ABSTRACT: PPAR? is a target for insulin-sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPAR?. Here we reveal that synthetic PPAR? ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPAR? hyperactivation in vivo, perhaps explaining why PPAR? full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPAR? activation by ligands and suggest that allosteric modulators could be designed to fine tune PPAR? activity without competing with endogenous ligands.
Project description:Crystal structures of peroxisome proliferator-activated receptor gamma (PPAR?) have revealed overlapping binding modes for synthetic and natural/endogenous ligands, indicating competition for the orthosteric pocket. Here we show that cobinding of a synthetic ligand to the orthosteric pocket can push natural and endogenous PPAR? ligands (fatty acids) out of the orthosteric pocket towards an alternate ligand-binding site near the functionally important omega (?)-loop. X-ray crystallography, NMR spectroscopy, all-atom molecular dynamics simulations, and mutagenesis coupled to quantitative biochemical functional and cellular assays reveal that synthetic ligand and fatty acid cobinding can form a 'ligand link' to the ?-loop and synergistically affect the structure and function of PPAR?. These findings contribute to a growing body of evidence indicating ligand binding to nuclear receptors can be more complex than the classical one-for-one orthosteric exchange of a natural or endogenous ligand with a synthetic ligand.
Project description:GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPAR?). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPAR? ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPAR? 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 PPAR? 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 PPAR? transcription by PPAR?-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 toward the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPAR? activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPAR? when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPAR? 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 PPAR?.
Project description:The nuclear receptor, peroxisome proliferator-activated receptor ? (PPAR?), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPAR? through conformational changes of the ? loop. Here, we report that serotonin metabolites act as endogenous agonists for PPAR? to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPAR? antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPAR?-target gene. Collectively, these results suggest a novel agonism, in which PPAR? functions as a multiple sensor in response to distinct metabolites.
Project description:Peroxisome proliferator-activated receptor ? (PPAR?) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte differentiation, insulin resistance, and inflammation. Here, we report the crystal structures of PPAR? in complex with lobeglitazone, a novel PPAR? agonist, and with rosiglitazone for comparison. The thiazolidinedione (TZD) moiety of lobeglitazone occupies the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) in ligand-binding domain as the TZD moiety of rosiglitazone does. However, the elongated p-methoxyphenol moiety of lobeglitazone interacts with the hydrophobic pocket near the alternate binding site of PPAR?. The extended interaction of lobeglitazone with the hydrophobic pocket enhances its binding affinity and could affect the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPAR? at Ser245 (in PPAR?1 numbering; Ser273 in PPAR?2 numbering). Lobeglitazone inhibited the phosphorylation of PPAR? at Ser245 in a dose-dependent manner and exhibited a better inhibitory effect on Ser245 phosphorylation than rosiglitazone did. Our study provides new structural insights into the PPAR? regulation by TZD drugs and could be useful for the discovery of new PPAR? ligands as an anti-diabetic drug, minimizing known side effects.
Project description:Peroxisome proliferator-activated receptor gamma (PPAR?) is a ligand-regulated transcription factor that plays crucial roles in adipogenesis, lipid metabolism, and glucose homeostasis. Several PPAR? ligands possess anti-diabetic activity and they commonly inhibit the phosphorylation of PPAR? at serine 273 (Ser273). The recently reported PPAR? ligand SR1664, which selectively blocks the phosphorylation of PPAR? without classical agonism, has potent anti-diabetic activity, indicating that the inhibition of Ser273 phosphorylation is sufficient to provoke anti-diabetic effects. In this study, we revealed the X-ray structure of PPAR? co-crystallized with SR1664 bound to the alternate binding site of PPAR? and confirmed that the alternate site binding of SR1664 blocks the phosphorylation of Ser273. Furthermore, using covalent inhibitors as chemical tools, we demonstrated that the inhibition of phosphorylation is attributed to the occupation of a specific site which is a hydrophobic region between helix 3 and ?3-?4 at the binding pocket of PPAR?. In high-fat diet-induced obese mice, we confirmed the anti-diabetic activity of our covalent inhibitor SB1453 that was designed to bind at the specific site in PPAR? for blocking the phosphorylation of Ser273. Lastly, the target selectivity of SB1453 was demonstrated by fluorescence-based visualization of target proteins complexed with the covalent probe 11 containing a bioorthogonal functional group.
Project description:Most triacylglycerol-lowering fibrates have been developed in the 1960s-1980s before their molecular target, peroxisome proliferator-activated receptor alpha (PPAR?), was identified. Twenty-one ligand-bound PPAR? structures have been deposited in the Protein Data Bank since 2001; however, binding modes of fibrates and physiological ligands remain unknown. Here we show thirty-four X-ray crystallographic structures of the PPAR? ligand-binding domain, which are composed of a "Center" and four "Arm" regions, in complexes with five endogenous fatty acids, six fibrates in clinical use, and six synthetic PPAR? agonists. High-resolution structural analyses, in combination with coactivator recruitment and thermostability assays, demonstrate that stearic and palmitic acids are presumably physiological ligands; coordination to Arm III is important for high PPAR? potency/selectivity of pemafibrate and GW7647; and agonistic activities of four fibrates are enhanced by the partial agonist GW9662. These results renew our understanding of PPAR? ligand recognition and contribute to the molecular design of next-generation PPAR-targeted drugs.
Project description:BACKGROUND:An alarming requirement for finding newer antidiabetic glitazones as agonists to PPAR? are on its utmost need from past few years as the side effects associated with the available drug therapy is dreadful. In this context, herein, we have made an attempt to develop some novel glitazones as PPAR? agonists, by rational and computer aided drug design approach by implementing the principles of bioisosterism. The designed glitazones are scored for similarity with the developed 3D pharmacophore model and subjected for docking studies against PPAR? proteins. Synthesized by adopting appropriate synthetic methodology and evaluated for in vitro cytotoxicity and glucose uptake assay. Illustrations about the molecular design of glitazones, synthesis, analysis, glucose uptake activity and SAR via 3D QSAR studies are reported. RESULTS:The computationally designed and synthesized ligands such as 2-(4-((substituted phenylimino)methyl)phenoxy)acetic acid derivatives were analysed by IR, 1H-NMR, 13C-NMR and MS-spectral techniques. The synthesized compounds were evaluated for their in vitro cytotoxicity and glucose uptake assay on 3T3-L1 and L6 cells. Further the activity data was used to develop 3D QSAR model to establish structure activity relationships for glucose uptake activity via CoMSIA studies. CONCLUSION:The results of pharmacophore, molecular docking study and in vitro evaluation of synthesized compounds were found to be in good correlation. Specifically, CPD03, 07, 08, 18, 19, 21 and 24 are the candidate glitazones exhibited significant glucose uptake activity. 3D-QSAR model revealed the scope for possible further modifications as part of optimisation to find potent anti-diabetic agents.
Project description:The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally, they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation.
Project description:In a previous study, a cocrystal structure of PPAR? bound to 2-chloro-N-(3-chloro-4-((5-chlorobenzo[d]thiazol-2-yl)thio)phenyl)-4-(trifluoromethyl)benzenesulfonamide (1, T2384) revealed two orthosteric pocket binding modes attributed to a concentration-dependent biochemical activity profile. However, 1 also bound an alternate/allosteric site that could alternatively account for the profile. Here, we show ligand aggregation afflicts the activity profile of 1 in biochemical assays. However, ligand-observed fluorine (19F) and protein-observed NMR confirms 1 binds PPAR? with two orthosteric binding modes and to an allosteric site.
Project description:Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD) of PPAR ? . This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPAR ? LBD. The role of L-FABP in the delivery of ligands directly to PPAR ? via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed.