Ternary crystal structure of human ROR? ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction.
ABSTRACT: Retinoic acid-related orphan receptor gamma (ROR?) plays pivotal roles in autoimmune diseases by controlling the lineage of interleukin 17 (IL-17)-producing CD4+ T cells (Th17 cells). Structure-based drug design has proven fruitful in the development of inhibitors targeting the ligand binding domain (LBD) of ROR?. Here, we present the crystal structure of a novel ROR? inhibitor co-complex, in the presence of a corepressor (CoR) peptide. This ternary complex with compound T reveals the structural basis for an inhibitory mechanism different from the previously reported inverse agonist. Compared to the inverse agonist, compound T induces about 2?Å shift of helix 5 (H5) backbone and side-chain conformational changes of Met365 on H5. These conformational changes correlate to reduced CoR peptide binding to ROR?-LBD in the presence of compound T, which suggests that the shift of H5 is responsible. This crystal structure analysis will provide useful information for the development of novel and efficacious drugs for autoimmune disorders.
Project description:Retinoic acid receptor-related-orphan-receptor-C (ROR?t) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of ROR?t in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in vitro and in vivo pharmacology of a potent and selective small-molecular-weight ROR?t inverse agonist. The compound binds to the ligand binding domain (LBD) of ROR?t leading to displacement of a co-activator peptide. We show for the first time that a ROR?t inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of ROR?t-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and ??T-cells and attenuated transcription of ROR?t target genes. The inhibitor showed good in vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex vivo recall assays. In summary, we demonstrate that inhibiting ROR?t by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.
Project description:The molecular recognition of the ROR? nuclear hormone receptor (NHR) ligand-binding domain (LBD) has been extensively studied with numerous X-ray crystal structures. However, the picture afforded by these complexes is static and does not fully explain the functional behavior of the LBD. In particular, the apo structure of the LBD seems to be in a fully active state, with no obvious differences to the agonist-bound structure. Further, several atypical in vivo inverse agonists have surprisingly been found to co-crystallize with the LBD in agonist mode (with co-activator), leading to a disconnection between molecular recognition and functional activity. Moreover, the experimental structures give no clues on how ROR? LBD binders access the interior of the LBD. To address all these points, we probe here, with a variety of simulation techniques, the fine structural balance of the ROR? LBD in its apo vs. holo form, the differences in flexibility and stability of the LBD in complex with agonists vs. inverse agonists and how binders diffuse in and out of the LBD in unbiased simulations. Our data conclusively point to the stability afforded by the so-called "agonist lock" between H479 and Y502 and the precise location of Helix 12 (H12) for the competence of the LBD to bind co-activator proteins. We observe the "water trapping" mechanism suggested previously for the atypical inverse agonists and discover a different behavior for the latter when co-activator is present or absent, which might help explain their conflicting data. Additionally, we unveil the same entry/exit path for agonists and inverse agonist into and out of the LBD for ROR?, suggesting it belongs to the type III NHR sub-family.
Project description:Biaryl amides as new ROR?t modulators were discovered. The crystal structure of biaryl amide agonist 6 in complex with ROR?t ligand binding domain (LBD) was resolved, and both "short" and "long" inverse agonists were obtained by removing from 6 or adding to 6 a proper structural moiety. While "short" inverse agonist (8) recruits a corepressor peptide and dispels a coactivator peptide, "long" inverse agonist (9) dispels both. The two types of inverse agonists can be utilized as potential tools to study mechanisms of Th17 transcriptional network inhibition and related disease biology.
Project description:As an attractive drug-target, retinoic acid receptor-related orphan receptor-gamma-t (ROR?t) has been employed widely to develop clinically relevant small molecular modulators as potent therapy for autoimmune disease and cancer, but its molecular mechanism of action (MOA) remains unclear. In the present study, we designed and discovered two novel ROR?t ligands that are similar in structure, but different in efficacy. Using fluorescence resonance energy transfer (FRET) assay, compound 1 was identified as an agonist with an EC50 of 3.7 ?M (max. act.: 78%), while compound 2 as an inverse agonist with an IC50 value of 2.0 ?M (max. inh.: 61%). We performed molecular dynamics (MD) simulations, and elucidated the MOA of ROR?t agonist and inverse agonist. Through the analyses of our MD results, we found that, after ROR?t is bound with the agonist 1, the side chain of Trp317 stays in the gauche- conformation, and thus helps to form the hydrogen bond, His479-Trp502, and a large hydrophobic network among H11, H11', and H12. All these interactions stabilize the H12, and helps the receptor recruit the coactivator. When the ROR?t is bound with the inverse agonist 2, the side chain of Trp317 is forced to adopt the trans conformation, and these presumed interactions are partially destroyed. Taken together, the critical role of residue Trp317 could be viewed as the driving force for the activation of ROR?t.
Project description:BACKGROUND:The nuclear hormone receptor ROR? regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of ROR? is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of ROR? and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a ROR? specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. RESULTS:We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of ROR? is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of ROR?. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for ROR? over ROR sub-family members ? and ?. CONCLUSION:The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of ROR?. The proteolytic sensitivity of the AF2 helix of ROR? demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on ROR? as the trigger for ROR? specific inverse agonism.
Project description:A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity. Compounds behaving as agonists in our biochemical assay also gave rise to an increased production of IL-17 in human PBMCs whereas inverse agonists led to significant suppression of IL-17 under the same assay conditions. The most potent inverse agonist compound showed >180-fold selectivity over the ROR isoforms as well as all other nuclear receptors that were profiled.
Project description:A novel series of tertiary amines as retinoid-related orphan receptor gamma-t (RORγt) inverse agonists was discovered through agonist/inverse agonist conversion. The level of RORγt inhibition can be enhanced by modulating the conformational disruption of H12 in RORγt LBD. Linker exploration and rational design led to the discovery of more potent indole-based RORγt inverse agonists.
Project description:The RAR-related orphan receptor gamma t (ROR?t) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as ROR?t agonists. The most potent and selective activator for ROR?t is 7?, 27-dihydroxycholesterol (7?, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an ROR?t antagonist, ursolic acid, in ROR?- or ROR?t-dependent cell-based reporter assays. These ligands bind directly to recombinant ROR? ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to ROR? LBD. In primary cells, 7?, 27-OHC and 7?, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an ROR?t-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7?, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and ??(+) T cells, similar to the deficiency observed in ROR?t knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these ROR?t agonist ligands, which we propose as ROR?t endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.
Project description:Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind ROR?, however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual ROR? and ROR? inverse agonist with no activity towards ROR? (Fig. 1). To our knowledge, this is one of only two small molecule ROR? inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design ROR?-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of ROR?.
Project description:The nuclear receptor retinoid acid receptor-related orphan receptor ?t (ROR?t) is a master regulator of the Th17/IL-17 pathway that plays crucial roles in the pathogenesis of autoimmunity. ROR?t has recently emerged as a highly promising target for treatment of a number of autoimmune diseases. Through high-throughput screening, we previously identified several classes of inverse agonists for ROR?t. Here, we report the crystal structures for the ligand-binding domain of ROR?t in both apo and ligand-bound states. We show that apo ROR?t adopts an active conformation capable of recruiting coactivator peptides and present a detailed analysis of the structural determinants that stabilize helix 12 (H12) of ROR?t in the active state in the absence of a ligand. The structures of ligand-bound ROR?t reveal that binding of the inverse agonists disrupts critical interactions that stabilize H12. This destabilizing effect is supported by ab initio calculations and experimentally by a normalized crystallographic B-factor analysis. Of note, the H12 destabilization in the active state shifts the conformational equilibrium of ROR?t toward an inactive state, which underlies the molecular mechanism of action for the inverse agonists reported here. Our findings highlight that nuclear receptor structure and function are dictated by a dynamic conformational equilibrium and that subtle changes in ligand structures can shift this equilibrium in opposite directions, leading to a functional switch from agonists to inverse agonists.