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

Abstract

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.

Serine Hydrolase Activity‐Based Probes for Use in Chemical Proteomics

“…Recent examples include the discovery of hydroxymethyl(MIDA)boronate inhibitors for predicted carboxypeptidase CPVL, a SH whose endogenous substrates and function were poorly annotated [88] (Figure 7A, right); succinimide-based sulfonyl ester and sulfonamide inhibitors for mitochondrial rhomboid protease PARL, a proteolytic regulator of mitophagy and cell death; [89] and a chymostatin inhibitor for protozoan carboxypeptidase and prolyloligopeptidase, two enzymes implicated in Leishmania mexicana parasitic diseases. [90] Cravatt and coworkers also further advanced their competitive ABPP strategy using kinetically-tuned, moderately-reactive triazole urea-based probes to confirm in vivo target engagement of reversible amido piperazine LYPLA1 and LYPLA2 inhibitors. [91] Since then, our laboratory [17] and others [92] have developed additional tunable probes with distinct reactivity profiles to further aid the discovery of reversible inhibitors.…”

Drug Discovery for Cutaneous Leishmaniasis: A Review of Developments in the Past 15 Years

“…Researchers have established several tools useful for broad-spectrum anti-leishmanial drug discovery, including parasites constitutively expressing fluorescent proteins [103,104], differential protein expression of different parasite life stages [49], automated image analysis protocols [105], proteome mining [106], and kinome mining [107]. These tools were useful in identifying several potential drug targets, including inositol phosphorylceramide synthase using purified Leishmania enzyme in a plate-based assay [108], serine proteases through activity-based protein profiling of promastigotes [109], the Lmj_04_BRCT protein domain first characterized via homology modeling and then validated in vitro using intracellular amastigotes [110], cysteine protease CPB2.8(∆)CTE via enzymatic screening followed by intramacrophage screening [111], and the Hsp90 chaperone of promastigote parasites [112].…”

Activity-Based Protein Profiling: A Graphical Review

“…Competitive ABPP and comparative ABPP have significantly impacted many stages of drug discovery. Comparative ABPP is particularly useful for target identification and validation (Morimoto and van der Hoorn, 2016), while competitive ABPP is a powerful tool for discovering new inhibitors for specific targets (Porta et al, 2022). By enabling target visualization in living cells or animals, fluorescent probes have enormous potential for future disease diagnosis and therapeutics.…”