Structural Basis of Altered Potency and Efficacy Displayed by a Major in Vivo Metabolite of the Antidiabetic PPARγ Drug Pioglitazone

Pioglitazone (Pio) is a Food and
Drug Administration-approved drug
for type-2 diabetes that binds and activates the nuclear receptor
peroxisome proliferator-activated receptor γ (PPARγ),
yet it remains unclear how in vivo Pio metabolites affect PPARγ
structure and function. Here, we present a structure–function
comparison of Pio and its most abundant in vivo metabolite, 1-hydroxypioglitazone
(PioOH). PioOH displayed a lower binding affinity and reduced potency
in co-regulator recruitment assays. X-ray crystallography and molecular
docking analysis of PioOH-bound PPARγ ligand-binding domain
revealed an altered hydrogen bonding network, including the formation
of water-mediated bonds, which could underlie its altered biochemical
phenotype. NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry
analysis coupled to activity assays revealed that PioOH better stabilizes
the PPARγ activation function-2 (AF-2) co-activator binding
surface and better enhances co-activator binding, affording slightly
better transcriptional efficacy. These results indicating that Pio
hydroxylation affects its potency and efficacy as a PPARγ agonist
contributes to our understanding of PPARγ–drug metabolite
interactions.