Cruzain Inhibitors Research Articles

Discovery of Novel Inhibitors of Cruzain Cysteine Protease of Trypanosoma cruzi.

Chagas disease (CD) is a parasitic disease endemic in several developing countries. According to the World Health Organization, approximately 6-8 million people worldwide are inflicted by CD. The scarcity of new drugs, mainly for the chronic phase, is the main reason for treatment limitation in CD. Therefore, there is an urgent need to discover new targets for which new therapeutical agents could be developed. Cruzain cysteine protease (CCP) is a promising alternative because this enzyme exhibits pleiotropic effects by acting as a virulence factor, modulating host immune cells, and interacting with host cells. This systematic review was conducted to discover new compounds that act as cruzain inhibitors, and their effects in vitro were studied through enzymatic assays and molecular docking. Additionally, the advances and perspectives of these inhibitors are discussed. These findings are expected to contribute to medicinal chemistry in view of the design of new, safe, and efficacious inhibitors against Trypanosoma cruzi CCP detected in the last decade (2013-2022) to provide scaffolds for further optimization, aiming toward the discovery of new drugs.

Thirty Years in the Design and Development of Cruzain Inhibitors

Cruzipain is the principal protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Since its discovery in the 1980s and the resolution of the crystal structure of cruzain (a truncated recombinant form of the enzyme) in 1995, this target has attracted the interest of many research groups for screening studies, structure-based and ligand-based drug design campaigns, which include peptide-like and non-peptide synthetic compounds. In this context, empirical and computational methods have proven to be valuable tools for the study of mechanisms of action, potential binding modes and structure-activity relationships for a diverse series of cruzain/cruzipain inhibitors. This paper, therefore, reviews some of the most relevant chemical groups reported as cruzain inhibitors over the last 30 years of research.

Trypanocidal potential of synthetic p-aminochalcones: In silico and in vitro evaluation

Chagas disease (CD) is a neglected tropical disease of worldwide health concern, caused by the flagellate protozoan Trypanosoma cruzi (T. cruzi), endemic in Latin America and present in North America and Europe. The WHO recommended drug for CD, benznidazole has low safety profile and several limitations. Therefore, an entity with better therapeutic potential to treat CD is required. Chalcones are an important class of compounds, which have shown antichagasic potential. Thus, the objective of this study was to evaluate the activity of synthetic p-aminochalcones against T. cruzi. Chalcones 1 and 2 were synthesized by Claisen-Schmidt condensation and characterized by both spectroscopic and theoretical methods. Initially, they were submitted to molecular docking simulations using cruzain and trypanothione reductase (TR) enzymes. It was expected to observe the possible interactions of chalcones with the catalytic site and other important regions of these main pharmacological targets of T. cruzi. Their cytotoxicity within host cells were assessed by MTT reduction assay using LLC-MK2 cells, with CC50 = 85.6 ± 9.2 µM and 1115 ± 381.7 µM for chalcones 1 and 2, respectively. These molecules were also tested against epimastigote and trypomastigote life forms of T. cruzi, causing reduction in the number of viable parasites. For the evaluation of the effect on intracellular amastigotes, infected LLC-MK2 cells were incubated with the chalcones for 24 h, causing reduction in the percentage of infected cells and the number of amastigotes/100 cells. Finally, flow cytometry assays were performed for analyzing cell death mechanisms (7-AAD/AxPE labelling), cytoplasmic ROS accumulation (DCFH-DA assay) and mitochondrial transmembrane potential disruption (Rho123 assay). Both chalcones (1 and 2) caused membrane damage, ROS accumulation and mitochondrial depolarization. In conclusion, the synthetic p-aminochalcones presented trypanocidal effect, causing membrane damage and oxidative stress. Their mechanism of action may be related to cruzain and TR inhibition.

Convergent QSAR Models for the Prediction of Cruzain Inhibitors.

Chagas disease is a parasitosis caused by Trypanosoma cruzi. Cruzain, the major cysteine protease from T. cruzi, is an excellent therapeutic target in the search for antichagasic drugs. It is important in the role of cell invasion, replication, differentiation, and metabolism of the parasite. In this work, we developed and assessed multiple quantitative structure-activity relationship (QSAR) models for a set of 61 cruzain inhibitors. These models include two-dimensional (2D) QSAR, three-dimensional (3D) QSAR, such as comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA), and Hologram QSAR (HQSAR). In total, we generated 10 major and 114 minor model variations. Molecular docking was used to successfully align the molecules. All CoMFA and CoMSIA models, which incorporate multiple fields, demonstrated robustness in our analysis. Steric fields exhibited satisfactory convergence in the contour maps, while the electrostatic field converged into a single small region. The HQSAR model taking into consideration only Atoms and Connectivity, with fragment sizes ranging from two to five atoms, was considered the best of the HQSAR variations, despite exhibiting a higher level of deviance. In total, 78 model variations meet the minimum requirements to be considered acceptable. We found that using as few as five descriptors it is possible to obtain robust results with 2D-QSAR. Models such as Random Forest, Tree Ensemble, Linear Regression, and HQSAR are recommended for working with large data sets, while the 3D-QSAR models are intended to study the geometry of the ligands, to optimize them into new and better performing antichagasics. Virtual Screening of a set of hydrazones, guided by the top-performing models, identified promising candidates for experimental validation. Among them, dv007 and dv015 exhibited consistently high predicted pIC50 values (7.26 and 7.24, respectively), making them compelling candidates for further drug development.

Structure-based discovery of novel cruzain inhibitors with distinct trypanocidal activity profiles

Over 110 years after the first formal description of Chagas disease, the trypanocidal drugs thus far available have limited efficacy and several side effects. This encourages the search for novel treatments that inhibit T. cruzi targets. One of the most studied anti-T. cruzi targets is the cysteine protease cruzain; it is associated with metacyclogenesis, replication, and invasion of the host cells. We used computational techniques to identify novel molecular scaffolds that act as cruzain inhibitors. First, with a docking-based virtual screening, we identified compound 8, a competitive cruzain inhibitor with a Ki of 4.6 μM. Then, aided by molecular dynamics simulations, cheminformatics, and docking, we identified the analog compound 22 with a Ki of 27 μM. Surprisingly, despite sharing the same isoquinoline scaffold, compound 8 presented higher trypanocidal activity against the epimastigote forms, while compound 22, against the trypomastigotes and amastigotes. Taken together, compounds 8 and 22 represent a promising scaffold for further development of trypanocidal compounds as drug candidates for treating Chagas disease.

Experimental and Computational Study of Aryl-thiosemicarbazones Inhibiting Cruzain Reveals Reversible Inhibition and a Stepwise Mechanism.

Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi, is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1). Additionally, we studied a semicarbazone (compound 2), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The Ki was estimated to be 36.3 μM and Ki* to be 11.5 μM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S- on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1, involving the proton transfer to the ligand, followed by the Cys25-S- attack at C═S. The ΔG and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.

Screening the Pathogen Box to Discover and Characterize New Cruzain and TbrCatL Inhibitors

Chagas disease and Human African Trypanosomiasis, caused by Trypanosoma cruzi and T. brucei, respectively, pose relevant health challenges throughout the world, placing 65 to 70 million people at risk each. Given the limited efficacy and severe side effects associated with current chemotherapy, new drugs are urgently needed for both diseases. Here, we report the screening of the Pathogen Box collection against cruzain and TbrCatL, validated targets for Chagas disease and Human African Trypanosomiasis, respectively. Enzymatic assays were applied to screen 400 compounds, validate hits, determine IC50 values and, when possible, mechanisms of inhibition. In this case, 12 initial hits were obtained and ten were prioritized for follow-up. IC50 values were obtained for six of them (hit rate = 1.5%) and ranged from 0.46 ± 0.03 to 27 ± 3 µM. MMV687246 was found to be a mixed inhibitor of cruzain (Ki = 57 ± 6 µM) while MMV688179 was found to be a competitive inhibitor of cruzain with a nanomolar potency (Ki = 165 ± 63 nM). A putative binding mode for MMV688179 was obtained by docking. The six hits discovered against cruzain and TbrCatL are of great interest for further optimization by the medicinal chemistry community.

Investigating the Lack of Translation from Cruzain Inhibition to Trypanosoma cruzi Activity with Machine Learning and Chemical Space Analyses.

Chagas disease is a neglected tropical disease caused by the protozoa Trypanosoma cruzi. Cruzain, its main cysteine protease, is commonly targeted in drug discovery efforts to find new treatments for this disease. Even though the essentiality of this enzyme for the parasite has been established, many cruzain inhibitors fail as trypanocidal agents. This lack of translation from biochemical to biological assays can involve several factors, including suboptimal physicochemical properties. In this work, we aim to rationalize this phenomenon through chemical space analyses of calculated molecular descriptors. These include statistical tests, visualization of projections, scaffold analysis, and creation of machine learning models coupled with interpretability methods. Our results demonstrate a significant difference between the chemical spaces of cruzain and T. cruzi inhibitors, with compounds with more hydrogen bond donors and rotatable bonds being more likely to be good cruzain inhibitors, but less likely to be active on T. cruzi. In addition, cruzain inhibitors seem to occupy specific regions of the chemical space that cannot be easily correlated with T. cruzi activity, which means that using predictive modeling to determine whether cruzain inhibitors will be trypanocidal is not a straightforward task. We believe that the conclusions from this work might be of interest for future projects that aim to develop novel trypanocidal compounds.

Cruzain Inhibitors: State-of-Art of Novel Synthetic Strategies

Abstract: Concerned about a million people are infected worldwide, and other millions are living at risk zones of infection. Chagas disease causes 10 000 deaths annually, and the discovery of safe and effective drugs on a nanomolar scale has been headlined as a crucial goal by the worldwide research community and international health agencies. Nifurtimox and Benznidazole are the only marketed drugs for Chagas disease therapy, with the mode of action depending on the formation of free radicals. Thus, diverse studies have revealed cysteine proteases from T. cruzi as well-established targets for drug discovery. Validation of cruzain as a promising target has been based on several findings on the molecular biology of T. cruzi. Hence, several inhibitor classes have been elucidated, consisting of peptidic and nonpeptidic, and covalent and noncovalent. Thus, we present a perspective for the synthesis of novel cruzain inhibitors from scaffolds both recently approached and well established with an IC50 range of micromolar to nanomolar and supported by computational techniques.

A structure-based virtual high-throughput screening, molecular docking, molecular dynamics and MM/PBSA study identified novel putative drug-like dual inhibitors of trypanosomal cruzain and rhodesain cysteine proteases.

Virtual screening a collection of ~ 25,000 ChemBridge molecule collection identified two nitrogenous heterocyclic molecules, 12 and 15, with potential dual inhibitory properties against trypanosomal cruzain and rhodesain cysteine proteases. Similarity search in DrugBank found the two virtual hits with novel chemical structures with unreported anti-trypanosomal activities. Investigations into the binding mechanism by molecular dynamics simulations for 100ns revealed the molecules were able to occupy the binding sites and stabilise the protease complexes. Binding affinities calculated using the MM/PBSA method for the last 20ns showed that the virtual hits have comparable binding affinities to other known inhibitors from literature suggesting both molecules as promising scaffolds with dual cruzain and rhodesain inhibition properties, i.e. 12 has predicted ΔGbind values of -38.1 and -38.2kcal/mol to cruzain and rhodesain, respectively, and 15 has predicted ΔGbind values of -34.4 and -25.8kcal/mol to rhodesain. Per residue binding free energy decomposition studies and visual inspection at 100ns snapshots revealed hydrogen bonding and non-polar attractions with important amino acid residues that contributed to the ΔGbind values. The interactions are similar to those previously reported in the literature. The overall ADMET predictions for the two molecules were favourable for drug development with acceptable pharmacokinetic profiles and adequate oral bioavailability.

From rational design to serendipity: Discovery of novel thiosemicarbazones as potent trypanocidal compounds

Chagas disease is a major public health problem caused by Trypanosoma cruzi, with an estimated 6–7 million people infected and 70 million at risk of infection. T. brucei gambiense and T. brucei rhodesiense are two subspecies of related parasites that cause human African trypanosomiasis, a neglected tropical disease with also millions of people at risk of infection. Pharmacotherapy for both diseases suffers from low efficacy, side effects, or drug resistance. Recently, we reported a noncovalent competitive inhibitor of cruzain (IC50 26 μM, Ki 3 μM) and TbrCatL (IC50 50 μM), two cysteine proteases considered promising drug targets for trypanosomiasis. Here, we describe the design and synthesis of derivatives of our lead compound. The new thiosemicarbazone derivatives showed potency in the nanomolar concentration range against the two enzymes, but they were later characterized as aggregators. Nevertheless, the thiosemicarbazone derivatives showed promising antiparasitic activities against T. b. brucei (EC50 13–49.7 μM) and T. cruzi (EC50 0.027–0.59 μM) under in vitro conditions. The most active thiosemicarbazone was 200-fold more potent than the current anti-chagasic drug, benznidazole, and showed a selectivity index of 370 versus myoblast cells. We have identified an excellent candidate for further optimization and in vivo studies.

Data and code for "Investigating the lack of translation from cruzain inhibition to Trypanosoma cruzi activity with machine learning and chemical space analyses"

Data and code for "Investigating the lack of translation from cruzain inhibition to Trypanosoma cruzi activity with machine learning and chemical space analyses"

Novel trypanocidal thiophen-chalcone cruzain inhibitors: structure- and ligand-based studies.

Background: Chagas disease is a neglected tropical disease that affects millions of people worldwide and for which no effective treatment is available. Materials & methods: 17chalcones were synthesized, for which the inhibition of cruzain and trypanocidal activity were investigated. Results: Chalcone C8 showed the highest cruzain inhibitory (IC50=0.536μm) and trypanocidal activity (IC50=0.990μm). Molecular docking studies showed interactions involving Asp161 and the thiophen group interacting with the S2 subsite. Furthermore, quantitative structure-activity relationship (q2=0.786; r2=0.953) and density functional theory studies were carried out, and a correlation between the lowest unoccupied molecular orbital surface and trypanocidal activity was observed. Conclusion: These results demonstrate that these chalcones are worthwhile hits to be further optimized in Chagas disease drug discovery programs.

Study of the dynamic behavior of the cruzain enzyme in free and complexed forms with competitive and noncovalent benzimidazole inhibitors

There are only two drugs for the treatment of Chagas disease, namely, nifurtimox and benznidazole, that can cause several adverse effects. Despite the effectiveness of these drugs in the disease's acute phase, they are not recognized as curative in the chronic phase, establishing the need for more effective treatment in all stages of the disease. Cruzain is an enzyme that plays a vital role in the life cycle of the etiologic agent, the protozoan Trypanosoma cruzi, being relevant as a therapeutic target in the planning of new drugs. Using molecular docking and dynamics simulations, we have investigated the structural and dynamic factors that can be involved in the enzyme inhibition process at the atomic-molecular level by benzimidazole compounds that are potent cruzain inhibitors with in vitro trypanocidal activity. The study suggests that these inhibitors bind cruzain through steric and hydrogen bonding interactions without altering its secondary structure content and protein compaction. Besides, we observed that these inhibitors decrease the correlation of movements between Cα-atoms of cruzain, increasing the number of atomic communities, mainly in the α-helix that presents the catalytic Cys25 residue. As expected, we also observed a correlation between the inhibitory activity of each inhibitor and their respective binding-free energies, reinforcing that the affinity of the complexes seems to be a relevant factor for enzymatic inhibition. Hence, the results presented in this work contribute to a better understanding of the cruzain enzyme inhibition mechanism through competitive and non-covalent inhibitors. Communicated by Ramaswamy H. Sarma

Isometric Stratified Ensembles: A Partial and Incremental Adaptive Applicability Domain and Consensus-Based Classification Strategy for Highly Imbalanced Data Sets with Application to Colloidal Aggregation.

Partial and incremental stratification analysis of a quantitative structure-interference relationship (QSIR) is a novel strategy intended to categorize classification provided by machine learning techniques. It is based on a 2D mapping of classification statistics onto two categorical axes: the degree of consensus and level of applicability domain. An internal cross-validation set allows to determine the statistical performance of the ensemble at every 2D map stratum and hence to define isometric local performance regions with the aim of better hit ranking and selection. During training, isometric stratified ensembles (ISE) applies a recursive decorrelated variable selection and considers the cardinal ratio of classes to balance training sets and thus avoid bias due to possible class imbalance. To exemplify the interest of this strategy, three different highly imbalanced PubChem pairs of AmpC β-lactamase and cruzain inhibition assay campaigns of colloidal aggregators and complementary aggregators data set available at the AGGREGATOR ADVISOR predictor web page were employed. Statistics obtained using this new strategy show outperforming results compared to former published tools, with and without a classical applicability domain. ISE performance on classifying colloidal aggregators shows from a global AUC of 0.82, when the whole test data set is considered, up to a maximum AUC of 0.88, when its highest confidence isometric stratum is retained.

Thiosemicarbazone derivatives: Evaluation as cruzipain inhibitors and molecular modeling study of complexes with cruzain

The development of cruzipain inhibitors represents one of the most attractive challenges in the search for drugs for the treatment of Chagas disease. A recombinant form of this enzyme, cruzain, has been crystallized with numerous inhibitors, excluding thiosemicarbazones. These compounds have been established as potent inhibitors of cruzain, although there is very little data in the literature of thiosemicarbazones tested on cruzipain. In this work, we present the results of the evaluation of eleven thiosemicarbazones on cruzipain, isolated from T. cruzi epimastigotes, six of them previously evaluated on cruzain. For these latter, we studied through computational methods, the mode of interaction with the active site of cruzain and the contribution of geometric parameters to the possible mechanism of action involved in the observed inhibition. Finally, from some geometric parameters analyzed on modeled TSC-cruzain complexes, a semi-quantitative relationship was established that could explain the inhibitory activity of thiosemicarbazones on cruzipain, the enzyme actually present in the parasite.

Impact of different protonation states on virtual screening performance against cruzain.

The cysteine protease cruzain is a Chagas disease target, exploited in computational studies. However, there is no consensus on the protonation states of the active site residues Cys25, His162, and Glu208 at the enzyme's active pH range. We evaluated the impact of different protonation states of these residues on docking calculations. Through a retrospective study with cruzain inhibitors and decoys, we compared the performance of virtual screening using four grids, varying protonation states of Cys25, His162, and Glu208. Based on enrichment factors and ROC plots, docking with the four grids affected compound ranking and the overall charge of top-ranking compounds. Different grids can be complementary and synergistic, increasing the odds of finding different ligands with diverse chemical properties.

Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics.

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite’s life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.

Computational approaches towards the discovery and optimisation of cruzain inhibitors

The need to develop safer and more efficacious drugs to treat Chagas disease has motivated the search for cruzain inhibitors. Cruzain is the recombinant, truncated version of cruzipain, a cysteine protease from Trypanosoma cruzi with important roles during the parasite life cycle. Several computational techniques have been applied to discover and optimise cruzain inhibitors, providing a molecular basis to guide this process. Here, we review some of the most recent computational studies that provided important information for the design of cruzain inhibitors. Moreover, we highlight the diversity of applications of in silico techniques and their impact.

PARA UMA DOENÇA TÃO ANTIGA, O VISLUMBRE DE NOVOS TRATAMENTOS PROMISSORES NA DOENÇA DE CHAGAS

It has been more than 100 years since the discovery of Chagas Disease (CD). However, the repertoire indicated for its treatment is still limited. Thus, this article aims to present a review of the new pharmacological strategies being studied for CD. This literature review, consisting of 68 articles, from 1957 to 2021, was carried out on several scientific platforms. Positive effects from benznidazole have been described in the acute and chronic phases, in addition to its association with itraconazole in the acute phase. Among the cruzain inhibitors, the compound K777 presented trypanocidal effects, although demonstrating major adverse effects, while its analogue WRR-483 demonstrated great beneficial effects in vivo and in vitro. As for the nitroheterocyclics, fexinidazole showed high rates of cure in animal model, in addition to low toxicity. Nifurtimox, in early chronic stages, was able to delay the progression of tissue damage and reduce the parasite load. The compound WC-9, a squalene synthase inhibitor, showed potential inhibition of T. cruzi replication. Regarding aromatic diamidines, many compounds were able to stop the trypanosome, both in vitro and in vivo models. It was concluded that there are favorable findings to improve the treatment of CD. However, the development of effective new drugs does not only depend on their effective action, but also on numerous variables that must be circumvented, such as the reduction of side effects, treatment time and adherence to the current medication of choice, as well as the investment in production and distribution to the population.