Mechanistic elucidation guided by covalent inhibitors for the development of anti-diabetic PPARγ ligands

Abstract

We revealed the X-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ and confirmed that this blocks the phosphorylation of Ser273.

Quantitative Structural Assessment of Graded Receptor Agonism

“…In contrast to these 8 ligands that adopt "canonical" binding modes, Ciglitazone and Mitoglitazone, two of the least potent ligands in the series, show alternate binding modes in the chain A active conformation where their TZD head groups associate near the flexible, solvent accessible Ω-loop and their relatively shorter extended tail groups insert into the orthosteric pocket along side the β-sheet surface. Ligand binding to this alternate site has been observed in several other structural studies (Hughes et al 2014, Bae et al 2016, Hughes et al 2016, Brust et al 2017, Jang et al 2017, Laghezza et al 2018. The alternate binding modes of Ciglitazone and Mitoglitazone may originate from their lower affinity, and in the case of Ciglitazone, a lack of atoms in its shorter tail group capable of forming water-mediated polar interactions with residues in the β-sheet (Mosure et al 2019).…”

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor

“…18–21 Alternate site ligand binding occurs when the orthosteric pocket is either blocked by GW9662 and T0070907 or bound to an endogenous ligand, indicating this alternate site can be classified as an allosteric site because it does not compete with endogenous ligand binding. 18 Notably, alternate or allosteric ligand-binding sites have been reported for other nuclear receptors, 22–31 and the site we identified was structurally visualized by x-ray crystallography for PPARγ and PPARα (Supplementary Figure 1).…”

“…18 Notably, alternate or allosteric ligand-binding sites have been reported for other nuclear receptors, 22–31 and the site we identified was structurally visualized by x-ray crystallography for PPARγ and PPARα (Supplementary Figure 1). 20,21,31 Structure-function studies have shown that ligands that form hydrogen bond contacts within the orthosteric subpocket near the AF-2 surface are associated with AF-2 stabilization and classical agonism. 32,33 However, this is not a strict rule as AF-2 stabilization and transcriptional activation can also occur upon ligand binding to the allosteric site, 18 or be reduced/inhibited when ligands occupy the orthosteric subpocket near the AF-2 surface without hydrogen bond contacts.…”

Cooperative Cobinding of Synthetic and Natural Ligands to the Nuclear Receptor PPARγ

“…However, recent studies have shown that synthetic PPARγ ligands that were originally designed to bind to the orthosteric pocket can also bind to an alternate site (20)(21)(22)(23)(24)(25). The alternate site that partially overlaps with 1 one of the arms of the T/Y-shaped orthosteric pocket near the β-sheet surface (branch II), but it uniquely occupies space in a solvent exposed pocket formed by the flexible omega (Ω)-loop that precedes helix 3 (Figure 1B).…”

“…In contrast, ligands that either do not form hydrogen bonds in the helix 12/ branch I subpocket, or bind to a combination of branch II and the alternate site, can stabilize the Ω-loop region and a nearby loop. Stabilization of this region near the Ω-loop can inhibit phosphorylation of a serine residue (S273 in PPARγ isoform 1 or S245 in isoform 2) by the Cdk5 kinase and is associated with anti-diabetic efficacy (24,25,27,28).…”