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.KEYWORDS: RORγt, agonists, inverse agonists, Th17 cell differentiation, cocrystal structure, structure-based design R etinoid-related orphan receptor gamma-t (RORγt) is a member of the nuclear receptor superfamily. RORγt is a key regulator of the development and functions of T-helper 17 (Th17) cells which are implicated in the pathology of a variety of human inflammatory and autoimmune disorders. 1,2 The RORγt inhibitors have potential utility in controlling the activity of Th17 cells and can be developed as therapeutic agents for treatment of Th17-related autoimmune diseases. A few small molecule inhibitors of RORγt have been reported in the literature. 3−10 In this paper, we report the discovery of tertiary amines and indoles as potent RORγt inverse agonists using structure-and knowledge-based compound design.A high-throughput screen (HTS) of the GSK in-house compound collection using a RORγ fluorescence resonance energy transfer (FRET) assay 11 resulted in identification of thiazole amide 1 as a RORγt inverse agonist with IC 50 of 1.0 μM. The binding of 1 to the RORγt ligand binding domain (LBD) was confirmed with a thermal shift of 7.1°C in a thermal shift assay. 11 SAR exploration on the left-hand side (LHS) of 1 led to the identification of tertiary amine 2 as a potent RORγt agonist with a EC 50 of 0.02 μM in dual FRET assay (Scheme 1). 12 Dual FRET assay, using the same technology as the FRET assay but without adding a surrogate agonist, only relies on the basal level of RORγt activity and is able to measure both agonists and inverse agonists. Peptide profiling study using dual FRET assay showed that coactivator peptide (e.g., steroid receptor coactivator 1 (SRC1)) was recruited upon binding of 2 to RORγt LBD whereas corepressor peptide (e.g., nuclear receptor corepressor 2 (NCOR2)) was not. 12 Given the structure similarity of 1 and 2, we assume that they adopt a similar binding mode within RORγt LBD despite their difference as agonist and inverse agonist. To understand the binding mode of the chemical series, an in-silico docking study for 2 based on a reported RORγt crystal structure 13 was conducted.A RORγt LBD crystal structure (PDB accession code: 3KYT) was selected and processed for the docking study. A total of 40 poses with the best scores were obtained and visually inspected after docking with Surflex-Dock v2.3 14−16 in Sybyl 8.1, 17 among which the top 10 poses were found to be representative and thus further ranked using MM/GBSA 18−20 affinity scores based on the VSGB2.0 solvent model. 21,22 As a
A novel series of biaryl amides was identified as RORγt inhibitors through core replacement of a starting hit 1. Structure−activity relationship exploration on the biaryl moiety led to discovery of potent RORγt inhibitors with good oral bioavailability and CNS penetration. Compounds 9a and 9g demonstrated excellent in vivo efficacy in EAE mice dose dependently with once daily oral administration. KEYWORDS: RORγt inhibitor, Th17 cell differentiation, biaryl amides, EAE, multiple sclerosis T helper (Th) 17 cells, a lineage of CD4 + effector T cells characterized by the production of IL-17A and IL-17F, are pathogenic in human autoimmune inflammatory diseases including multiple sclerosis (MS). 1−4 The presence of IL-17 was detected in MS lesions, and Th17 cells were observed in the infiltrations of mouse experimental autoimmune encephalomyelitis (EAE) central nervous system (CNS). 5,6 Differentiation and function of Th17 cells are controlled by the transcription factor retinoic acid receptor-related orphan receptor-gamma-t (RORγt). 7−9,11 It has been shown that the genetic deficiency of RORγt in mice severely impaired Th17 cell differentiation and conferred resistance to EAE. 10 RORγt inhibitors has potential utility in reducing the activity of Th17 cells and therefore can be developed as therapeutic agents for the treatment of Th17 cell mediated autoimmune diseases. 12−18 A few small molecule RORγt inhibitors have been reported in literature. 19 Digoxin, 20 SR1001, 21 and ursolic acid 22 were first reported to inhibit RORγt and ameliorate EAE in mice via intraperitoneal administration. Other small molecular RORγt inhibitors 23−31 were later disclosed. Recently, we reported discovery of thiazole ketone amides (e.g., 2) and thiophene ketone amides (e.g., 3) as novel RORγt inhibitors based on a high throughput screening (HTS) hit 1 (Figure 1). 32 These ketones, especially the thiophene ketones, showed good RORγt activities but were poorly orally bioavailable and lack of CNS penetration that is believed to be important for developing an effective oral MS drug. In this Letter, we report the discovery of novel biaryl amides as first potent, orally bioavailable, and CNS penetrant RORγt inhibitors, which demonstrated EAE in vivo efficacy dose dependently via oral administration.The lack of CNS penetration of thiazole/thiophene ketones was attributed to their ketone moiety as the nonketone thiazole amide 1 is CNS penetrant with a brain-to-blood ratio (Br/Bl) of 1.5 in a mouse CNS study (i.p., 2 mg/kg). 33 Encouraged by
HDAC inhibitors are an emerging class of anticancer agents with the approved indications of multiple myeloma, cutaneous and/or peripheral T cell lymphoma. However, non-selective pan-HDAC inhibitors are often reported associated with heart, hematologic and gastrointestinal toxicities. Among HDAC subtypes, inhibiting HDAC6 selectively may not only enhance potency, but may also reduce the toxicity related to off-target effects of pan-HDACis. In previous studies, pioneer HDAC6 selective inhibitors have been demonstrated with the potential to treat both non-solid tumor and solid tumors along or in combination with chemotherapy, proteasome inhibitors or checkpoint inhibitors. In this work, we report the design, in vitro and in vivo evaluation a class of novel HDAC6 selective inhibitors, series CVL608, with potent antitumor profiles in non-solid tumor such as multiple myeloma, and solid tumors including breast cancer, melanoma and NSCLC. Method:1.The HDAC enzymes inhibition screening were performed using HDAC Caliper assay, 12-sipper chip and Caliper EZ Reader II systems.2.The anti-proliferative effects of compounds were assessed in variety cancer cell lines including MM.1S, REC-1, Mino, SK-MEL-5, A375, MCF-7, A549 and OVCAR-3 by quantifying cell proliferation by standard CellTiter-Glo® assay after a 72-h incubation.3.The in vivo efficacy of compounds as monotherapy and in combination with chemotherapy and proteasome inhibitors were examined in MM.1s and MCF-7 CDX models in BALB/c nude mice (n=6/group); compounds in combination with checkpoint inhibitors were examined in LL/2(LLC1) and B16F10 CDX models in C57BL/6 mice (n=6/group). Conclutions:1.According to the HDAC Caliper assay, CVL608 series compounds were identified as HDAC6 selective inhibitors bearing 5-20X selectivity over HDAC3, 20-100X selectivity over HDAC1 and HDAC8, with the HDAC6 potency upto less than 5 nM. The compounds exhibited a priority selectivity than clinical candidates ACY-1215 and ACY-241.2.Lead compounds CVL608 inhibited MM.1S, REC-1, Mino, SK-MEL-5, A375, MCF-7, A549 and OVCAR-3 with the IC50 less than 10 micromole, which were equal or better compared with ACY-241.3.In MM.1s CDX models, lead compounds CVL608 was more potent than ACY-241 at 30mg/kg in tumor proliferation as monotherapy. Moreover, CVL608 in combination with Bortezomib (0.5 mg/kg) significate inhibit tumor growth compared with monotherapy and ACY-241+Bortezomib (p<0.05). CVL608 monotherapy and in combination with PTX in MCF-7 CDX, and in combination with PD-1 inhibitor in LL/2(LLC1) and B16F10 CDX models are under investigating.These data may support interest in the clinical development of novel HDAC6 selective inhibitors in both non-solid tumor and solid tumors. Citation Format: Zeng Li, Yaobang Cheng, Xiaokun Shen, Yonghui Wang. Preclinical evaluation of novel HDAC6 selective inhibitors CVL608 with potent in vitro and in vivo profiles in tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 513.
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