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

Authors: Ming-cheng Yu1, Feng Yang1,2, Xiao-yu Ding2,3, Nan-nan Sun1,4, Zheng-yuan Jiang1, Ya-fei Huang1, Yu-rong Yan1, Chen Zhu1, Qiong Xie1,5, Zhi-feng Chen2,3, Si-qi Guo2, Hua-liang Jiang2,3,6, Kai-xian Chen2,3, Cheng Luo2,3,6, Xiao-min Luo2,3, Shi-jie Chen2,3, Yong-hui Wang1
1 Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
2 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
3 University of Chinese Academy of Sciences, Beijing 100049, China
4 Key Laboratory of Metabolism and Molecular Medicine, The Ministry of Education, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China
5 Fudan Zhangjiang Institute, Shanghai 201203, China
6 School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, UCAS, Hangzhou 310024, China
Correspondence to: Xiao-min Luo:, Shi-jie Chen:, Yong-hui Wang:,
DOI: 10.1038/s41401-020-00552-w
Received: 21 July 2020
Accepted: 2 October 2020
Advance online: 25 November 2020


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.
Keywords: RORγt; cocrystal structures; MD simulation; “short” inverse agonists; agonists; “long” inverse agonists

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