Development of Selective Phosphatidylinositol 5-Phosphate 4-Kinase γ Inhibitors with a Non-ATP-competitive, Allosteric Binding Mode

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kβ. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

Figure 1

Structures of PI5P4Kγ inhibitors.

Figure 2

Crystal structure of PI5P4Kγ bound to 40 at 2.4 Å (pdb: 7QIE). (a) Dimer of chains A (orange) and B (green) with 40 bound superposed onto the dimer of chain B and C of the apo PI5P4Kγ structure 2GK9 (in pink) and (b) two binding sites for 40: chain A (orange, 40 in allosteric binding pocket) and chain B (green, 40 in ATP site) superposed with 40 in the stick. The binding sites are mutually exclusive.

Figure 3

Focus on chain A of the crystal structure of PI5P4Kγ bound to 40 at 2.4 Å with 40 in the allosteric binding pocket (pdb: 7QIE; orange). (a) AMP-binding site of PI5P4Kβ (3X01, gray) superposed onto chain A (orange). Tyr379 of PI5P4Kγ overlays onto the pyrimidine ring of AMP; (b) electron density at 1 σ for the allosteric binding pocket in chain A of the 40-PI5P4Kγ complex; (c) novel binding pocket in chain A of the 40-PI5P4Kγ complex with key interactions highlighted; and (d) allosteric binding pocket in chain A of the 40-PI5P4Kγ complex with ligand and receptor molecular surfaces.

Figure 4

Focus on chain B of the crystal structure of PI5P4Kγ bound to 40 at 2.4 Å (pdb: 7QIE; green). This chain has 40 bound in the ATP pocket. (a) Allosteric binding pocket observed in chains A, C, and D of PI5P4Kγ bound to 40 is (partially) occluded in the apo structure 2GK9 (pink) and chain B of the complex with 40 (green) by residues Ile375, Leu376, and Asn165 (apo only). 40 (orange sticks) from chain A of the PI5P4Kγ-40 complex is superposed onto apo PI5P4Kγ (pink) and chain B of the PI5P4Kγ-40 complex (green). (b) 40 bound to the ATP pocket of chain B of the PI5P4Kγ complex in green with AMP as bound to PI5P4Kβ (pdb: 3X01) superposed in gray. The pyrimidine rings do not superpose. (c) Electron density at 1 σ for the ATP-binding pocket in chain B of the 40-PI5P4Kγ complex. (d) 40 bound to the ATP pocket of chain B of the PI5P4Kγ complex in green with apo PI5P4Kγ (pdb: 2GK9) superposed in pink. Orange dotted lines indicate clashes between the ligand and the apo structure.

Figure 5

(a) Chain A in orange (allosteric binding site occupied) and (b) chain B in green (ATP site occupied) for 7QIE. HDX-MS sites identified are shown in cyan (site 1: residues 158–162) and blue (site 2: residues 373–407). Lys383A-Val403A and Thr377B-His404B, which connect the two regions of site 2, are not visible in the crystal structure. The N-terminal part of site 2 adopts different conformations in chains A and B. (c) Close-up of chain A with 40 bound highlighting the location of Asn165. The N165I mutation was found to remove the sensitivity of PI5P4Kγ to 40.