Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds

Shirai, Junya; Tomata, Yoshihide; Kono, Mitsunori; Ochida, Atsuko; Fukase, Yoshiyuki; Sato, Ayumu; Masada, Shinichi; Kawamoto, Tadashi; Yonemori, Kazuko; Koyama, Ryoukichi; Nakagawa, Hideyuki; Nakayama, Masaharu; Uga, Keiko; Shibata, Akira; Koga, Keiko; Okui, Toshitake; Shirasaki, Mikio; Skene, R.J.; Sang, Bi‐Ching; Hoffman, Isaac; Lane, Wes; Fujitani, Yasushi; Yamasaki, Masashi; Yamamoto, Satoshi

doi:10.1016/j.bmc.2017.12.006

Cited by 21 publications

(23 citation statements)

References 36 publications

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“…Novel compounds with higher FRET efficacy and higher LUC activity may be synthesized from compound T if the conformational disadvantage can be overcome by structure-activity relation (SAR) analysis. Actually, it has been reported recently that some related compounds can be improved without the movement of H5 26 .…”

Section: Discussionmentioning

confidence: 99%

Ternary crystal structure of human RORγ ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction

Noguchi

Nomura

Doi

et al. 2018

Sci Rep

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Retinoic acid-related orphan receptor gamma (RORγ) 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 RORγ. Here, we present the crystal structure of a novel RORγ 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 RORγ-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.

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Section: Discussionmentioning

confidence: 99%

Ternary crystal structure of human RORγ ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction

Noguchi

Nomura

Doi

et al. 2018

Sci Rep

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“…In summary, we identified compound 1 as a cholesterolcompetitive RORgt inverse agonist and showed that the inverse agonist effect of compound 1 depends on whether cholesterol is saturated in the receptor or not. Further compound optimization study of compound 1 has been reported by Shirai et al 19 This study emphasizes the importance of characterizing the recombinant protein and the optimization of cell-based assay conditions for high-throughput screening, and should impact the investigation of other orphan NRs for obtaining new lead compounds. Matsui and Naoki Tarui for their support in this study.…”

Section: Discussionmentioning

confidence: 87%

“…TAK-828F identified through further optimization of compound 1 has higher potency, and selectivity against NRs, including ROR isoforms. [19][20][21] The compound also inhibited IL-17 secretion from mouse splenocytes and human primary cells, and inhibited Th17 cell differentiation from naive T cells and memory CD4 + T cells. 22 Furthermore, the compound showed significant efficacy in naive T cell transfer mouse colitis model.…”

Section: Introductionmentioning

confidence: 92%

“…Of these hit compounds, compound 1 (Fig. 3D) was selected as a lead compound because the compound had 15-fold greater selectivity against RORa and RORb, 19 has good physical properties, including water solubility and cLogP of 2.8, and did not have cytotoxity and CYP inhibition. Table 3 summarizes the IC 50 values of compound 1 and TO901317 for RORgt from three functional assays, demonstrating that compound 1, similar to TO901317, was effective in the TopFluor cholesterol binding assay (IC 50 = 200 nM), the SRC1-2 recruitment assay (IC 50 = 91 nM), and the RORgt-RORE reporter gene assay (IC 50 = 1,600 nM), although the potency of compound 1 was slightly weaker than TO901317.…”

Section: High-throughput Screeningmentioning

confidence: 99%

“…Figure 5B showed that compound 1 inhibited TopFluor cholesterol binding of RORgt protein in a competitive manner, same as TO901317, and further structural analysis of compound 1 derivative revealed that this chemotype binds to cholesterol binding site. 19 To confirm the effect of cholesterol for RORgt transcriptional activity, we used RORE reporter gene assay (Fig. 1B), detecting inhibition of RORgt's constitutive active transcriptional activity by inverse agonist.…”

Section: The Effect Of Cholesterol On the Potency Of Compound 1 In Cementioning

confidence: 99%

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Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening

Koyama

Fukuda

Kamada

et al. 2018

ASSAY and Drug Development Technologies

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The retinoic acid-related orphan receptor gamma T (RORγt) plays an important role in Th17 cell proliferation and functionality. Thus, RORγt inverse agonists are thought to be potent therapeutic agents for Th17-mediated autoimmune diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and psoriasis. Although RORγt has constitutive activity, it is recognized that the receptor is physiologically regulated by various cholesterol derivatives. In this study, we sought to identify RORγt inverse agonists through a high-throughput screening campaign. To this end, we compared an apo-RORγt protein from Escherichia coli and a cholesterol-bound RORγt protein from insect cells. The IC of the known RORγt inverse agonist TO901317 was significantly lower for the apoprotein than for the cholesterol-bound RORγt. Through high-throughput screening using a fluorescence-based cholesterol binding assay with the apoprotein, we identified compound 1 as a novel cholesterol-competitive RORγt inverse agonist. Compound 1 inhibited the RORγt-TopFluor cholesterol interaction, coactivator recruitment, and transcriptional activity of RORγt. Cell-based reporter gene assay demonstrated that compound 1 showed higher potency by lipid depletion treatment. Collectively, our findings indicate that eliminating cholesterol from the RORγt protein is suitable for sensitive high-throughput screening to identify RORγt inverse agonists.

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Design and Synthesis of Conformationally Constrained RORγt Inverse Agonists

et al. 2019

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Retinoic-acid-related orphan receptor γt (RORγt) inverse agonists could be used for the treatment of autoimmune diseases. Previously, we reported a novel quinazolinedione 1 a with a flexible linear linker as a novel RORγt inverse agonist. A Ushaped conformation in the complex structure of 1 a with RORγt protein was confirmed. Further improvement of the pharmacokinetic (PK) profiles was required because of the low drug exposure in mice upon oral administration (mouse AUC of 1 a: 27 ng · h · mL À 1 at 1 mg · kg À 1 , p.o.). To improve the PK profiles, conformationally constrained U-shaped scaffolds were investigated. As a result, morpholine analogues with improved PK profiles and high potency were successfully identified. The substituent at the N1 position of the quinazoline moiety was also modified, leading to an enhancement of reporter activity. Consequently, compound 43 (N 2 -(3-chloro-4-cyanophenyl)-N 4 -(3-(cyclopropylmethyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)morpholine-2,4-dicarboxamide) exhibited improved drug exposure (mouse AUC: 1289 ng · h · mL À 1 at 1 mg · kg À 1 , p.o.). In addition, suppression of IL-17A gene expression by IL-23 stimulation in a mouse pharmacodynamics model was observed for 43. The conformation of 43 with RORγt protein was also confirmed as U-shape by X-ray co-crystal structure analysis. The key interaction that boosts potency is also discussed. . Conversion of the substituent on quinazoline ring. Reagents and conditions:(a) 1-bromo-2-methylpropane, K 2 CO 3 , DMF, 70°C, 51 % (for 40 a); (b) 2iodopropane, Cs 2 CO 3 , DMF, 50°C, 39 %; (for 40 b); (c) Pd/C, H 2 , MeOH or MeOH-THF, rt, 80-87 %; (d) (1) 4-nitrophenyl carbonochloridate, pyridine, THF, rt; (2) 33 (scheme 3), DIEA, DMF, rt, 37-78 %. Scheme 5. The preparation of the optically pure compound 43 and 44. Reagents and conditions:(a) chiral column separation by HPLC. crystal was collected by filtration and washed with cold hexane. 3-(cyclopropylmethyl)-2-isopropoxy-6-nitroquinazolin-4(3H)-one: 1 H NMR (300 MHz, CDCl 3 ) δ 0.42-0.57 (4H, m), 1.17-1.32 (1H, m), 1.47 (6H, d, J = 6.0 Hz), 3.99 (2H, d, J = 7.2 Hz), 5.59 (1H, m), 7.52 (1H, d, J = 8.7 Hz), 8.42 (1H, dd, J = 9.1, 2.6 Hz), 9.06 (1H, d, J = 2.6 Hz); MS (ESI) m/z: 304.2 [M + H] + . 40 b: 1 H NMR (300 MHz, CDCl 3 ) δ 0.42-0.55 (4H, m), 1.24-1.36 (1H, m), 1.65 (6H, d, J = 6.8 Hz), 3.98 (2H, d, J = 7.2 Hz), 5.12 (1H, brs), 7.48 (1H, d, J = 9.1 Hz), 8.45 (1H, dd, J = 9.3, 2.8 Hz), 9.10 (1H, d, J = 2.6 Hz); MS (ESI) m/z: 304.3 [M + H] + . -3-(cyclopropylmethyl)-1-isobutylquinazoline-2,4(1H,3H)dione (41 a). To a solution of 40 a (300 mg, 0.95 mmol) in MeOH (6.0 mL) was added PdÀ C (30 mg, 0.28 mmol) and the mixture was stirred at rt for 5 h under hydrogen atmosphere (1 atm). The mixture was filtered through Celite pad. The filtrate was concentrated in vacuo to give 41 a (235.2 mg, 0.818 mmol, 87 %) as a gray solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 0.23-0.37 (2H, m), 0.36-0.45 (2H, m), 0.89 (6H, d, J = 6.8 Hz), 1.05-1.30 (1H, m), 2.07 (1H, dt, J = 13.7, 6.8 Hz), ...

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Discovery of orally efficacious RORγt inverse agonists, part 1: Identification of novel phenylglycinamides as lead scaffolds

Cited by 21 publications

References 36 publications

Ternary crystal structure of human RORγ ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction

Ternary crystal structure of human RORγ ligand-binding-domain, an inhibitor and corepressor peptide provides a new insight into corepressor interaction

Cholesterol Unbound RORγt Protein Enables a Sensitive Inverse Agonist Screening

Design and Synthesis of Conformationally Constrained RORγt Inverse Agonists

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