RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-ß signaling in cancer.

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-ß1 (TGF-ß1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

Generation of Aryl Radicals from Aryl Halides: Rongalite-Promoted Transition-Metal-Free Arylation.

A new and practical method for the generation of aryl radicals from aryl halides is reported. Rongalite as a novel precursor of super electron donors was used to initiate a series of electron-catalyzed reactions under mild conditions. These transition-metal-free radical chain reactions enable the efficient formation of C-C, C-S, and C-P bonds through homolytic aromatic substitution or SRN1 reactions. Moreover, the synthesis of antipsychotic drug Quetiapine was performed on gram scale through the described method. This protocol demonstrated its potential as a promising arylation method in organic synthesis.

Discovery, synthesis, biological evaluation and structure-based optimization of novel piperidine derivatives as acetylcholine-binding protein ligands.

The homomeric α7 nicotinic receptor (α7 nAChR) is widely expressed in the human brain that could be activated to suppress neuroinflammation, oxidative stress and neuropathic pain. Consequently, a number of α7 nAChR agonists have entered clinical trials as anti-Alzheimer's or anti-psychotic therapies. However, high-resolution crystal structure of the full-length α7 receptor is thus far unavailable. Since acetylcholine-binding protein (AChBP) from Lymnaea stagnalis is most closely related to the α-subunit of nAChRs, it has been used as a template for the N-terminal domain of α-subunit of nAChR to study the molecular recognition process of nAChR-ligand interactions, and to identify ligands with potential nAChR-like activities.Here we report the discovery and optimization of novel acetylcholine-binding protein ligands through screening, structure-activity relationships and structure-based design. We manually screened in-house CNS-biased compound library in vitro and identified compound 1, a piperidine derivative, as an initial hit with moderate binding affinity against AChBP (17.2% inhibition at 100 nmol/L). During the 1st round of optimization, with compound 2 (21.5% inhibition at 100 nmol/L) as the starting point, 13 piperidine derivatives with different aryl substitutions were synthesized and assayed in vitro. No apparent correlation was demonstrated between the binding affinities and the steric or electrostatic effects of aryl substitutions for most compounds, but compound 14 showed a higher affinity (Ki=105.6 nmol/L) than nicotine (Ki=777 nmol/L). During the 2nd round of optimization, we performed molecular modeling of the putative complex of compound 14 with AChBP, and compared it with the epibatidine-AChBP complex. The results suggested that a different piperidinyl substitution might confer a better fit for epibatidine as the reference compound. Thus, compound 15 was designed and identified as a highly affinitive acetylcholine-binding protein ligand. In this study, through two rounds of optimization, compound 15 (Ki=2.8 nmol/L) has been identified as a novel, piperidine-based acetylcholine-binding protein ligand with a high affinity.

Discovery of novel triazine derivatives as potent retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonists.

Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) has been exploited as a promising target for the new small molecule therapeutics to treat inflammatory and autoimmune diseases via modulating the interleukin-17 (IL-17) production by T helper 17 (Th17) cells. Herein, we reported a series of triazine-based derivatives as novel RORγt inverse agonists. By screening of our in-house compound library, the hit compound 1 was identified with weak RORγt inhibitory activity. Subsequently, we engineered detailed structural modifications to explore the structure-activity relationships (SARs) of triazines derivatives, which led to discovery of a number of potent RORγt inverse agonists with IC50 values in the range of 7 nM-50 nM in RORγt dual FRET assay. Among them, compound 14g displayed potent RORγt inverse agonistic activity with an IC50 value of 22.9 nM in dual FRET assay. In a cell-based reporter gene assay, compound 14g showed an IC50 value of 0.428 µM and maximum inhibition rate of 108.9%. Compound 14g also exhibited good metabolic stability and a decent pharmacokinetic profile with a low clearance (CL = 0.229 L/h/kg) and a reasonable oral exposure (AUC0-Last = 5058 ng/mL*h). Most importantly, 14g alleviated the severity of imiquimod-induced psoriasis in mice. Taken together, triazine-based derivatives represent a new chemical class of RORγt inverse agonists as potential therapeutic agents against autoimmune diseases.

Optimization of carbazole carboxamide RORγt agonists: Challenges in improving the metabolic stability and maintaining the agonistic activity.

Based on two previously discovered carbazole carboxamide retinoic acid receptor-related orphan receptor-γt (RORγt) agonists 6 and 7 (t1/2 = 8.7 min and 16.4 min in mouse liver microsome, respectively), new carbazole carboxamides were designed and synthesized according to the molecular mechanism of action (MOA) and metabolic site analysis with the aim of identifying novel RORγt agonists with optimal pharmacological and metabolic profiles. By modifying the "agonist lock" touching substitutions on carbazole ring, introducing heteroatoms into different parts of the molecule and attaching a side chain to the sulfonyl benzyl moiety, several potent RORγt agonists were identified with greatly improved metabolic stability. Best overall properties were achieved in compound (R)-10f with high agonistic activities in RORγt dual FRET (EC50 = 15.6 nM) and Gal4 reporter gene (EC50 = 141 nM) assays and greatly improved metabolic stability (t1/2 > 145 min) in mouse liver microsome. Besides, the binding modes of (R)-10f and (S)-10f in RORγt ligand binding domain (LBD) were also studied. Altogether, the optimization of carbazole carboxamides led to the discovery of (R)-10f as a potential small molecule therapeutics for cancer immunotherapy.

Discovery of Biaryl Amide Derivatives as Potent, Selective, and Orally Bioavailable RORγt Agonists for Cancer Immunotherapy.

The master transcription factor receptor retinoic acid receptor-related orphan receptor γt (RORγt) regulates the differentiation of T-helper 17 (Th17) cells and the production of interleukin-17 (IL-17). Activation of RORγt+ T cells in the tumor microenvironment promotes immune infiltration to more effectively inhibit tumor growth. Therefore, RORγt agonists provide a reachable approach to cancer immunotherapy. Herein, a series of biaryl amide derivatives as novel RORγt agonists were designed, synthesized, and evaluated. Starting from the reported RORγt inverse agonist GSK805 (1), "functionality switching" and structure-based drug optimization led to the discovery of a promising RORγt agonist lead compound 14, which displayed potent and selective RORγt agonist activity and significantly improved metabolic stability. With excellent in vivo pharmacokinetic profiles, compound 14 demonstrated robust efficacy in preclinical tumor models of mouse B16F10 melanoma and LLC lung adenocarcinoma. Taken together, current studies indicate that 14 deserves further investigation as a potential lead RORγt agonist for cancer immunotherapy.

RORγt agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers.

BACKGROUND: The overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive. METHODS: The expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression. RESULTS: We designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti­PD­1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration. CONCLUSION: Our results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

Ontogeny of RORγt+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis.

BACKGROUND: Neonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated. RESULTS: We examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity. CONCLUSIONS: Our data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

Discovery of tert-amine-based RORγt agonists.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma-t (RORγt) is a transcription factor regulating Th17 cell differentiation and proliferation from naive CD4+ T cells. Since Th17 cells have demonstrated the antitumor efficacy by eliciting remarkable activation of CD8+ T cells, RORγt agonists could be applied as potential small molecule therapeutics for cancer immunotherapy. Based on the previously reported RORγt agonist 1 and its resolved co-crystal structure, a series of new tertiary amines were designed, synthesized and biologically evaluated, yielding optimal moieties with improved chemical properties and biological responses. The combination of these optimal moieties resulted in identification of novel RORγt agonists such as 8b with further elevated RORγt agonism responses at a target-based level as well as in cell-based assays, which provided some structural knowledge for further optimization of RORγt agonists as small molecule therapeutics for cancer immunotherapy.

Discovery of tetrahydroquinolines and benzomorpholines as novel potent RORγt agonists.

The retinoic acid receptor-related orphan receptor γt (RORγt) is an important nuclear receptor that regulates the differentiation of Th17 cells and production of interleukin 17(IL-17). RORγt agonists increase basal activity of RORγt and could provide a potential approach to cancer immunotherapy. Herein, hit compound 1 was identified as a weak RORγt agonist during in-house library screening. Changes in LHS core of 1 led to the identification of tetrahydroquinoline compound 6 as a partial RORγt agonist (max. act. = 39.3%). Detailed structure-activity relationship on substituent of the LHS core, amide linker and RHS arylsulfonyl moiety was explored and a novel series of tetrahydroquinolines and benzomorpholines was discovered as potent RORγt agonists. Tetrahydroquinoline compound 8g (EC50 = 8.9 ± 0.4 nM, max. act. = 104.5%) and benzomorpholine compound 9g (EC50 = 7.5 ± 0.6 nM, max. act. = 105.8%) were representative compounds with high RORγt agonistic activity in dual FRET assay, and they showed good activity in cell-based Gal4 reporter gene assay and Th17 cell differentiation assay (104.5% activation at 300 nM of 8g; 59.4% activation at 300 nM of 9g). The binding modes of 8g and 9g as well as the two RORγt inverse agonists accidentally discovered were also discussed.

Discovery of carboxyl-containing biaryl ureas as potent RORγt inverse agonists.

GSK805 (1) is a potent RORγt inverse agonist, but a drawback of 1 is its low solubility, leading to a limited absorption in high doses. We have explored detailed structure-activity relationship on the amide linker, biaryl and arylsulfonyl moieties of 1 trying to improve solubility while maintaining RORγt activity. As a result, a novel series of carboxyl-containing biaryl urea derivatives was discovered as potent RORγt inverse agonists with improved drug-like properties. Compound 3i showed potent RORγt inhibitory activity and subtype selectivity with an IC50 of 63.8 nM in RORγ FRET assay and 85 nM in cell-based RORγ-GAL4 promotor reporter assay. Reasonable inhibitory activity of 3i was also achieved in mouse Th17 cell differentiation assay (76% inhibition at 0.3 µM). Moreover, 3i had greatly improved aqueous solubility at pH 7.4 compared to 1, exhibited decent mouse PK profile and demonstrated some in vivo efficacy in an imiquimod-induced psoriasis mice model.

Discovery of aryl-substituted indole and indoline derivatives as RORγt agonists.

A series of aryl-substituted indole and indoline derivatives were discovered as novel RORγt agonists by a scaffold-based hybridization of the reported RORγt agonists 1 and 2. SAR studies on the core structures, the RHS hydrophilic side chains and the LHS hydrophobic aryl groups of a hybrid compound 3 led to the identification of potent RORγt agonists with improved drug-like properties. Compound 14 represented a high potency lead with an EC50 of 20.8 ±â€¯1.5 nM, the (S)-enantiomer (EC50 = 16.1 ±â€¯4.5 nM) of which was 17 times more potent than the (R) counterpart (EC50 = 286 ±â€¯30.4 nM) in RORγ dual FRET assay. The cell-based GAL4 reporter gene assay also suggested 14 as the most active compound which exhibited an EC50 of 247 ±â€¯33.1 nM and a maximum activation percentage of 133%. Moreover, 14 showed high metabolic stability (t1/2 = 113 min) in mouse liver microsome and had improved aqueous solubility at pH 7.4 compared to the parent compounds. Furthermore, 14 was found to be orally bioavailable and demonstrated excellent in vivo pharmacokinetics in mice. Present studies indicate that 14 deserves further investigation in tumor animal models as a potential candidate of RORγt agonist for cancer immunotherapy.

Discovery of N-indanyl benzamides as potent RORγt inverse agonists.

The retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is a promising therapeutic target for treatment of Th17 cell-mediated autoimmune diseases. Based on a scaffold hopping/conformational restriction strategy, a series of N-indanyl benzamides as novel RORγt inverse agonists was discovered. Exploration of structure-activity relationship on the piperazine ring, benzoyl moiety and cyclopentyl moiety of N-indanyl benzamides 2a and 2d led to identification of potent RORγt inverse agonists. Compound 5c with (S)-enantiomer was found having an IC50 of 153.7 nM in Fluorescence Resonance Energy Transfer (FRET) assay, and an IC50 of 47.1 nM in mouse Th17 cell differentiation assay, which represents a promising starting point for developing potent small molecule RORγt inverse agonists. Binding modes of the two enantiomers 5c and 5d in RORγt ligand binding domain were also discussed.

Discovery of carbazole carboxamides as novel RORγt inverse agonists.

A novel series of carbazole carboxamides was discovered as potent RORγt inverse agonists using a scaffold hybridization strategy. Structure-activity relationship exploration on the amide linker, carbazole ring and arylsulfone moiety of the hybrid amide 3a led to identification of potent RORγt inverse agonists. Compound 6c was found to have a good RORγt activity with an IC50 of 58.5 nM in FRET assay, and reasonable inhibitory activity in mouse Th17 cell differentiation assay (58.8% inhibition at 0.3 µM). The binding mode of carbazole carboxamides in RORγt ligand binding domain was discussed.

Discovery, cocrystallization and biological evaluation of novel piperidine derivatives as high affinity Ls-AChBP ligands possessing α7 nAChR activities.

A series of novel pyridine-substituted piperidine derivatives were discovered as low nanomolar Ls-AChBP ligands with α7 nAChR partial agonism or antagonism activities. A high-resolution antagonist-bound Ls-AChBP complex was successfully resolved with a classic Loop C opening phenomenon and unique sulfur-π interactions which deviated from our previous docking mode to a large extent. With the knowledge of the co-complex, 27 novel piperidine derivatives were designed and synthesized. The structure-activity relationships (SARs) of the aromatic and pyridine regions were well established and binding modes were illustrated with the help of molecular docking which indicated that interactions with Trp 143 and the "water bridge" are essential for the high binding affinities. Halogen bonding as well as the space around 5'- or 6'- position of the pyridine ring was also proposed to influence the binding conformation of the compounds. Notably, two enantiomers of compound 2 showed opposite functions toward α7 nAChR and compound (S)-2 showed sub-nanomolar affinity (Ki = 0.86 nM) on Ls-AChBP and partial agonism (pEC50 = 4.69 ±â€¯0.11,Emax = 36.1%) on α7 nAChR with reasonable pharmacokinetics (PK) properties and fine ability of blood-brain-barrier (BBB) penetration. This study provided promising hits to develop candidates targeting nAChR-related CNS diseases.