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.

Keywords: ABPP; Leishmania; fluorophosphonate; proteomics; serine protease inhibitors. Discovery of Leishmania druggable serine proteases; serine proteases.

FIGURE 1

(A) ABPP workflow of serine hydrolases in Leishmania spp. (B): Structures of TAMRA-FP (red) and FP-Biotin (green).

FIGURE 2

In-gel fluorescence analysis (emission at 560 nm)—serinome fingerprint of Leishmania spp. lysates (1 mg/ml) revealed by TAMRA-FP (1 μM). Lane 1: Markers; Lane 2: Log-phase L. mexicana; Lane 3: Log-phase L. major; Lane 4: Log-phase L. amazonensis; Lane 5: Markers; Lane 6: Stationary-phase L. mexicana; Lane 7: Stationary-phase L. major; Lane 8: Stationary-phase L. amazonensis; Lane 9: Markers; Lane 10: Log-phase L. mexicana; Lane 11: Stationary-phase L. mexicana; Lane 12: Axenic amastigotes L. mexicana. DMSO was used as a negative control and pre-heated (95°C for 5 min) proteome solutions were used to verify FP-TAMRA probe specificity with no labelling being observed (Supplementary Figures S2–S4).

FIGURE 3

(A) In-gel fluorescence analysis (emission at 560 nm) of the competitive ABPP between FP-biotin conducted to compare affinity. Lane 1: Protein ladder. Lane 2: FP-biotin (4 μM) was incubated in a L. mexicana promastigote lysate (1 mg/ml) for 60 min at rt. Subsequently, TAMRA-FP (1 μM) was added, and the mixture was incubated for a further 15 min followed by SDS-PAGE and fluorescent imaging. Lane 3: Labelling of TAMRA-FP (1 μM) in L. mexicana promastigote lysate (1 mg/ml). (B) A representative MS/MS spectrum of a tryptic peptide of carboxypeptidase LmxM.18.0450 (B1) and prolyl oligopeptidase LmxM.36.675 (B2) identified by database search using MaxQuant showing the observed y and b ion fragmentation patterns (values in tables).

FIGURE 4

Structure of chymostatin (1), ZPP (2), lactacystin (3), and omuralide (4).

FIGURE 5

Left: In-gel fluorescence analysis (emission at 560 nm) of the competitive ABPP between increasing concentration of serine protease inhibitors and TAMRA-FP (1 μM) in L. mexicana promastigotes lysates (1 mg/ml). Right: Relative fluorescence units (RFU) vs. competitor concentration inhibition curves of bands POP (80 kDa) and Carboxypeptidase (60 kDa). In details, Gel (A) Chymostatin (0–500 μM); Gel (B) ZPP (0–100 μM); and Gel (C) Lactacystin (0–50 μΜ). For experiments and analysis performed in L. mexicana amastigote lysates, Supplementary Figure S9.