Carlos Alberto Montanari

Cysteine proteases are recognized as relevant therapeutic targets in both parasitic diseases and cancer due to their essential biological functions. Among these enzymes, cruzain from Trypanosoma cruzi is an important molecular target for the treatment of Chagas disease. Likewise, human cathepsin L (hCatL) is a potential target for anticancer drug development. Both enzymes share conserved catalytic features, particularly the cysteine–histidine catalytic dyad, which enables interaction with nitrile‐based reversible covalent inhibitors. Here, six nitrile‐derived inhibitors ( Neq1171.1, Neq1172.1, Neq1181.1, Neq1182.1, Neq1268, and Neq1276 ) had their cruzain and hCatL inhibition constants, K _i , determined. Besides, we have used molecular dynamics simulation and alchemical free energy calculations for evaluating the overall relative binding free energies of these inhibitors. The calculated free energy differences closely reproduced experimental trends, confirming the reliability of the computational protocol. These findings demonstrate that thermodynamic integration is an effective strategy for predicting relative binding affinities and provides valuable molecular‐level insights for the rational optimization of cysteine protease inhibitors with potential applications in Chagas disease and cancer therapy.

Background: Prostate cancer is one of the most prevalent and deadly neoplasias in the male population. Despite the availability of therapies that increase the long-term survival of patients with localized tumors, metastatic prostate cancer is challenging to treat. A previous study revealed that the 2-aminopyridine derivative (named Neq0440) inhibited the PI3K-AKT-mTOR pathway and presented selective cytotoxicity toward the metastatic prostate cancer cell line PC-3. Methods: Here, we further analyzed the mechanism of action of these molecules by using cell-based colorimetric, fluorometric, epifluorescence microscopy, and Western blot assays. Results: Mitochondrial depolarization increased the AMPK level at 24 h inhibition with Neq0440, which led to the PI3K-AKT-mTOR pathway downregulation after 48 h. The phosphorylation was inhibited for AKT and the downstream quinases (S6RP and 4EBP1) from the PI3K-AKT-mTOR pathway, which can work together with the mitochondrial depolarization, lowering the pH of the medium, increasing ROS levels, and translocating the lysosomes toward the nucleus to trigger cell death. Conclusions: Therefore, Neq0440 can be used as a lead compound to obtain derivatives with a novel anticancer mechanism of action.

Drug repurposing and repositioning are concepts that involve identifying alternative therapeutic uses for existing drug candidates or molecular series. During the COVID‐19 pandemic, hundreds of antivirals were developed, many of which remain unexplored for other diseases. Concurrently, machine learning (ML) has become a valuable tool in early drug discovery for screening the most promising compounds for a target. In this work, an ExtraTrees ML model is developed to predict inhibitory activity against cruzain, the main cysteine protease of Trypanosoma cruzi , the causative agent of Chagas disease. The model is used to screen a proprietary library of peptidomimetic compounds originally designed to target SARS‐CoV‐2 M ^pro and human cathepsin L. High‐affinity cruzain inhibitors are identified, some containing P1 moieties not previously reported in cruzain inhibitors, expanding the known chemical space for this target. Selected hits are validated using isothermal titration calorimetry and some compounds display more favorable enthalpic and entropic contributions to binding than similar peptidomimetic nitrile‐based inhibitors. Notably, this is achieved without highly lipophilic R‐groups, preserving drug‐like properties. This work also highlights how compound libraries derived from global health efforts can be effectively repurposed for neglected tropical diseases with ML models.

Prostate cancer is one of the most prevalent and deadly neoplasias in the male population. Despite the availability of therapies that increase the long-term survival of patients with localized tumors, metastatic prostate cancer is challenging to treat. A previous study revealed that the 2-aminopyridine derivative (named Neq0440) inhibited the PI3K-AKT-mTOR pathway and presented selective cytotoxicity toward the metastatic prostate cancer cell line PC-3. Here, we further analyzed the mechanism of action of these molecules by using cell-based colorimetric, fluorometric, epifluorescence microscopy, and Western blot assays. Mitochondrial depolarization increased the AMPK level at 24 h inhibition with Neq0440, which led to the PI3K-AKT-mTOR pathway downregulation after 48 h. The phosphorylation was inhibited for AKT and the downstream quinases (S6RP and 4EBP1) from the PI3K-AKT-mTOR pathway, which can work together with the mitochondrial depolarization, lowering the pH of the medium, increasing ROS levels, and translocating the lysosomes toward the nucleus to trigger cell death. Therefore, Neq0440 can be used as a lead compound to obtain derivatives with a novel anticancer mechanism of action.

The COVID‐19 pandemic underscored the urgent need for effective antiviral agents, particularly against coronaviruses, which pose a continuing threat of future outbreaks. Targeting the main protease (M ^pro ), a key enzyme in viral replication, represents a promising therapeutic strategy. This study investigates structural modifications to known M ^pro inhibitors, focusing on substitutions at the P2 position to explore alterations in both inhibitory potency and metabolic stability. Computational modeling and biochemical assays revealed that incorporating large, hydrophobic, and π‐rich groups, such as the 4‐phenylproline, significantly enhances binding affinity. Additionally, we evaluated warheads that have not yet been explored in the context of SARS‐CoV‐2 M ^pro inhibition. Among these, fluoro‐vinylsulfone and nitrile groups demonstrated superior inhibitory activity. A fragment‐merging strategy combining an optimized P2 substituent with the nitrile warhead yielded a hybrid molecule with binding affinity comparable to nirmatrelvir. However, other analogs incorporating individual warhead optimizations displayed similar potency. These findings generate valuable insights into the design of robust M ^pro inhibitors and support their potential development as broad‐spectrum antiviral agents.

Cysteine proteases are pivotal enzymes involved in diverse physiological and pathological processes, with cruzain and human cathepsin L emerging as key therapeutic targets. We report the design, synthesis, and characterization of a series of dipeptidyl inhibitors incorporating engineered modifications at the P1, P2, and P3 positions. Among these, Neq1276, featuring an indole substituent at P2, displayed the highest affinity for hCatL (pKi = 9.9) while maintaining activity against Cz (pKi = 7.4), whereas Neq1277 showed enhanced affinity for Cz (ΔpKi = +1.0) with preserved selectivity. Thermodynamic analyses by isothermal titration calorimetry uncovered a rare synergistic additivity of enthalpic and entropic contributions in Neq1153, Neq1260, and Neq1265, deviating from the widely observed entropy–enthalpy compensation. This phenomenon provides a unique framework for rational ligand design by enabling simultaneous ΔH and ΔS gains in binding. Selectivity patterns highlighted the key role of P2 substituents, particularly the indole group, in fine-tuning recognition. Retrospective database analysis further identified Neq1276 as a novel chemical entity, underscoring its potential as a promising lead compound for future development.

The closo -1,2-C ₂ B ₁₀ H ₁₂ carborane is a recognized 3D aromatic icosahedral building block, with an electron distribution governed by the outer hydridic BH and acidic CH vertices.

Promiscuous fragments as part of frequent-hitters can certainly be seen as problematic in fragment-based design. In contrast, previous work indicated that promiscuous scaffolds can be part of ligands binding to unrelated targets, whereas the ligands can still show selectivity. In analogy to this work, we present a scaffold-based analysis of bioactivity data with the focus to identify promiscuous scaffolds or fragments on a broader scale. A scaffold network was created based on the ChEMBL database, followed by the analysis of the scaffold decoration using a python based workflow and target similarity calculations. In the end, 3124 promiscuous scaffolds belonging to ligands binding to different unrelated proteins were identified. Within these scaffolds, a phenylthiophene-like scaffold was identified in ligands binding to five unrelated protein targets that show similar hydrophobic subpockets. Interestingly, a subsequent docking study revealed different binding modes of the corresponding ligands, which indicates also a promiscuous subpocket. Subsequent screening by binding site comparisons of the protein ligand complexes available in the PDB revealed Cruzipain of T. cruzi as potential new target for this specific scaffold. The binding site similarity to Cruzipain was confirmed by biochemical validation of the phenylthiophene-like scaffold as well as grown derivatives. A subsequent analysis of the REAL Space showed that these promiscuous scaffolds are also part of the ultra-large chemical space of Enamine. Together with the available commercial derivatives, these promiscuous scaffolds can be seen as a promising sociable fragment library for fragment-based design by catalogue. In contrast, this library can also be excluded from fragment libraries depending on the viewpoint if such promiscuous scaffolds are foe or friends.

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.