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

A Maltose-Binding Protein Fusion Construct Yields a Robust Crystallography Platform for MCL1

<div><p>Crystallization of a maltose-binding protein MCL1 fusion has yielded a robust crystallography platform that generated the first apo MCL1 crystal structure, as well as five ligand-bound structures. The ability to obtain fragment-bound structures advances structure-based drug design efforts that, despite considerable effort, had previously been intractable by crystallography. In the ligand-independent crystal form we identify inhibitor binding modes not observed in earlier crystallographic systems. This MBP-MCL1 construct dramatically improves the structural understanding of well-validated MCL1 ligands, and will likely catalyze the structure-based optimization of high affinity MCL1 inhibitors.</p></div

The structure of Apo MBP-MCL1 determined at 1.90 Å.

<p>(A) The MBP domain (red) is connected by a short GS linker (orange) to MCL1 173–321 (blue). A portion of alpha helix four is not ordered in the structure (red dashed-line). Maltose ligand is shown in yellow. (B) The MCL1 domain is structurally very similar to the NMR structure of Apo-MCL1 (gray).</p

The structure of MBP-MCL1 bound to fragment 5 determined at 1.9 Å.

<p>Fragment <b>5</b> (yellow) binds similarly in comparison to the elaborated ligand from PDB ID 4OQ6 (gray).</p

The conformational flexibility of the binding pocket of MCL1.

<p>Surface representations are shown as side views and ligands are shown as yellow sticks. (A and B) Fragment 4 maps onto L78 of NoxaB from PDB ID 2NLA, with only minor structural perturbation of the BH3-binding groove of MCL1. In contrast, binding of fragment 6 creates a significant pocket (C) which is further expanded upon binding of ligand 1 (D).</p

Comparison of PDB 4HW3 and MBP-MCL1 with fragment 4.

<p>The structure of MBP-MCL1 with fragment <b>4</b> (yellow) determined to 2.4 Å (blue) overlaid with the structure of MCL1 171–323 determined at 2.4 Å (PDB ID 4HW3, gray). The carboxylic acid of 4HW3 adopts multiple conformations depending on the chain; only chain A is shown for clarity.</p

Binding affinity (K_D) of ligands to MCL1 and MBP-MCL1.

<p>All experiments are n ≥ 3, and averaged values for K_D are reported.</p><p>Binding affinity (K_D) of ligands to MCL1 and MBP-MCL1.</p

MCL1 ligands used in co-crystallization experiments.

<p>MCL1 ligands used in co-crystallization experiments.</p

Structure of MBP-MCL1 bound to fragment 6 determined at 2.0 Å.

<p>(A) The surface side-view shows that fragment <b>6</b> shifts and makes water mediated hydrogen bond contacts with the peptide backbone of R263. (B) The elaborated ligand of fragment <b>6</b> (PDB ID 4OQ5) shifts to allow the methyl-naphthalene to bind in the hydrophobic pocket, requiring the carboxylic acid to make a single hydrogen bond with the sidechain of R263. (C) Overlay of crystallized fragment <b>6</b> and the elaborated ligand in PDB ID 4OQ5 reveals a distinct binding pose for <b>6</b>.</p

Crystal packing of MCL1 173–321 is mediated by zinc and pyrophosphate.

<p>(A) The structure of MCL1 173–321 was determined to 1.70 Å. (B) In the ligand-bound MCL1 173–321 structure, the imidazole group of <b>1</b> coordinates with zinc along with H224 and pyrophosphate.</p