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

Abstract

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.

Keywords: Leishmania; M17 leucyl-aminopeptidase; RapidFire-MS; drug discovery; target validation.

Figure 1

Optimization of RapidFire-MS for rLmLAP with LSTVIVR. (A) Relationship between initial velocity (v₀) and enzyme concentration. (B) Michaelis–Menten curve with LSTVIVR substrate within the range 0.19 μM to 1 mM. (C) Determination of the reaction interval in which the initial rate is maintained. (D) Concentration-dependent inhibition for the general LAP inhibitor bestatin.

Figure 2

Compound library inhibition potency toward rLmLAP, and single-point effect against L. donovani intracellular amastigotes. (A) Percentage of inhibition of rLmLAP by the synthetic compounds. Compounds were tested at 30 μM by RapidFire-MS. The light-red dotted line indicates the hit selection threshold and dot color indicates the percentage effect; dark: ≥30%, light: ≥22%, while the two different sizes indicate the concentrations at which the referred effect is observed. The structures of the two selected compounds are shown. (B) IC₅₀ values for rLmLAP inhibition, concentration–response curves, and inhibition percentages at 50 μM on L. donovani amastigotes by the two selected compounds.

Figure 3

Kinetic and bioinformatic analyses of rLmLAP:inhibitor interactions for DDD00057570 and DDD00097924. (A) Predicted binding mode of the rLmLAP:DDD00057570 complex. For clarity, the interacting residues within 4 Å are represented as lines and van der Waals interactions between inhibitors and interacting residues are not indicated. Color codes: Inhibitor atoms: carbon in salmon, oxygen in red, nitrogen in blue and sulfur in yellow. LAP residues interacting with Zn²⁺ ions: carbon in gray, oxygen in red, and nitrogen in blue. LAP inhibitor’s interacting residues: carbon in orange, oxygen in red, nitrogen in blue and sulfur in yellow. The Zn²⁺ ions are represented as gray spheres. Zn²⁺ coordination is represented by gray dotted lines. (B) Lineweaver–Burk plot for diagnosis of the modality of rLmLAP inhibition by DDD00057570. Data are reported as mean, n = 4, for 0, 15, 30, and 60 μM of the inhibitor in the presence of 300, 150, 75, 37.5, 4.7, 2.3, and 0.59 μM of the substrate (l-Leu-pNA). (C) Predicted binding mode of the rLmLAP:DDD00097924 complex. Representation as described above. (D) Lineweaver–Burk plot for the diagnosis of the modality of rLmLAP inhibition by DDD00097924 under the same conditions as for DDD00057570. ΔH_b^MM/GBSA: effective binding free energy by the MM/GBSA method.

Figure 4

Impact of LmLAP overexpression and drug susceptibility on L. major growth. The potency of compounds DDD00057570 (A) and DDD00097924 (B) was assessed against parental (WT, open circles) and LmLAP-overexpressing (closed circles) L. major promastigotes. L. major representative proliferation curves for each compound are shown and the EC₅₀ values corresponding to each specific curve shown in the figure. EC₅₀ values (weighted means) of 2 ± 0.2 and 7 ± 1 μM were determined with DDD00057570 for parental and overexpressing cells, respectively; while values of 3 ± 0.2 and 11 ± 1 μM were determined with DDD00097924. Collated data from biological replicates are reported in Table S2.

Figure 5

Target deconvolution utilizing iTPP. Plots show protein abundance log₂ fold change between compound-treated and untreated lysates subjected to thermal shock at 47 °C. Lysates were exposed to DDD00057570 (A) and DDD00097924 (B). Data from biological replicates A and B are represented on the x- and y-axes, respectively. Two of the most highly stabilized proteins are shown in red (LmjF.11.0630) and green (LmjF.33.2570). The cutoff value for stabilization was log₂-fold shift >1 in both replicates. See also Tables S3 and S4 for hit identification data.

Figure 6

Cell cycle and morphology of LmLAP-overexpressing cells. (A) Schematic of the L. major cell cycle. Number of flagella, nuclei and kinetoplasts in interphase and mitotic cells are shown (scheme is based on Ambit et al., 2011). (B) L. major cells across the cell cycle as depicted in (A). Images of light field and DAPI are shown for each stage of the cell cycle. Cells were fixed, DAPI-stained, and visualized by confocal microscopy. Scale bar: 2 μm. Cells with dividing nucleus and kinetoplast (1N^m1K^d2F) can be detected in normal L. major populations and are present in low percentages and have been described a minority configuration. Moreover, aberrant cells were observed, namely 2N1K2F cells and cells without a kinetoplast, 1N0K1F, the latter only occurring in LmLAP OE cells. (C) Impact of DDD00057570 and DDD00097924 on the cell cycle. Cells were exposed to DDD00057570 and DDD00097924 at the corresponding EC₅₀ concentrations for 72 h. n = 70 cells per condition. Cell cycle stages as in (A) and (B). N: nucleus. K: kinetoplast. F: flagellum. LAP: leucyl-aminopeptidase. WT: wild-type. OE: overexpressing. 570: DDD00057570. 924: DDD00097924. DAPI: 4′,6-diamidino-2-phenylindole.

Figure 7

Cytotoxicity of DDD00057570 and DDD00097924 in mammalian cells. Compounds DDD00057570 (A and C) and DDD00097924 (B and D) were tested in murine RAW 264.7 (A and B) and human THP-1 macrophages (C and D). Cells were exposed to the compounds for 48 h and viability was determined with the MTT method. Data are presented as mean ± SD (n = 3).