in developing CYP1B1 inhibitors (flavonoids, trans-stilbenes, estradiol derivatives, and carbazoles) and CYP1B1-activated prodrugs (a chalcone DMU-135 and an oxime DMAKO-20). Finally, we have analyzed their possible molecular interactions with this enzymatic target by molecular docking, which can help to design new active substances.

Synergistic antifungal interaction of N-(butylcarbamothioyl) benzamide and amphotericin B against Cryptococcus neoformans.

Introduction: Cryptococcus neoformans is one of the leading causes of invasive fungal infections worldwide. Cryptococcal meningoencephalitis is the main challenge of antifungal therapy due to high morbidity and mortality rates, especially in low- and middle-income countries. This can be partly attributed to the lack of specific diagnosis difficulty accessing treatment, antifungal resistance and antifungal toxicity. Methods: In the present study, the effect of the synthetic thiourea derivative N-(butylcarbamothioyl) benzamide (BTU-01), alone and combined with amphotericin B (AmB), was evaluated in planktonic and sessile (biofilm) cells of C. neoformans. Results: BTU-01 alone exhibited a fungistatic activity with minimal inhibitory concentrations (MICs) ranging from 31.25 to 62.5 µg/mL for planktonic cells; and sessile MICs ranging from 125.0 to 1000.0 µg/mL. BTU-01 caused a concentration-dependent inhibitory activity on cryptococcal urease and did not interfere with plasma membrane fluidity. Molecular docking was performed on Canavalia ensiformis urease, and BTU-01 showed relevant interactions with the enzyme. The combination of BTU-01 and AmB exhibited synergistic fungicidal activity against planktonic and sessile cells of C. neoformans. Microscopic analysis of C. neoformans treated with BTU-01, alone or combined with AmB, revealed a reduction in cell and capsule sizes, changes in the morphology of planktonic cells; a significant decrease in the number of cells within the biofilm; and absence of exopolymeric matrix surrounding the sessile cells. Neither hemolytic activity nor cytotoxicity to mammalian cells was detected for BTU-01, alone or combined with AmB, at concentrations that exhibited antifungal activity. BTU-01 also displayed drug-likeness properties. Conclusion: These results indicate the potential of BTU-01, for the development of new strategies for controlling C. neoformans infections.

Thiohydantoins and hydantoins derived from amino acids as potent urease inhibitors: Inhibitory activity and ligand-target interactions.

We report the investigation of hydantoins and thiohydantoins derived from L and d-amino acids as inhibitors against the Canavalia ensiformis urease (CEU). The biochemical in vitro assay against CEU revealed a promising inhibitory potential for most thiohydantoins with six of them showing %I higher than the reference inhibitor thiourea (56.5%). In addition, thiohydantoin derived from l-valine, 1b, as well as the hydantoin 2d, derived from l-methionine, were identified as the most potent inhibitors with %I = 90.5 and 85.9 respectively. Enzyme kinetic studies demonstrated a mixed and uncompetitive inhibition profile for these compounds with Ki values of 0.42 mM for 1b and 0.99 mM for 2d. These kinetic parameters, obtained from traditional colorimetric assay, were strictly related to the KD values measured spectroscopically by the Saturation Transfer Difference (STD) technique for the urease complex. STD was also used to evince the moieties of the ligands responsible for the binding with the enzyme. Molecular docking studies showed that the thiohydantoin and hydantoin rings can act as a pharmacophoric group due to their binding affinity by hydrogen bonding interactions with critical amino acid residues in the enzyme active and/or allosteric site. These findings agreed with the experimental alpha values, demonstrating that 1b has affinity by free enzyme, and 2d derivative, an uncompetitive inhibitor, has great binding affinity at the allosteric site. The results for the thiohydantoin 1a, derived from d-valine, demonstrated a drastic stereochemical influence on inhibition, kinetics, and binding parameters in comparison to its enantiomer 1b.

Thiohydantoins as anti-leishmanial agents: n vitro biological evaluation and multi-target investigation by molecular docking studies.

Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. The first-line treatment of this disease is still based on pentavalent antimonial drugs that have a high toxicity profile, which could induce parasitic resistance. Therefore, there is a critical need to discover more effective and selective novel anti-leishmanial agents. In this context, thiohydantoins are a versatile class of substances due to their simple synthesis and several biological activities. In this work, thiohydantoins 1a-l were evaluated in vitro for antileishmania activity. Among them, four derivatives (1c, 1e, 1h and 1l) showed promising IC50 values around 10 µM against promastigotes forms of Leishmania amazonensis and low cytotoxicity profile for peritoneal macrophages cells. Besides, these compounds induce oxidative stress through an increase in ROS production and the labeling of annexin-V and propidium iodide, indicating that promastigotes were undergoing a late apoptosis-like process. Additionally, molecular consensual docking analysis was carried out against two important targets to L. amazonensis: arginase and trypanothione reductase enzymes. Docking results suggest that thiohydantoin ring could be a pharmacophoric group due to its binding affinity by hydrogens bond interactions with important amino acid residues at the active site of both enzymes. These results demonstrate that compounds 1c, 1e, 1h and 1l may are promising in future advance studies.Communicated by Ramaswamy H. Sarma.