Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17

Leishmaniasis is a neglected tropical disease; there is currently no vaccine and treatment is reliant upon a handful of drugs suffering from multiple issues including toxicity and resistance. There is a critical need for development of new fit-for-purpose therapeutics, with reduced toxicity and targeting new mechanisms to overcome resistance. One enzyme meriting investigation as a potential drug target in Leishmania is M17 leucyl-aminopeptidase (LAP). Here, we aimed to chemically validate LAP as a drug target in L. major through identification of potent and selective inhibitors. Using RapidFire mass spectrometry, the compounds DDD00057570 and DDD00097924 were identified as selective inhibitors of recombinant Leishmania major LAP activity. Both compounds inhibited in vitro growth of L. major and L. donovani intracellular amastigotes, and overexpression of LmLAP in L. major led to reduced susceptibility to DDD00057570 and DDD00097924, suggesting that these compounds specifically target LmLAP. Thermal proteome profiling revealed that these inhibitors thermally stabilized two M17 LAPs, indicating that these compounds selectively bind to enzymes of this class. Additionally, the selectivity of the inhibitors to act on LmLAP and not against the human ortholog was demonstrated, despite the high sequence similarities LAPs of this family share. Collectively, these data confirm LmLAP as a promising therapeutic target for Leishmania spp. that can be selectively inhibited by drug-like small molecules.

A mock screen was performed with RapidFire-MS (see methods).Recombinant LmLAP enzyme was tested at 5 nM and 10 M LSTVIVR peptide substrate for a 60 min reaction time.S/B: Signal-to-noise ratio.LmLAP is highlighted in red.Data represents the weighted mean ± SD (n = 5).

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Table S3: iTPP hits stabilized in the presence of DDD00057570.
Cut-off value log 2 enrichment > 1 in both replicates A and B Table S4: iTPP hits stabilized in the presence of DDD00097924.
Cut-off value log 2 enrichment > 1 in both replicates A and B

Figure S1 :
Figure S1: Bioinformatics analyses of rLmLAP:bestatin interactions using PyMol.Predicted binding mode of the rLmLAP:bestatin complex.For clarity, the interacting residues within 4Å of the inhibitor are represented as lines and van der Waals interactions between inhibitor and interacting residues are not indicated.Color codes: Inhibitor atoms: Carbon in salmon, oxygen in red, and nitrogen in blue; LAP residues interacting with Zn 2+ ions: carbon in gray, oxygen in red, and nitrogen in blue; LAP's inhibitor interacting residues: carbon in orange, oxygen in red, nitrogen in blue and sulphur in yellow.The Zn 2+ ions are represented as gray spheres.Zn 2+ coordination is represented by dotted lines.

Figure S2 :
Figure S2: Optimization of RapidFire-MS for HsLAP.(A) Relationship between v 0 and enzyme concentration at 60, 120 and 180min reaction times.The selected enzyme concentration (150 nM) is shown with a dashed line.(B) Typical curves at 37, 75 and 150 nM enzyme for a 180 min reaction time.The selected reaction time (180 min) is indicated by a dashed line.In both experiments 2 mM (3.33  appK M ) LSTVIVR peptide substrate was used.Straight line fit equations and coefficients for determination of the linear fits (R 2 ) are shown.(C) Determination of appK M for HsLAP and LSTVIVR peptide substrate.Enzyme was assayed at 150 nM towards ten LSTVIVR peptide substrate concentrations, prepared by serial dilution in water between 3.9 and 2000 M.The reaction time was 180 min.All assays were performed with 1 mM ZnCl 2 .A Michaelis-Menten rectangular hyperbola function was fitted to the experimental data using OriginPro8 SR0 software.R 2 : coefficient of

Figure S3 :
Figure S3: Concentration response for inhibition of HsLAP by DDD00057570 and DDD00097924.HsLAP was assayed at 150 nM with 600 M LSTVIVR peptide substrate, 1 mM ZnCl 2 and 180 min reaction time by RapidFire-MS.IC 50 was calculated by non-linear fit of the dose-response to experimental data using OriginPro8 SR0 software.R 2 : Determination coefficient.Experimental data are presented as mean ± standard deviations (n = 3).

Figure S4 :
Figure S4: Confirmation of LmLAP overexpression in transgenic promastigotes by TMT quantitation.Relative levels of proteins in wild-type (WT) versus transgenic overexpressing (OE) cell lines were directly compared.

Figure S5 :
Figure S5: Cumulative growth of wild-type (WT) and LmLAP overexpressing (OE) parasites.Parasites were grown in the presence of

Figure S6 :
Figure S6: Ultrastructure morphology of L. major promastigotes after treatment with compounds DDD00057570 and DDD00097924.(A-B) Wildtype (WT) cells without any treatment.Notice the elongated cellular form.(C-F) Cells overexpressing LAP (OE).A combination of normal (panel C and D) and abnormal (panel E and F) morphologies were observed, including swollen kinetoplast (C), swollen cell morphology (E) and the presence of vesicle-like

Figure S7 :
Figure S7: EC 50 values for DDD00057570 against Leishmania donovani intracellular amastigotes.For determination of anti-leishmanial activity parasites were exposed to compounds for 96 h, after which parasite and host cells were stained with 5 g/mL Hoechst 33342 dye, and parasite numbers measured by imaging.For cytotoxicity, EC 50 values were determined by exposing macrophages to test compounds for 48 h, with MTT assays used to determine cell viability.Data are presented as mean of three replicates.Standard deviations are within 10% of the mean values.EC 50 : half-maximum effective concentration.CSI: cellular selectivity index (EC 50 macrophage / EC 50 L. donovani).