Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography

We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein−protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors

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

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

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

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

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

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

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