Discovery of Leishmania Druggable Serine Proteases by Activity-Based Protein Profiling

Leishmaniasis are a group of diseases caused by parasitic protozoa of the genus Leishmania. Current treatments are limited by difficult administration, high cost, poor efficacy, toxicity, and growing resistance. New agents, with new mechanisms of action, are urgently needed to treat the disease. Although extensively studied in other organisms, serine proteases (SPs) have not been widely explored as antileishmanial drug targets. Herein, we report for the first time an activity-based protein profiling (ABPP) strategy to investigate new therapeutic targets within the SPs of the Leishmania parasites. Active-site directed fluorophosphonate probes (rhodamine and biotin-conjugated) were used for the detection and identification of active Leishmania serine hydrolases (SHs). Significant differences were observed in the SHs expression levels throughout the Leishmania life cycle and between different Leishmania species. Using iTRAQ-labelling-based quantitative proteomic mass spectrometry, we identified two targetable SPs in Leishmania mexicana: carboxypeptidase LmxM.18.0450 and prolyl oligopeptidase LmxM.36.6750. Druggability was ascertained by selective inhibition using the commercial serine protease inhibitors chymostatin, lactacystin and ZPP, which represent templates for future anti-leishmanial drug discovery programs. Collectively, the use of ABPP method complements existing genetic methods for target identification and validation in Leishmania

Synthesis and interaction of terminal unsaturated chemical probes with Mycobacterium tuberculosis CYP124A1

A series of C15–C20 isoprenyl derivatives bearing terminal alkenyl and alkynyl groups were synthesized as possible substrates of the methyl-branched lipid ω-hydroxylase CYP124A1 from Mycobacterium tuberculosis. The interactions of each compound with the enzyme active site were characterized using UV–vis spectroscopy. We found that C10 and C15 analogs bind with similar affinity to the corresponding parent C10 and C15 substrates geraniol and farnesol, respectively. Three analogs (C10-ω-ene, C10-ω-yne, C15-ω-yne) interact with the proximal side of the heme iron by coordinating to the oxygen atom of the ferric heme, as judged by the appearance of typical Type-IA binding spectra. On the other hand, the C15-ω-ene analog interacts with the ferric heme by displacing the bound water that generates a typical Type I binding spectrum. We were unable to detect P450-mediated oxidation of these probes following extended incubations with CYP124A1 in our reconstituted assay system, whereas a control reaction containing farnesol was converted to ω-hydroxy farnesol under the same conditions. To understand the lack of detectable oxidation, we explored the possibility that the analogs were acting as mechanism-based inhibitors, but we were unable to detect time-dependent loss of enzymatic activity. In order to gain insight into the lack of detectable turnover or time-dependent inhibition, we examined the interaction of each compound with the CYP124A1 active site using molecular docking simulations. The docking studies revealed a binding mode where the terminal unsaturated functional groups were sequestered within the methyl-binding pocket, rather than positioned close to the heme iron for oxidation. These results aid in the design of specific inhibitors of Mtb-CYP124A1, an interesting enzyme that is implicated in the oxidation of methyl-branched lipids, including cholesterol, within a deadly human pathogen

Mapping the Evolution of Activity-Based Protein Profiling: A Bibliometric Review

Activity-based protein profiling (ABPP) is a chemoproteomic approach that employs small-molecule probes to directly evaluate protein functionality within complex proteomes. This technology has proven to be a potent strategy for mapping ligandable sites in organisms and has significantly impacted drug discovery processes by enabling the development of highly selective small-molecule inhibitors and the identification of new therapeutic molecular targets. Despite being nearly a quarter of a century old as a chemoproteomic tool, ABPP has yet to undergo a bibliometric analysis. In order to gauge its scholarly impact and evolution, a bibliometric analysis was performed, comparing all 1919 reported articles with the articles published in the last five years. Through a comprehensive data analysis, including a 5-step workflow, the most influential articles were identified, and their bibliometric parameters were determined. The 1919 analyzed articles span from 1999 to 2022, providing a comprehensive overview of the historical and current state of ABPP research. This analysis presents, for the first time, the characteristics of the most influential ABPP articles, offering valuable insight into the research conducted in this field and its potential future directions. The findings underscore the crucial role of ABPP in drug discovery and novel therapeutic target identification, as well as the need for continued advancements in the development of novel chemical probes and proteomic technologies to further expand the utility of ABPP

Navigating drug repurposing for Chagas disease: advances, challenges, and opportunities

Chagas disease is a vector-borne illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). It poses a significant public health burden, particularly in the poorest regions of Latin America. Currently, there is no available vaccine, and chemotherapy has been the traditional treatment for Chagas disease. However, the treatment options are limited to just two outdated medicines, nifurtimox and benznidazole, which have serious side effects and low efficacy, especially during the chronic phase of the disease. Collectively, this has led the World Health Organization to classify it as a neglected disease. To address this problem, new drug regimens are urgently needed. Drug repurposing, which involves the use of existing drugs already approved for the treatment of other diseases, represents an increasingly important option. This approach offers potential cost reduction in new drug discovery processes and can address pharmaceutical bottlenecks in the development of drugs for Chagas disease. In this review, we discuss the state-of-the-art of drug repurposing approaches, including combination therapy with existing drugs, to overcome the formidable challenges associated with treating Chagas disease. Organized by original therapeutic area, we describe significant recent advances, as well as the challenges in this field. In particular, we identify candidates that exhibit potential for heightened efficacy and reduced toxicity profiles with the ultimate objective of accelerating the development of new, safe, and effective treatments for Chagas disease

Systematic study of 1,2,3-triazolyl sterols for the development of new drugs against parasitic Neglected Tropical Diseases

A series of thirty 1,2,3-triazolylsterols, inspired by azasterols with proven antiparasitic activity, were prepared by a stereocontrolled synthesis. Ten of these compounds constitute chimeras/hybrids of 22,26-azasterol (AZA) and 1,2,3-triazolyl azasterols. The entire library was assayed against the kinetoplastid parasites Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei, the causatives agents for visceral leishmaniasis, Chagas disease, and sleeping sickness, respectively. Most of the compounds were active at submicromolar/nanomolar concentrations with high selectivity index, when compared to their cytotoxicity against mammalian cells. Analysis of in silico physicochemical properties were conducted to rationalize the activities against the neglected tropical disease pathogens. The analogs with selective activity against L. donovani (E4, IC50 0.78 μM), T brucei (E1, IC50 0.12 μM) and T. cruzi (B1– IC50 0.33 μM), and the analogs with broad-spectrum antiparasitic activities against the three kinetoplastid parasites (B1 and B3), may be promising leads for further development as selective or broad-spectrum antiparasitic drugs