Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2013 Nov 22;5(1):65–68. doi: 10.1021/ml4003875

Discovery of Tertiary Amine and Indole Derivatives as Potent RORγt Inverse Agonists

Ting Yang †, Qian Liu †, Yaobang Cheng †, Wei Cai †, Yingli Ma †, Liuqing Yang †, Qianqian Wu †, Lisa A Orband-Miller ‡, Ling Zhou †, Zhijun Xiang †, Melanie Huxdorf †, Wei Zhang †, Jing Zhang †, Jia-Ning Xiang †, Stewart Leung †, Yang Qiu †, Zhong Zhong †, John D Elliott †, Xichen Lin †, Yonghui Wang †,*
PMCID: PMC4027777  PMID: 24900774

Abstract

graphic file with name ml-2013-003875_0010.jpg

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

Retinoid-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) assay11 resulted in identification of thiazole amide 1 as a RORγt inverse agonist with IC50 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 EC50 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 structure13 was conducted.

Scheme 1. From HTS Hit (1) to Tertiary Amine RORγt Agonist (2).

Scheme 1

Open in a new tab

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 v2.314−16 in Sybyl 8.1,17 among which the top 10 poses were found to be representative and thus further ranked using MM/GBSA18−20 affinity scores based on the VSGB2.0 solvent model.21,22 As a result, the binding mode with the most favored MM/GBSA binding energy was selected and illustrated in Figure 1.12 In this binding mode, two H-bondings were observed: One from the sulfone moiety with Arg367 and a backbone amide, and the other between the linker amide and a backbone carbonyl. In addition, two π–π stacking interactions are formed: one between the sulfone-substituted phenyl ring and Phe377 and the other between the middle phenyl ring and Phe378. The LHS benzyl group of the ligand occupies the hydrophobic site near Tyr502 and Trp316, which is believed to be important for activating RORγt by stabilizing the activation function 2 (AF2) domain (H12) essential for the downstream peptide recruitment.23

Figure 1.

Figure 1

Open in a new tab

Predicted binding mode of 2 in RORγt LBD is illustrated, where 2 is in cyan stick and RORγt LBD in ribbon expression. Residues involved in key molecular interactions with 2 are highlighted and labeled.

On the basis of literature evidence of agonist/inverse agonist conversion reported for estrogen receptor ligands,24 we decided to design RORγt inverse agonists based on 2 and its predicted binding mode by introducing substituents to the para-position of the LHS phenyl ring near the AF2 domain to interfere H12’s packing and thus affect the downstream peptide recruitment.

A series of compounds with different size and/or shape of 4-substituents on the LHS phenyl ring were designed and synthesized.12 Potency (IC50 or XC50) and maximum response (max %) were measured in FRET and dual FRET assays (Table 1). As expected, the compounds showed a range of maximum responses depending on the properties (size, shape, and electrostatics) of the 4-substituents. The maximum inhibition in the FRET assay improved in an order of Me < Et <t-Bu < CF3 < O-c-Pen ∼ Ph. In the dual FRET assay, maximum % activation of compounds 2–4 decreased from 125% to 65% with the increase in size of the substituents. With a larger substituent than 4, compound 5 showed no effect in modulating RORγt basal activity and is considered as a neutral antagonist. Enlarging 4-substituents further from that of 5 resulted in inverse agonists with an increased level of inhibition. Shape and electrostatic effect also play a role for the substituents with similar size (5 vs 6; 7 vs 8). The results of dual FRET assay together with the structural insight from the docking study revealed the molecular mechanism of action for the compounds. The strong correlation between bulk of the substituent and level of inhibition can be interpreted as severity of the structural interference of the substituent to H12 based on the predicted binding mode. The severity of the structural interference is translated to the magnitude of conformational change of H12 and thus the ability of coactivator and corepressor recruitment. In contrast to the notable change in the level of inhibition, potency of the compounds was merely affected by bulk of the substituents. Compound 7 was further characterized by peptide recruitment profiling based on the dual FRET assay.12 As a result, neither coactivator peptide nor corepressor peptide tested was recruited. This was the first time to observe that a corepressor peptide was not recruited by a type of inverse agonists. Further biological investigation is needed to interpret the results.

Table 1. SAR of 4-Substituents on LHS Phenyl of Amines.

graphic file with name ml-2013-003875_0006.jpg

    FRET
 dual FRET
compd R IC50 (nM) max % XC50 (nM)a max %
2 H >25 000   20b 125
3 Me >25 000   25b 89
4 Et 13 38 25b 65
5 t-Bu 40 84 >10 000  
6 CF3 25 92 6 25
7 O-c-Pen 20 115 32 85
8 Ph 50 115 20 94
Open in a new tab
a

IC50 (nM) unless indicated.

b

EC50 (nM).

A crystal structure of RORγt LBD in a complex with 2 was later resolved at a resolution of 2.01 Å (PDB accession code: 4NIE).12 To our delight, the overall binding mode of 2 in RORγt LBD is essentially the same as the predicted one. With the established binding mode, we explored SAR of the amine containing linker with the LHS fixed as 4-CF3–Ph (Table 2). Removal of the propyl group from the tertiary amine lowered the potency by 50-fold (9), indicating that the hydrophobic interaction between the propyl group and RORγt LBD has significant impact on compound potency. Interestingly, shifting the NH by one position (10) regained RORγt potency by more than 12-fold from that of 9. Attaching a propyl moiety to the amine did not improve much of the potency (11 vs 10). While introducing a Cl group on the ortho-position of the middle phenyl ring led to the RORγt potency increased by more than 6-fold (12 vs 10). The potency improvement of 12 can be attributed to both molecular interaction and conformational effect. Compounds, 6, 9, 11, and 12, were also tested in mouse Th17 cell differentiation assay,11 and their IC50 values were more than 100 nM (Table 2). Therefore, further optimization was needed for achieving better Th17 potency.

Table 2. SAR of Amine Linkers.

graphic file with name ml-2013-003875_0007.jpg

graphic file with name ml-2013-003875_0008.jpg

Open in a new tab

To mimic the aniline geometry of 11 and 12, we designed a more rigid indole compound 13 as a potential RORγt inverse agonist. Figure 2 shows the predicted binding mode of 13, compared with that of 6 in RORγt crystal structure. The 4-CF3–Ph moiety of 13 extends naturally toward H12 in a low energy conformation, where CF3 is in close contact with the side chain of Tyr502 on H12. Comparing to phenyl, indole seems to be more efficient for the design of inverse agonists with AF2 domain disrupting mechanism. With this assessment, 13 was synthesized and confirmed to be a potent RORγt inverse agonist with FRET IC50 of 5 nM (max % = 104) (Table 3).

Figure 2.

Figure 2

Open in a new tab

Structural overlay of 13 (in brown stick) and 6 (in cyan stick) in RORγt LBD.

Table 3. SAR of Substituents on LHS Phenyl of Indoles.

graphic file with name ml-2013-003875_0009.jpg

    FRET
 Th17
compd R IC50 (nM) max % IC50 (nM)
13 4-CF3 5 104 31
14 4-H 3 65  
15 4-F 8 92  
16 4-Cl 4 99 164
17 4-CN 6 108 457
18 4-OiPr 6 119 70
19 2-CF3 6 83  
20 3-CF3 4 57  
21 2-Cl, 4-Cl 6 100 9
22 3-Cl, 4-Cl 6 98 40
Open in a new tab

A series of indole analogues were designed and prepared subsequently to explore SAR of the LHS phenyl substitution. Synthetic procedures for preparation of indole analogues 13–21 and 22 were shown in Scheme 2. Reaction of bromides 13a–21a and 5-nitro-1H-indole gave 1-substituted-5-nitro-indoles 13b–21b. Reduction of the nitro-indoles to amino-indoles 13c–21c, followed by amide formation with acid A or activated ester B produced the targeted compounds 13–21. Synthesis of compound 22 started with reaction of 2-(3,4-dichlorophenyl)acetic acid and 5-nitro-indoline, which led to formation of amide 22a. Reduction of the amide, followed by oxidation of indoline gave the nitro-indole 22b, which upon nitro reduction and amide formation with B afforded the targeted compound 22.

Scheme 2. Synthetic Procedures for Indole Analogues 13–21 and 22.

Scheme 2

Open in a new tab

Reagents and conditions: (a) for X = Br: 5-nitro-1H-indole, Cs2CO3, KI, DMF, 90 °C. For X = I: 5-nitro-1H-indole, K2CO3 (or Cs2CO3), DMF, RT; or 5-nitro-1H-indole, NaH, DMF, 0–90 °C. (b) SnCl2·2H2O, ethanol, reflux. (c) For acid A, HATU, DIPEA, DCM, RT; for perfluorophenyl ester B, DIPEA, DCM, RT; (d) HATU, DIPEA, DCM; (e) NaBH4, BF3·Et2O, THF, RT; (f) toluene, 4,5-dichloro-3,6-dioxocyclohexa-1,4-diene-1,2-dicarbonitrile, 80 °C; (g) SnCl2·2H2O, ethanol, reflux; (h) perfluorophenyl ester B, DIPEA, DCM, RT.

All indole-containing compounds tested showed strong RORγt inhibition with FRET IC50 values below 10 nM. Similar to the tertiary amine, the bulk of the para-substitution clearly impacts the level of RORγt inhibition. The nonsubstituted analogue 14 showed only 65% of inhibition, and the level of inhibition generally improved with the increase in size of the substituted moieties (14–18), provided that shape and electrostatic effect of the substituents are taken into consideration when comparing compounds with similar size of the substituents. Substitution on other positions of the LHS phenyl was also explored. The reduced maximum response of 19 and 20 suggests that 4-substitution is more effective on H12 disruption than 2- or 3-substitution. The RORγt potencies of the disubstituted compounds 21 and 22 are similar to that of 16 but the Th17 potencies are higher. This suggests that lipophilicity might have played a role in cellular potency. With proper substituents on LHS phenyl of the indoles, we were able to achieve high Th17 potency with IC50s equal or below 100 nM (13, 18, 21, and 22).

In summary, we identified a novel series of tertiary amines as RORγt inverse agonists using structure- and knowledge-based design. Relationship between ligand/H12 structural disruption and the level of RORγt inhibition was established for the first time. Linker exploration and rational design led to a series of indole-based analogues as more potent RORγt inverse agonists. Compound 21 was discovered as a potent RORγt lead with both FRET and Th17 IC50s lower than 10 nM. Further optimization of the PK profile of the aryl amide series is ongoing and will be reported in due course.

Acknowledgments

We thank Guifeng Zhang, Shuai Wang, Gang An, Bruce Wisely, Tom Consler, Kevin Meng, Hui Lei, Feng Ren and Hongtao Lu for their helpful discussions.

Supporting Information Available

RORγt FRET, dual FRET and Th17 assays description; peptide profiling studies; modeling studies; cocrystal structure data; synthetic procedures; and compound characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ml4003875_si_001.pdf (1,007.8KB, pdf)

References

  1. Huh J. R.; Littman D. R. Small molecule inhibitors of RORγt: targeting Th17 cells and other applications. Eur. J. Immunol. 2012, 42, 2232–2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burris T. P.; Busby S. A.; Griffin P. R. Targeting orphan nuclear receptors for treatment of metabolic diseases and autoimmunity. Chem. Biol. 2012, 19, 51–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Huh J. R.; Leung M. W. L.; Huang P.; Ryan D. A.; Krout M. R.; Malapaka R. R. V.; Chow J.; Manel N.; Ciofani M.; Kim S. V.; Cuesta A.; Santori F. R.; Lafaille J. J.; Xu H. E.; Gin D. Y.; Rastinejad F.; Littman D. R. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. Nature 2011, 472, 486–490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Solt L. A.; Kumar N.; Nuhant P.; Wang Y.; Lauer J. L.; Liu J.; Istrate M. A.; Kamenecka T. M.; Roush W. R.; Vidovic D.; Schürer S. C.; Xu J.; Wagoner G.; Drew P. D.; Griffin P. R.; Burris T. P. Suppression of Th17 Differentiation and autoimmunity by a synthetic ROR ligand. Nature 2011, 472, 491–494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Xu T.; Wang X.; Zhong B.; Nurieva R. I.; Ding S.; Dong C. Ursolic acid suppresses interleukin-17 (IL-17) production by selectively antagonizing the function of RORγt protein. J. Biol. Chem. 2011, 286, 22707–22710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Solt L. A.; Kumar N.; He Y.; Kamenecka T. M.; Griffin P. R.; Burris T. P. Identification of a selective RORγ ligand that suppresses Th17 cells and stimulates T regulatory cells. ACS Chem. Biol. 2012, 7, 1515–1519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kumar N.; Lyda B.; Chang M. R.; Lauer J. L.; Solt L. A.; Burris T. P.; Kamenecka T. M.; Griffin P. R. Identification of SR2211: a potent synthetic RORγ-selective modulator. ACS Chem. Biol. 2012, 7, 672–677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Huh J. R.; Englund E. E.; Wang H.; Huang R.; Huang P.; Rastinejad F.; Inglese J.; Austin C. P.; Johnson R. L.; Huang W.; Littman D. R. Identification of potent and selective diphenylpropanamide RORγ inhibitors. ACS Med. Chem. Lett. 2013, 4, 79–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kamenecka T. M.; Lyda B.; Chang M. R.; Griffin P. R. Synthetic modulators of the retinoic acid receptor-related orphan receptors. Med. Chem. Commun. 2013, 4, 764–776. [Google Scholar]
  10. Murali Dhar T. G.; Zhao Q.; Markby D. W. Targeting the nclear hormone receptor RORγt for the treatment of autoimmune and inflammatory disorders. Annu. Rep. Med. Chem. 2013, 48, 169–182. [Google Scholar]
  11. Zhang W.; Zhang J.; Fang L.; Zhou L.; Wang S.; Xiang Z.; Li Y.; Wisely B.; Zhang G.; An G.; Wang Y.; Leung S.; Zhong Z. Increasing human Th17 differentiation through activation of orphan nuclear receptor retinoid acid-related orphan receptor γ (RORγ) by a class of aryl amide compounds. Mol. Pharmacol. 2012, 82, 583–590. [DOI] [PubMed] [Google Scholar]
  12. See the Supporting Information for details.
  13. Jin L.; Martynowski D.; Zheng S.; Wada T.; Xie W.; Li Y. Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor RORg. Mol. Endocrinol. 2010, 24, 923–929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pham T. A.; Jain A. N. Parameter estimation for scoring protein ligand interactions using negative training data. J. Med. Chem. 2006, 49, 5856–5868. [DOI] [PubMed] [Google Scholar]
  15. Jain A. N. Virtual screening in lead discovery and optimization. Curr. Opin. Drug Discovery Dev. 2004, 7, 396–403. [PubMed] [Google Scholar]
  16. Jain A. N. Surflex: Fully automatic flexible molecular docking using a molecular similarity-based search engine. J. Med. Chem. 2003, 46, 499–511. [DOI] [PubMed] [Google Scholar]
  17. SYBYL 8.1; Tripos International: St. Louis, MO.
  18. Greenidge P. A.; Kramer C.; Mozziconacci J.-C.; Wolf R. M. MM/GBSA Binding Energy Prediction on the PDB bind Data Set: Successes, Failures, and Directions for Further Improvement. J. Chem. Inf. Model. 2013, 53, 201–209. [DOI] [PubMed] [Google Scholar]
  19. Kollman P. A.; Massova I.; Reyes C.; Kuhn B.; Huo S. H.; Chong L.; Lee M.; Lee T.; Duan Y.; Wang W.; Donini O.; Cieplak P.; Srinivasan J.; Case D. A.; Cheatham T. E. Calculating structures and free energies of complex molecules: Combining molecular mechanics and continuum models. Acc. Chem. Res. 2000, 3312889–897. [DOI] [PubMed] [Google Scholar]
  20. Wang J. M.; Hou T. J.; Xu X. J. Recent advances in free energy calculations with a combination of molecular mechanics and continuum models. Curr. Comput.-Aided Drug Des. 2006, 23287–306. [Google Scholar]
  21. Li J.; Abel R.; Zhu K.; Cao Y.; Zhao S.; Friesner R. A. The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. Proteins 2011, 79, 2794–2812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maestro, ver. 9.2.109; Schrodinger, LLC: New York.
  23. Huang Z.; Xie H.; Wang R.; Sun Z. Retinoid-related orphan receptor gt is a potential therapeutic target for controlling inflammatory autoimmunity. Expert Opin. Ther. Targets 2007, 116737–743. [DOI] [PubMed] [Google Scholar]
  24. Shiau A. K.; Barstad D.; Loria P. M.; Cheng L.; Kushner P. J.; Agard D. A.; Greene G. L. The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen. Cell 1998, 95, 927–937. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml4003875_si_001.pdf (1,007.8KB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES