Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity

Background: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery.Methodology/Principal findings: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 &lt; 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of &gt;500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.Conclusions/Significance: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.</p

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a <i>Leishmania major</i> M17-aminopeptidase

The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appKM = 30 μM, appkcat = 14.7 s−1). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ but is insensitive towards the protease inhibitors PMSF, TLCK, E−64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity.

BackgroundChagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery.Methodology/principal findingsA RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.Conclusions/significanceOur data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease

Potency determination for LAPTc screening hits.

Compound structures and pIC50 values against LAPTc as determined by dose-response studies with the RapidFire-MS enzymatic assay. Data from four independent replicates. * = one replicate was deemed inactive (maximum effect < 50%), average and standard deviation are for the three active replicates.</p

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.

RF LAPTc hits.xlsx: Structures, compounds identifier and percent inhibition from primary screen for the 30 hits.</p

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].

Structures of previously described M17 LAP inhibitors [14,16,21,22,27–31].</p

Potency against intracellular <i>T</i>. <i>cruzi</i> amastigotes, VERO host cells and HepG2 cells.

Potency against intracellular T. cruzi amastigotes, VERO host cells and HepG2 cells.</p

Determination of the mode of inhibition of LAPTc by compound 4, with respect to the LSTVIVR peptide substrate using the RapidFire-MS enzymatic assay.

Double reciprocal Lineweaver-Burk plots are shown. Determination coefficient values (R2) for the linear fittings are shown, and data are presented as means ± standard deviations (n = 3). v0: initial velocity. AU: arbitrary units. appKM: apparent KM. appvmax: apparent maximal velocity. [S]0: initial substrate concentration.</p

Modelling of LAPTc inhibition by compound 4 by molecular docking <i>in silico</i> and molecular dynamic simulations.

(A) Structure of the selected conformer of the compound 4-LAPTc complex, according to its binding energy value, obtained by molecular docking in silico (AutoDock Vina software). (B) Energy optimization of the compound 4-LAPTc complex by vacuum molecular dynamics simulations. Compound 4 (in sticks) in its initial conformation (beige) and after 1 ns of vacuum molecular dynamics simulations (magenta) is shown. The enzyme active site is shown in cartoon (light grey), with the two Mn2+ atoms represented as purple spheres. (C) Temporal course of RMSD variation in the solvated molecular dynamics simulations for the compound 4-LAPTc complex. 500 frames = 1 ns. (D) 2D representation of predicted stabilizing interactions between compound 4 and LAPTc after 100 ns of solvated molecular dynamics simulations. Compound 4 and R374 residue (carbons represented in black) are shown in balls and sticks model. Hydrophobic interactions between amino acid residues and compound 4 are shown with red curve lines, and the hydrogen bond with a green dashed line. In (A), (B) and (D) nitrogen atoms are represented in blue, oxygens in red, sulfurs in yellow and hydrogen in white.</p

HepG2 pEC50 DRC.xlsx: Potency data against HepG2 cells.

HepG2 pEC50 DRC.xlsx: Potency data against HepG2 cells.</p