Peroxisome proliferator-activated receptor gamma (PPARγ) regulates gene expression programs that influence cell differentiation and insulin sensitization in response to ligand binding. In 1995, PPARγ was discovered to be a molecular target for thiazolidinedione (TZD)-containing synthetic ligands (Lehman et al., 1995), which was first reported in 1983 as an insulin sensitizer through phenotypic screening (Takeda Pharmaceutical’s ciglitazone).Fujita et al., 1983) has sparked great interest in pharmacologically modulating PPARγ for therapeutic intervention in patients with type 2 diabetes. Several TZDs have been approved by the U.S. Food and Drug Administration (FDA) for use in patients with type 2 diabetes, including pioglitazone (Takeda’s Actos), rosiglitazone (GSK’s Avandia), and troglitazone (Daiichi Sankyo and Parke-Davis’ Rezulin). However, in the 2000s, patient reports of undesirable side effects and adverse events associated with clinical use of TZDs, including edema, congestive heart failure, and risk of bone fractures, led to FDA black box warnings or withdrawal from the market.Soccio et al., 2014).

TZDs act as pharmacological agonists to activate PPARγ-mediated transcription by stabilizing the surface structure of the active ligand binding domain (LBD) activating function 2 (AF-2) coregulator (Shan et al., 2019), promote the recruitment of coactivators to PPARγ-binding regions of chromatin and increase the expression of PPARγ target genes (Håkonsson et al., 2013). Recent mechanism-of-action studies have also revealed potential pathways to separate the insulin-sensitizing effects of PPARγ-binding ligands from undesirable side effects. The field now recognizes that ligand binding can affect the structure and function of two different surfaces of the PPARγ LBD (Frkic et al., 2021). Ligand binding can stabilize the interaction surface of the AF-2 coregulator in a conformation that promotes the recruitment of transcriptional coactivator complexes that regulate the adipogenic gene program, or, in a non-mutually exclusive manner, can inhibit obesity-associated phosphorylation of Ser273 by Cdk5, thereby affecting the expression of the insulin-sensitizing gene program (Choi et al., 2010). Subsequent studies have focused on the development of so-called next-generation selective PPARγ modulators that contain a different compound scaffold than TZDs and include a wide range of pharmacological activities: PPARγ partial agonists (Choi et al., 2010), transcriptional neutral antagonist (Choi et al., 2011), and transcriptional repressor inverse agonists (Marciano et al., 2015; Steckschulte et al., 2016) Can inhibit Cdk5-mediated Ser273 phosphorylation.

To determine whether off-target effects are responsible for the beneficial effects or side effects of TZDs and other PPARγ-binding compounds, the researchers used two compounds first reported in 2002, GW9662 by GlaxoSmithKline (Riesnitzer et al., 2002) and T0070907 by Tularik (Lee et al., 2002) – as a covalent inhibitor antagonist of ligand binding to the PPARγ ligand-binding domain (LBD). GW9662 and T0070907 bind to reactive cysteine ​​residues (Cys285 or Cys313 PPARγ isoforms 1 or 2, respectively) that point to the orthosteric ligand binding pocket of PPARγ via a halogen exchange reaction; [³H]In a radiolabeled ligand binding assay – Rosiglitazone is used to inhibit agonist-induced transcription and adipocyte differentiation in cells co-treated with a covalent inhibitor and rosiglitazone. Crystal structures show overlapping orthosteric ligand binding modes of covalent inhibitors (Brust et al., 2018; Chandra et al., 2008) and rosiglitazone (Nolte et al., 1998) or partial agonists containing MRL-24 and nTZDpa (Bruning et al., 2007), provided additional support to the field of using GW9662 and T0070907 as covalent inhibitors. However, in 2014, we showed that GW9662 and T0070907 do not block the binding of all ligands to the PPARγ LBD. This phenomenon was originally called “alternate site” ligand binding (Hughes et al., 2014). Other studies have confirmed that non-covalent synthetic ligands and cellular metabolites can co-bind to the PPARγ LBD in the presence of GW9662 or T0070907 when used as covalent inhibitors (Arifi et al., 2023; Brust et al., 2017; Hughes et al., 2016; Jiang et al., 2017; Laguezza et al., 2018; Leiten van de Gevel et al., 2022; Shan et al., 2018).

To gain structural insight into the cobinding mechanism of noncovalent and covalent ligands, we recently reported seven X-ray crystal structures of PPARγ LBD coconjugated with covalent inhibitors (GW9662 or T0070907) and different synthetic non-TZD PPARγ modulators (Shan and Kojetin, 2024). We surprisingly found that the non-covalent ligand binding events previously described at alternative/allosteric sites close to the orthosteric ligand binding pocket can instead correspond to ligands adopting the original orthosteric binding mode where the covalent inhibitor adopts a binding mode that allows co-binding of the ligand (Figure 1a) There is some variation depending on the particular covalent and noncovalent ligand pair. Furthermore, biochemical and protein NMR studies suggested a potential mechanism explaining why T0070907, a corepressor-selective pharmacological inverse agonist, is a more effective covalent inhibitor of ligand binding than GW9662, a transcriptionally neutral pharmacological antagonist. GW9662 stabilizes the active-like LBD conformation, whereas the LBD bound to T0070907 exchanges between two long-lived conformations corresponding to the active-like and inhibitory-like states (Brust et al., 2018). The crystal structure of the PPARγ LBD reveals that in the transcriptionally repressed state, upon cobinding to T0070907 and the NCoR1 corepressor peptide, a key regulatory element called helix 12 adopts a solvent-occluded conformation within the orthosteric ligand-binding pocket (Figure 1b; Shan et al., 2020). When T0070907 was used as a covalent inhibitor, cobinding of the ligand abolished the inhibition-like state and stabilized an activation-like state similar to the cobound ligand state of GW9662 (Shan and Kojetin, 2024). These data suggested a mechanism by which helix 12 and noncovalent ligands compete to occupy the orthosteric ligand binding pocket (Figure 1) – and the ligand binding in the presence of the pharmacological inverse agonist T0070907, when used as a covalent inhibitor, selects or induces an active LBD conformation in which helix 12 adopts the active conformation exposed to solvent. This mechanism is consistent with the two-step mechanism described for agonist binding to PPARγ (Shan and Kojetin, 2021).

In the discussion of previous studies (Shan and Kojetin, 2024), we suggested that covalent inhibitors with improved pharmacological corepressor-selective inverse agonist function (more stabilizing the inhibitory-like LBD conformation in which helix 12 adopts a solvent-occluded conformation within the orthosteric pocket) may more effectively inhibit binding of the ligand to the orthosteric pocket. Here, we test this hypothesis using two covalent inverse agonists, SR33065 and SR36708, that we recently reported to have improved efficacy over T0070907 (McTavish et al., 2025). Biochemical and NMR-based structural biology ligand binding assays show that although SR33065 and SR36708 have similar inverse agonist effects, they exhibit ligand-specific differences when used as covalent inhibitors and still cannot completely block ligand binding to PPARγ. Finally, we also show that noncovalent ligand binding is not blocked by another previously described covalent inhibitor, SR16832 (Brust et al., 2017), which appears to function through a different mechanism that does not involve corepressor-selective inverse agonism and helix 12 occupancy of the orthosteric pocket.