Homology model of human mitochondrial MetRS overlaid with the crystal structure of <i>Bm</i>MetRS in complex with 1433.

<p>Very few differences could be observed between amino acid residues within the inhibitor binding site and interaction of the compound in the 2 enzymes. This fits well with experimental data showing similar level of inhibition between <i>Bm</i>MetRS and human mitochondrial MetRS enzymes.</p

Structures of MetRS:

<p><b>A. MetRS bound to SeMet.</b> Structural features of <i>Bm</i>MetRS include a catalytic domain formed by a Rossmann fold (red), inserted connective peptide (CP) zinc finger domain (blue), a stem-contact fold (SCF) domain (magenta), and an anti-codon binding α-helix bundle (green). <b>B. Overlay of MetRS bound to SeMet and MetRS bound to 1312.</b> MetRS bound to <b>1312</b> is colored in yellow. Domains in MetRS bound to SeMet are colored identically to Fig 3a above. Movement of the CP domain is highlighted by the arrow.</p

Comparative analysis of thermal stability plots of recombinant <i>Bm</i>MetRS with ligands.

<p>Thermal shift assays T_m curves for apo form (control) of the <i>Bm</i>MetRS (red plot) and inhibitors bound with <b>1312</b> (amber), <b>1433</b> (blue) and <b>1415</b> (green) plots. A significant shift in melting temperature (ΔT_m) of 7.8°C was observed when compound <b>1312</b> was complexed with <i>Bm</i>MetRS compared to the Apo <i>Bm</i>MetRS (unbound). Two other compounds, <b>1415</b> and <b>1433</b>, exhibited Δ<i>T</i>_ms of 3.1°C and 2.5°C respectively.</p

A superposition of <i>Bm</i>MetRS (PDB ID 4PLY2 Chain B) and <i>Tb</i>MetRS (PDB ID 4MVW Chain) bound to compound 1433.

<p>A key difference is the interaction of <i>Bm</i>MetRS Phe213 which is functionally equivalent to Leu456 in <i>Tb</i>MetRS but led to different protein geometry. The <i>Tb</i>MetRS structure is shown in blue and the 2 different residues in orange.</p

Amino acid sequence alignments within the benzyl and quinolone pocket of <i>Bm</i>MetRS, <i>Tb</i>MetRS and <i>Lm</i>MetRS.

<p>Inhibitor binding residues in <i>Bm</i>MetRS compared to <i>Tb</i>MetRS and <i>Lm</i>MetRS are highlighted in red boxes. The most significant differences can be found in residues 211–213 (TTF) of the <i>Bm</i>MetRS which are analogous to a larger run of residues (<i>Tb</i>MetRS: KRETLH <i>Lm</i>MetRS: KRESVM) in the trypanosome structures. Inhibitor interaction with Phe213 within the <i>Bm</i>MetRS complex led to different protein geometry relative to the <i>Tb</i>MetRS complex. Table shows list of residues within the benzyl and quinolone pockets interacting with inhibitors relative to other MetRS. Sequence numbers refer to the <i>Bm</i>MetRS sequence. # LR = linker region, BP = benzyl pocket, QP = quinolone pocket.</p

Data collection and model refinement statistics.

<p>Data collection and model refinement statistics.</p

Crystal structure of <i>Bm</i>MetRS in-complex with selenomethione and three different inhibitors to define structural pockets.

<p>The binding of each ligand; selenomethionine (Fig 4a), <b>1312</b> (Fig 4b), <b>1415</b> (Fig 4c) and <b>1433</b> (Fig 4d) in the crystal structure at the active site causes similar conformational changes to the residues surrounding the methionine-binding site.</p

Compound 1312 additional hydrogen bonding of its ketone group with ASP51.

<p>The crucial hydrogen bonds in all 3 inhibitors with Asp51 are conserved in all <i>Bm</i>MetRS inhibitor complexes. In addition, the 4-ketone group in the aminoquinolone compound <b>1312</b> makes water-mediated interactions (water molecule is shown as red sphere) with the phenol hydroxyl of Tyr228.</p

Growth inhibition.

<p>The lowest effective concentration of a methionyl-tRNA synthetase inhibitor required to inhibit the growth of <i>B</i>. <i>melitensis</i> strain 16M (MIC) was assessed from values observed over concentrations ranging from 0.716–71.6 μg/mL for compound <b>1433,</b> 0.696–69.6 μg/mL for <b>1415</b> while <b>1312</b> was determined at inhibitor concentration 0.865–37.6 μg/mL. Average growth (OD₄₅₀ nm) at 28 hours (late log phase), 37°C, in <i>Brucella</i> minimal medium was measured. Inhibitor <b>1415</b> MIC = 53.93μg/mL; Inhibitor <b>1433</b> MIC = 38.67 μg/mL; Inhibitor <b>1312</b>, MIC = 28.96 μg/mL. Control = gentamicin, no growth, 0.7–50 μg/mL; DMSO control has no inhibition of bacterial growth. Data table shows MICs from <i>B</i>. <i>melitensis</i> growth inhibition and IC₅₀s of inhibitors (Kinase-Glo^®, aminoacylation assays). The characteristics of each inhibitor experimentally measured against human mitochondria MetRS, <i>Ec</i>MetRS, human liver hepatocellular (Hep G2) and lymphocytic (CRL-8155) cell lines are also shown.</p

OD₄₅₀ counts and growth inhibitory effects on <i>B</i>. <i>melitensis</i> exposed to MetRS inhibitors.

<p>OD₄₅₀ counts and growth inhibitory effects on <i>B</i>. <i>melitensis</i> exposed to MetRS inhibitors.</p