Antibiotics | Novel Chalcone Derivatives as Anti-Leishmania infantum Agents

This study designed and synthesized a series of novel 4-hydroxychalcone-derived acetamide compounds, evaluated their anti-Leishmania activity, cytotoxicity, potential synergistic effects with amphotericin B, and predicted their mechanisms of action through in silico analysis. Compound 4b exhibited the best anti-Leishmania activity and selectivity, and effectively reduced drug doses when used synergistically with amphotericin B (AmB). It also demonstrated favorable pharmacokinetic and safety profiles in predictions.

Literature Overview
The article 'Novel Chalcone Derivatives as Anti-Leishmania infantum Agents with Potential Synergistic Activity and In Silico Insights', published in the journal Antibiotics, reviews and summarizes recent research on novel therapeutic strategies for visceral leishmaniasis (VL). The paper focuses on the anti-Leishmania activity of chalcone-acetamide compounds and their synergistic effects with amphotericin B (AmB). Furthermore, the authors predict that these compounds may exert their therapeutic effects by inhibiting key parasitic enzymes through in silico analysis.

Background Knowledge
Visceral leishmaniasis (VL) is a deadly tropical disease caused by Leishmania parasites, primarily transmitted by sandflies. Current treatments, such as amphotericin B and sodium stibogluconate, are associated with high toxicity, high costs, and drug resistance. Chalcone compounds are considered promising anti-Leishmania candidates due to their simple structure, ease of synthesis, and broad biological activities. Chemical modifications to the chalcone scaffold, such as introducing an acetylamino group, may improve pharmacological properties. Additionally, combination therapy strategies (e.g., co-administration with AmB) are emerging as important approaches to enhance efficacy and reduce toxicity. Molecular docking and pharmacokinetic (ADMET) predictions further aid in elucidating the potential mechanisms of action of these compounds, providing theoretical support for drug development.

Research Methods and Experiments
The study synthesized 4-hydroxychalcones via Claisen-Schmidt condensation, followed by alkylation reactions to produce chalcone-acetamides (compounds 3a–c and 4a–c), which were characterized using IR, NMR, and HRMS techniques. Anti-Leishmania activity was evaluated in vitro against L. infantum promastigotes and amastigotes, while cytotoxicity was assessed in human red blood cells and peripheral blood mononuclear cells (PBMCs). Synergistic effects with amphotericin B were analyzed using combination index methodology. Molecular docking studies were performed on key parasitic enzymes (DUB16 and TXNPx), and pharmacokinetic and safety profiles were assessed using ADMET prediction tools.

Key Conclusions and Perspectives

• Phenyl-substituted compounds 3a–c showed no activity, whereas cyclohexyl-substituted compounds 4a–c exhibited anti-Leishmania activity.
• Compound 4b demonstrated IC50 values of 7.02 µM and 3.4 µM against promastigotes and amastigotes, respectively, with low cytotoxicity and a high selectivity index (SI).
• 4b showed significant synergy with AmB, achieving a dose reduction index (DRI) of 2.87, thereby enhancing the therapeutic window.
• Molecular docking analyses revealed that 4b forms hydrogen bonds and hydrophobic interactions with DUB16 and TXNPx, suggesting it may exert anti-Leishmania effects by inhibiting these key enzymes.
• ADMET predictions indicated that 4b has favorable absorption and low toxicity, supporting its potential as a drug candidate.

Conclusion
This study successfully synthesized and evaluated a series of chalcone-acetamide derivatives, among which cyclohexyl-substituted 4b exhibited the strongest anti-Leishmania activity and the lowest cytotoxicity. The synergistic interaction between 4b and AmB further highlights its potential in reducing drug dosage and improving the therapeutic index. In silico analyses support its mechanism of action involving inhibition of key parasitic enzymes (DUB16 and TXNPx). These findings provide a solid foundation for the development of safer and more effective anti-Leishmania agents and offer direction for the optimization of novel acetylated chalcone-based drugs.

Folding Stability

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Prediction of absolute protein stability ΔG by protein sequence inverse folding model ESM-IF. Traditional physical methods (e.g., FoldX, Rosetta, etc.) for predicting protein stability ΔG rely on high-confidence structural pdb, and if there are too many mutations, the structural confidence decreases and the prediction results are poor. Benchmark results at ProteinGym show that the generative model ESM-IF predicts protein mutation stability ΔΔG of DMS data at best-in-class level in zero-shot. The method is an extension of mutation prediction by using the ESM-IF model to directly predict the absolute ΔG value of intact protein folding stability. It was tested with a prediction error RMSE ≈ 1.5 kcal/mol and a correlation coefficient of 0.7, representing a major breakthrough in predicting the folding stability ΔΔG of proteins.