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.

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.