Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement.

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to 'sense' the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

Promoting activity of (α4)3(ß2)2 nicotinic cholinergic receptors reduces ethanol consumption.

There is increasing interest in developing drugs that act at α4ß2 nicotinic acetylcholine receptors (nAChRs) to treat alcohol use disorder. The smoking cessation agent varenicline, a partial agonist of α4ß2 nAChRs, reduces alcohol intake, but its use can be limited by side effects at high therapeutic doses. There are two stoichiometric forms of α4ß2 nAChRs, (α4)3(ß2)2 and (α4)2(ß2)3. Here we investigated the hypothesis that NS9283, a positive allosteric modulator selective for the (α4)3(ß2)2 form, reduces ethanol consumption. NS9283 increased the potency of varenicline to activate and desensitize (α4)3(ß2)2 nAChRs in vitro without affecting other known targets of varenicline. In male and female C57BL/6J mice, NS9283 (10 mg/kg) reduced ethanol intake in a two-bottle choice, intermittent drinking procedure without affecting saccharin intake, ethanol-induced incoordination or ethanol-induced loss of the righting reflex. Subthreshold doses of NS9283 (2.5 mg/kg) plus varenicline (0.1 mg/kg) synergistically reduced ethanol intake in both sexes. Finally, despite having no aversive valence of its own, NS9283 enhanced ethanol-conditioned place aversion. We conclude that compounds targeting the (α4)3(ß2)2 subtype of nAChRs can reduce alcohol consumption, and when administered in combination with varenicline, may allow use of lower varenicline doses to decrease varenicline side effects.

Discovery of an intrasubunit nicotinic acetylcholine receptor-binding site for the positive allosteric modulator Br-PBTC.

Nicotinic acetylcholine receptor (nAChR) ligands that lack agonist activity but enhance activation in the presence of an agonist are called positive allosteric modulators (PAMs). nAChR PAMs have therapeutic potential for the treatment of nicotine addiction and several neuropsychiatric disorders. PAMs need to be selectively targeted toward certain nAChR subtypes to tap this potential. We previously discovered a novel PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which selectively potentiates the opening of α4ß2*, α2ß2*, α2ß4*, and (α4ß4)2α4 nAChRs and reactivates some of these subtypes when desensitized (* indicates the presence of other subunits). We located the Br-PBTC-binding site through mutagenesis and docking in α4. The amino acids Glu-282 and Phe-286 near the extracellular domain on the third transmembrane helix were found to be crucial for Br-PBTC's PAM effect. E282Q abolishes Br-PBTC potentiation. Using (α4E282Qß2)2α5 nAChRs, we discovered that the trifluoromethylated derivatives of Br-PBTC can potentiate channel opening of α5-containing nAChRs. Mutating Tyr-430 in the α5 M4 domain changed α5-selectivity among Br-PBTC derivatives. There are two kinds of α4 subunits in α4ß2 nAChRs. Primary α4 forms an agonist-binding site with another ß2 subunit. Accessory α4 forms an agonist-binding site with another α4 subunit. The pharmacological effect of Br-PBTC depends both on its own and agonists' occupancy of primary and accessory α4 subunits. Br-PBTC reactivates desensitized (α4ß2)2α4 nAChRs. Its full efficacy requires intact Br-PBTC sites in at least one accessory and one primary α4 subunit. PAM potency increases with higher occupancy of the agonist sites. Br-PBTC and its derivatives should prove useful as α subunit-selective nAChR PAMs.

Discovery and Optimization of a Series of Sulfonamide Inverse Agonists for the Retinoic Acid Receptor-Related Orphan Receptor-α.

BACKGROUND: Despite a massive industry endeavor to develop RORγ-modulators for autoimmune disorders, there has been no indication of efforts to target the close family member RORα for similar indications. This may be due to the misconception that RORα is redundant to RORγ, or the inherent difficulty in cultivating tractable starting points for RORα. RORα-selective modulators would be useful tools to interrogate the biology of this understudied orphan nuclear receptor. OBJECTIVE: The goal of this research effort was to identify and optimize synthetic ligands for RORα starting from the known LXR agonist T0901317. METHODS: Fourty-five analogs of the sulfonamide lead (1) were synthesized and evaluated for their ability to suppress the transcriptional activity of RORα, RORγ, and LXRα in cell-based assays. Analogs were characterized by 1H-NMR, 13C-NMR, and LC-MS analysis. The pharmacokinetic profile of the most selective RORα inverse agonist was evaluated in rats with intraperitoneal (i.p.) and per oral (p.o.)dosing. RESULTS: Structure-activity relationship studies led to potent dual RORα/RORγ inverse agonists as well as RORα-selective inverse agonists (20, 28). LXR activity could be reduced by removing the sulfonamide nitrogen substituent. Attempts to improve the potency of these selective leads by varying substitution patterns throughout the molecule proved challenging. CONCLUSION: The synthetic RORα-selective inverse agonists identified (20, 28) can be utilized as chemical tools to probe the function of RORα in vitro and in vivo.

REV-ERBα Regulates TH17 Cell Development and Autoimmunity.

RORγt is well recognized as the lineage-defining transcription factor for T helper 17 (TH17) cell development. However, the cell-intrinsic mechanisms that negatively regulate TH17 cell development and autoimmunity remain poorly understood. Here, we demonstrate that the transcriptional repressor REV-ERBα is exclusively expressed in TH17 cells, competes with RORγt for their shared DNA consensus sequence, and negatively regulates TH17 cell development via repression of genes traditionally characterized as RORγt dependent, including Il17a. Deletion of REV-ERBα enhanced TH17-mediated pro-inflammatory cytokine expression, exacerbating experimental autoimmune encephalomyelitis (EAE) and colitis. Treatment with REV-ERB-specific synthetic ligands, which have similar phenotypic properties as RORγ modulators, suppressed TH17 cell development, was effective in colitis intervention studies, and significantly decreased the onset, severity, and relapse rate in several models of EAE without affecting thymic cellularity. Our results establish that REV-ERBα negatively regulates pro-inflammatory TH17 responses in vivo and identifies the REV-ERBs as potential targets for the treatment of TH17-mediated autoimmune diseases.

Identification of potent RORß modulators: Scaffold variation.

We sought to develop RORß-selective probe molecules in order to investigate the function of the receptor in vitro and in vivo and its role in the pathophysiology of disease. To accomplish this, we modified a potent dual RORß/RORγ inverse agonist from the primary literature with the goal of improving selectivity for RORß vs RORγ. Truncation of the Western portion of the molecule ablated activity at RORγ and led to a potent series of RORß modulators. Continued exploration of this series investigated alternate replacement cores for the aminothiazole ring. Numerous suitable replacements were found during the course of our SAR investigations and are reported herein.

Development of novel NEMO-binding domain mimetics for inhibiting IKK/NF-κB activation.

Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.

Identification of an aminothiazole series of RORß modulators.

Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORß, however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual RORß and RORγ inverse agonist with no activity towards RORα (Fig. 1). To our knowledge, this is one of only two small molecule RORß inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design RORß-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of RORß.

Design, synthesis, and evaluation of simple phenol amides as ERRγ agonists.

Herein we report the design and synthesis of a series of simple phenol amide ERRγ agonists based on a hydrazone lead molecule. Our structure activity relationship studies in this series revealed the phenol portion of the molecule to be required for activity. Attempts to replace the hydrazone with more suitable chemotypes led to a simple amide as a viable alternative. Differential hydrogen-deuterium exchange experiments were used to help understand the structural basis for binding to ERRγ and aid in the development of more potent ligands.

N-Arylsulfonyl Indolines as Retinoic Acid Receptor-Related Orphan Receptor†γ (RORγ) Agonists.

The nuclear retinoic acid receptor-related orphan receptor γ (RORγ; NR1F3) is a key regulator of inflammatory gene programs involved in T 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.

Development of Dipeptidic hGPR54 Agonists.

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH2 was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimize the N terminus. Using two successive Sonogashira cross-coupling reactions on a solid-supported peptide, we were able to introduce various alkynes at the N terminus to afford compounds with sub-micromolar affinities for hGPR54. However, functional assays indicated the benzoylated dipeptide Bz-Arg-Trp-NH2 as the most promising compound in terms of agonistic properties. Interestingly, this compound appeared much more stable than the endogenous neuropeptide kisspeptin, both in serum and in liver microsomes of rats. This compound was also found to be able to induce a significant in vivo increase in testosterone levels in male rats.

Synthetic RORγt Agonists Enhance Protective Immunity.

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.

Multigram-scale Synthesis of Enantiopure 3,3-Difluoroproline.

An efficient route for the synthesis of enantiopure 3,3-difluoroproline on multigram-scale is described herein. The deoxofluorination can be achieved with DAST on the corresponding racemic pyrrolidinone in good yield. Resolution of the racemate by crystallization with D- and L-tyrosine hydrazide provides both enantiomers of 3,3-difluoroproline in high yield and ee%.

Effects of systematic N-terminus deletions and benzoylations of endogenous RF-amide peptides on NPFF1R, NPFF2R, GPR10, GPR54 and GPR103.

Mammalian RF-amide peptides including RF-amide-related peptides-1 and -3, neuropeptides AF and FF, Prolactin releasing peptides, Kisspeptins and RFa peptides are currently considered endogenous peptides for the GPCRs NPFF1R, NPFF2R, GPR10, GPR54 and GPR103, respectively. While NPFF1R and NPFF2R displayed high affinity for all the RF-amide peptides, GPR10, GPR54 and GPR103 only bind their cognate ligands. Through a systematic and sequential N-terminus deletion and benzoylation of either RF-amide neuropeptide (RFRP-3, NPFF, Kp-10, PrRP20, and 26RFa), we report the corresponding impact on affinity and activity towards all the RF-amide receptors (NPFF1R, NPFF2R, GPR10, GPR54 and GPR103). Our results highlight the difficulty to develop selective peptide ligands for GPR10, GPR54 or GPR103 without a modification of the C-terminus RF-amide signature, but open the door to the design of new RF-amide peptides acting as agonist for one receptor and antagonist for another one.

Fully regiocontrolled polyarylation of pyridine.

Starting from commercially available 2-chloro-3-hydroxypyridine, a new route leading to the first protypical pentaarylpyridine bearing five different substituents is reported. This strategy involves a set of five sequential but fully regiocontrolled Suzuki-Miyaura reactions and highlights the 2-OBn pyridine protecting group as a key intermediate. The 2-OBn group played a double role: (i) it allowed additional bromination at position 5 and (ii) it could afford the reactive OTf species for the last C-arylation step at the less hindered 2 position of the tetraarylpyridine. The photophysical properties of the novel compounds are also described. The synthesized pentaarylpyridine derivative exhibit a large Stokes shift, strong solvatochromism, and quantum yield values up to 0.47; thus, they constitute promising building blocks for the design of environment-sensitive probes.

Nucleophilic substitution of azide acting as a pseudo leaving group: one-step synthesis of various aza heterocycles.

The reaction of 3-azidopropanoic acid with the carbodiimide-based coupling reagent DIC leads to a six-membered-ring intermediate acting as a versatile precursor to a diverse set of aza heterocycles, including mono-, bi-, and tricyclic compounds.