RESUMEN
The nuclear retinoic acid receptor-related orphan receptorâ γ (RORγ; NR1F3) is a key regulator of inflammatory gene programs involved in Tâ helper 17 (TH 17) cell proliferation. As such, synthetic small-molecule repressors (inverse agonists) targeting RORγ have been extensively studied for their potential as therapeutic agents for various autoimmune diseases. Alternatively, enhancing TH 17 cell proliferation through activation (agonism) of RORγ may boost an immune response, thereby offering a potentially new approach in cancer immunotherapy. Herein we describe the development of N-arylsulfonyl indolines as RORγ agonists. Structure-activity studies reveal a critical linker region in these molecules as the major determinant for agonism. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) analysis of RORγ-ligand complexes help rationalize the observed results.
Asunto(s)
Indoles/química , Receptores de Ácido Retinoico/agonistas , Sitios de Unión , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Indoles/síntesis química , Indoles/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Simulación del Acoplamiento Molecular , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Ácido Retinoico/metabolismo , Relación Estructura-Actividad , Células Th17/citología , Células Th17/inmunología , Células Th17/metabolismo , Receptor de Ácido Retinoico gammaRESUMEN
The T cell specific RORγ isoform RORγt has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 and TC17 cells, cells that have demonstrated antitumor efficacy. RORγt controls gene networks that enhance immunity including increased IL17 production and decreased immune suppression. Both synthetic and putative endogenous agonists of RORγt have been shown to increase the basal activity of RORγt enhancing TH17 cell proliferation. Here, we show that activation of RORγt using synthetic agonists drives proliferation of TH17 cells while decreasing levels of the immune checkpoint protein PD-1, a mechanism that should enhance antitumor immunity while blunting tumor associated adaptive immune resistance. Interestingly, putative endogenous agonists drive proliferation of TH17 cells but do not repress PD-1. These findings suggest that synthetic agonists of RORγt should activate TC17/TH17 cells (with concomitant reduction in the Tregs population), repress PD-1, and produce IL17 in situ (a factor associated with good prognosis in cancer). Enhanced immunity and blockage of immune checkpoints has transformed cancer treatment; thus such a molecule would provide a unique approach for the treatment of cancer.
Asunto(s)
Inmunidad Celular/efectos de los fármacos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Proliferación Celular , HumanosRESUMEN
The retinoid x receptors (RXRs) are the pharmacological target of Bexarotene, an antineoplastic agent indicated for the treatment of cutaneous T cell lymphoma (CTCL). The RXRs form heterodimers with several nuclear receptors (NRs), including peroxisome proliferator-activated receptor gamma (PPARγ), to regulate target gene expression through cooperative recruitment of transcriptional machinery. Here we have applied hydrogen/deuterium exchange (HDX) mass spectrometry to characterize the effects of Bexarotene on the conformational plasticity of the intact RXRα:PPARγ heterodimer. Interestingly, addition of Bexarotene to PPARγ in the absence of RXRα induced protection from solvent exchange, suggesting direct receptor binding. This observation was confirmed using a competitive binding assay. Furthermore, Bexarotene functioned as a PPARγ antagonist able to alter rosiglitazone induced transactivation in a cell based promoter:reporter transactivation assay. Together these results highlight the complex polypharmacology of lipophilic NR targeted small molecules and the utility of HDX for identifying and characterizing these interactions.
RESUMEN
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.
RESUMEN
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently, we reported the development of PPARγ antagonist SR1664, designed to block the obesity-induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin-sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow-derived mesenchymal stem cells with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand-mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.
Asunto(s)
Compuestos de Bifenilo/farmacología , Huesos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Hipoglucemiantes/farmacología , Indoles/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Células 3T3-L1 , Adipogénesis/efectos de los fármacos , Animales , Huesos/metabolismo , Cristalografía , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Osteoblastos/metabolismo , PPAR gamma/agonistas , Fosforilación/efectos de los fármacosRESUMEN
The orphan nuclear receptor RORγ is a key regulator for T helper 17 (TH17) cell differentiation, which regulates metabolic and circadian rhythm genes in peripheral tissues. Previously, it was shown that the small molecule inverse agonist of RORγ SR1555 [1-(4-((4'-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-[1,1'-biphenyl]-4-yl)methyl)piperazin-1-yl) ethanone] suppressed TH17 differentiation and stimulated induced T regulatory (iTreg) cells. Here, we show that treatment of cultured pre-adipocyctes with SR1555 represses the expression of RORγ while leading to increased expression of FGF21 and adipoQ. Chronic administration of SR1555 to obese diabetic mice resulted in a modest reduction in food intake accompanied with significant reduction in fat mass, resulting in reduced body weight and improved insulin sensitivity. Analysis ex vivo of treated mice demonstrates that SR1555 induced expression of the thermogenic gene program in fat depots. Further studies in cultured cells showed that SR1555 inhibited activation of hormone-sensitive lipase and increased fatty acid oxidation. Combined, these results suggest that pharmacological repression of RORγ may represent a strategy for treatment of obesity by increasing thermogenesis and fatty acid oxidation, while inhibition of hormone-sensitive lipase activity results in a reduction of serum free fatty acids, leading to improved peripheral insulin sensitivity.
Asunto(s)
Fármacos Antiobesidad/administración & dosificación , Compuestos de Bifenilo/administración & dosificación , Insulina/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Obesidad/tratamiento farmacológico , Piperazinas/administración & dosificación , Células 3T3-L1 , Adipogénesis/efectos de los fármacos , Adiponectina/metabolismo , Animales , Fármacos Antiobesidad/síntesis química , Fármacos Antiobesidad/farmacología , Compuestos de Bifenilo/síntesis química , Compuestos de Bifenilo/farmacología , Peso Corporal/efectos de los fármacos , Células Cultivadas , Dieta Alta en Grasa , Factores de Crecimiento de Fibroblastos/metabolismo , Masculino , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Piperazinas/síntesis química , Piperazinas/farmacologíaRESUMEN
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.
Asunto(s)
PPAR gamma/metabolismo , Sitios de Unión , Ligandos , PPAR gamma/químicaRESUMEN
Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators.
Asunto(s)
Modelos Moleculares , PPAR gamma/agonistas , PPAR gamma/química , PPAR gamma/metabolismo , Conformación Proteica , Medición de Intercambio de Deuterio , Ligandos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Rosiglitazona , TiazolidinedionasRESUMEN
PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of antidiabetes drugs including rosiglitazone and pioglitazone. These drugs are full classical agonists for this nuclear receptor, but recent data have shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk5. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent antidiabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of antidiabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Hipoglucemiantes/farmacología , PPAR gamma/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Líquidos Corporales/efectos de los fármacos , Células COS , Chlorocebus aethiops , Grasas de la Dieta/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/química , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Moleculares , Obesidad/inducido químicamente , Obesidad/metabolismo , Osteogénesis/efectos de los fármacos , PPAR gamma/agonistas , PPAR gamma/química , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Rosiglitazona , Tiazolidinedionas/efectos adversos , Tiazolidinedionas/farmacología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Aumento de Peso/efectos de los fármacosRESUMEN
Retinoids are potent forms of vitamin A and are involved in a broad range of physiological processes and the pharmacological effects of retinoids are primarily mediated by the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Several natural and synthetic RAR modulators have proven to be clinically useful for a number of therapeutic indications including cancer, psoriasis, and diabetes. Unfortunately, these agents lead to a number of significant side effects. Most synthetic retinoid ligands are based on the retinoid scaffold and thus have similarities to the natural ligand with all previously disclosed RAR ligands having a carboxylic acid that makes a critical ionic bridge within the ligand binding domain of the receptors. The potential therapeutic value offered from RAR modulation provides the impetus to identify novel ligands based on unique scaffolds that may offer improved toxicity and pharmacokinetic profiles. Here we describe the identification of an atypical RAR inverse agonist that represents the first non-acid, non-retinoid direct modulator of RAR receptor subfamily. SR-0065 functions as a pan-RAR inverse agonist suppressing the basal activity of RARα, RARß, and RARγ, as well as inhibiting agonist-induced RAR activity. SR-0065 treatment enhanced receptor interaction with a peptide representative of the corepressor SMRT, and in cells SR-0065 enhances recruitment of SMRT to the promoter of the RARγ dependent gene, Cyp26A1. The acid form of SR-0065, SR-1758, was inactive in all assays. Thus, SR-0065 represents a new class of non-acid, non-retinoid RAR modulator that may be used as a point to initiate development of improved RAR-targeted drugs.