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While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.

The repressive states of nuclear receptors (i.e., apo or bound to antagonists or inverse agonists) are poorly defined, despite the fact that nuclear receptors are a major drug target. Most ligand bound structures of nuclear receptors, including peroxisome proliferator-activated receptor γ (PPARγ), are similar to the apo structure. Here we use NMR, accelerated molecular dynamics and hydrogen-deuterium exchange mass spectrometry to define the PPARγ structural ensemble. We find that the helix 3 charge clamp positioning varies widely in apo and is stabilized by efficacious ligand binding. We also reveal a previously undescribed mechanism for inverse agonism involving an omega loop to helix switch which induces disruption of a tripartite salt-bridge network. We demonstrate that ligand binding can induce multiple structurally distinct repressive states. One state recruits peptides from two different corepressors, while another recruits just one, providing structural evidence of ligand bias in a nuclear receptor.

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor (PPAR) gamma converts a transcriptionally neutral antagonist (GW9662) into a repressive inverse agonist (T0070907) relative to basal cellular activity. X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for corepressor-selective inverse agonism. NMR spectroscopy reveals that PPARγ exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPARγ for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide routes for developing corepressor-selective repressive PPARγ ligands.

Peroxisome proliferator-activated receptors (PPARs) are pharmacological targets for the treatment of metabolic disorders. Previously, we demonstrated the anti-diabetic effects of SR1664, a PPARγ modulator lacking classical transcriptional agonism, despite its poor pharmacokinetic properties. Here, we report identification of the antagonist SR11023 as a potent insulin sensitizer with significant plasma exposure following oral administration. To determine the structural mechanism of ligand-dependent antagonism of PPARγ, we employed an integrated approach combining solution-phase biophysical techniques to monitor activation helix (helix 12) conformational dynamics. While informative on receptor dynamics, hydrogen/deuterium exchange mass spectrometry and nuclear magnetic resonance data provide limited information regarding the specific orientations of structural elements. In contrast, label-free quantitative crosslinking mass spectrometry revealed that binding of SR11023 to PPARγ enhances interaction with co-repressor motifs by pushing H12 away from the agonist active conformation toward the H2-H3 loop region (i.e., the omega loop), revealing the molecular mechanism for active antagonism of PPARγ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The nuclear receptor ligand-binding domain (LBD) is a highly dynamic entity. Crystal structures have defined multiple low-energy LBD structural conformations of the activation function-2 (AF-2) co-regulator-binding surface, yet it remains unclear how ligand binding influences the number and population of conformations within the AF-2 structural ensemble. Here, we present a nuclear receptor co-regulator-binding surface structural ensemble in solution, viewed through the lens of fluorine-19 ( 19 F) nuclear magnetic resonance (NMR) and molecular simulations, and the response of this ensemble to ligands, co-regulator peptides and heterodimerization. We correlate the composition of this ensemble with function in peroxisome proliferator-activated receptor-γ (PPARγ) utilizing ligands of diverse efficacy in co-regulator recruitment. While the co-regulator surface of apo PPARγ and partial-agonist-bound PPARγ is characterized by multiple thermodynamically accessible conformations, the full and inverse-agonist-bound PPARγ co-regulator surface is restricted to a few conformations which favor coactivator or corepressor binding, respectively.

Peroxisome proliferator activated receptor γ (PPARγ) is a nuclear receptor and target for antidiabetics that increase insulin sensitivity. Owing to the side effects of PPARγ full agonists, research has recently focused on non-activating ligands of PPARγ, which increase insulin sensitivity with decreased side effects. Here, we present the crystal structures of inverse agonist SR10171 and a chemically related antagonist SR11023 bound to the PPARγ ligand-binding domain, revealing an allosteric switch in the activation helix, helix 12 (H12), forming an antagonist conformation in the receptor. H12 interacts with the antagonists to become fixed in an alternative location. Native mass spectrometry indicates that this prevents contacts with coactivator peptides and allows binding of corepressor peptides. Antagonists of related nuclear receptors act to sterically prevent the active configuration of H12, whereas these antagonists of PPARγ alternatively trap H12 in an inactive configuration, which we have termed the tumble and trap mechanism., Competing Interests: DECLARATIONS OF INTERESTS The authors declare no competing interests.

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The thiazolidinediones (TZD) typified by rosiglitazone are the only approved therapeutics targeting PPARγ for the treatment of type-2 diabetes (T2DM). Unfortunately, despite robust insulin sensitizing properties, they are accompanied by a number of severe side effects including congestive heart failure, edema, weight gain, and osteoporosis. We recently identified PPARγ antagonists that bind reversibly with high affinity but do not induce transactivation of the receptor, yet they act as insulin sensitizers in mouse models of diabetes (SR1664).1 This Letter details our synthetic exploration around this novel series of PPARγ antagonists based on an N-biphenylmethylindole scaffold. Structure-activity relationship studies led to the identification of compound 46 as a high affinity PPARγ antagonist that exhibits antidiabetic properties following oral administration in diet-induced obese mice.

Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hormone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored. We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring. Herein we report the design and the structure-activity relationships study of these new ghrelin receptor ligands., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The only peripherally released orexigenic hormone, ghrelin, plays a key role in food intake and body weight regulation. Antagonizing the ghrelin receptor, GHS-R1a, represents a promising approach for anti-obesity therapy. In our study, two novel GHS-R1a antagonists JMV4208 and JMV3002, which are trisubstituted 1,2,4-triazoles, decreased food intake in fasted lean mice in a dose-dependent manner, with ED50 values of 5.25 and 2.05 mg/kg, respectively. Both compounds were stable in mouse blood, with half-lives of 90 min (JMV4208) and 60 min (JMV3002), and disappeared from the blood 8h after administration. Fourteen days of treatment with the ghrelin antagonists (20 mg/kg twice a day) decreased food intake, body weight and adipose tissue mass in mice with diet-induced obesity (DIO). These results are likely attributable to an impact on food intake reduction and an attenuated expression of the lipogenesis-promoting enzymes (acetyl-CoA carboxylase 1 in subcutaneous fat and fatty acid synthase in subcutaneous and intraperitoneal fat). The decrease in fat mass negatively impacted circulating leptin levels. These data suggest that JMV4208 and JMV3002 could be useful therapeutic agents for the treatment of obesity., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

PPARγ 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 PPARγ. Here we reveal that synthetic PPARγ 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 PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands.