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

Abstract

GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPARγ). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPARγ ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPARγ LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists. Here, we developed a series of analogs based on the orthosteric covalent antagonist scaffold with the goal of inhibiting both orthosteric and allosteric cellular activation of PPARγ by MRL20, an orthosteric agonist that also binds to an allosteric site. Our efforts resulted in the identification of SR16832 (compound 22), which functions as a dual-site covalent inhibitor of PPARγ transcription by PPARγ-binding ligands. Molecular modeling, protein NMR spectroscopy structural analysis, and biochemical assays indicate the inhibition of allosteric activation occurs in part through expansion of the 2-chloro-5-nitrobenzamidyl orthosteric covalent antagonist towards the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPARγ activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPARγ when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPARγ ligands such as docosahexaenoic acid (DHA) compared to orthosteric covalent antagonists. Compounds such as SR16832 may be useful chemical tools to use as a dual-site bitopic orthosteric and allosteric covalent inhibitor of ligand binding to PPARγ.

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).…”

“…The structurally diverse ligand set includes natural/endogenous PPARγ ligands (arachidonic acid, decanoic acid, docosahexaenoic acid, dodecanoic acid, linoleic acid, nonanoic acid, oleic acid, palmitoleic acid) and synthetic ligands (BVT.13, GQ-16, GW1929, MRL20, MRL24, nTZDpa, SR1663). All ligands except MRL20 and SR1663 were obtained from commercial sources, including BioVision (MRL24), Cayman Chemical, Sigma-Aldrich, and Tocris Bioscience; MRL20 (Bruning et al 2007, Hughes et al 2012, Hughes et al 2014, Brust et al 2017) and SR1663 (Marciano et al 2015) were synthesized and characterized previously. A peptide containing an LXXLL nuclear receptor interaction motif from TRAP220/MED1/ DRIP205 (residues 638-656; NTKNHPMLMNLLKDNPAQD) was synthesized by LifeTein with an N-terminal FITC label with a six-carbon linker (Ahx) and an amidated C-terminus for stability.…”

Arylation Chemistry for Bioconjugation

“…[63,64] Both antagonists (GW9662 and T0070907) block ligand binding to PPARg through selective arylation of Cys-285 located in the orthosteric ligand-binding pocket of PPARg. [66] Together,2chloro-5-nitrobenzamidyl-basedcompounds are useful chemical tools to modulate the PPARg function in biological processes. [65] By enhancing the size of the 2-chloro-5-nitrobenzamidyl scaffold, Kojetin reported in 2017 ad ual-site PPARg antagonist (Table 1, entry 3) that simultaneously blocked the ligand from binding to both the orthosteric pocket and the allosteric site.…”

“…[65] By enhancing the size of the 2-chloro-5-nitrobenzamidyl scaffold, Kojetin reported in 2017 ad ual-site PPARg antagonist (Table 1, entry 3) that simultaneously blocked the ligand from binding to both the orthosteric pocket and the allosteric site. [66] Together,2chloro-5-nitrobenzamidyl-basedcompounds are useful chemical tools to modulate the PPARg function in biological processes.…”

Arylierungschemie für die Biokonjugation

“…PPARg reguliert mehrere in die Adipogenese involvierte Gene und stellt das molekulare Ziel fürD iabetes-Arzneimittel vom Thiazolidindion-Typ dar. [66] Daher kçnnen 2-Chlor-5-nitrobenzamidyl-basierte Verbindungen als nützliche Verbindungen fürd ie Modulation der Funktion von PPARg in biologischen Prozessen betrachtet werden. Obwohl die genannten Verbindungen eine Aktivität in vitro und in der Zellkultur aufwiesen, konnten sie die Ligandenbindung an PPARg zu einer von der orthosterischen Tasche verschiedenen, allosterischen Position nicht verhindern.…”