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Identification of Potent and Selective RORγ Antagonists

, , , , , , and .

Author Information

,a,* ,a ,a ,a ,a ,b and b.

a NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Rockville, MD.
b Skirball Institute of Biomolecular Medicine, New York University, New York, NY.
* To whom correspondence should be addressed: NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Building B, Bethesda, MD 20892-3370. Phone: 301-217-5740. Fax: 301-217-5736. Email: vog.hin.liam@ewgnauh

Received: ; Last Update: February 25, 2013.

Retinoic acid-related orphan receptor RORγt plays a pivotal role in the differentiation of Th17 cells. Antagonizing RORγt transcriptional activity is a potential means to treat Th17-related autoimmune diseases. In this report, we present the identification of a series of diphenylpropanamides as novel and selective RORγt antagonists. ML209 inhibited transcriptional activity of RORγt, but not RORα, in cells. In addition, it suppressed Th17 cell differentiation at submicromolar concentrations.

Assigned Assay Grant #: R03 DA026211

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: Dan Littman, New York University

PubChem Summary Bioassay Identifier (AID): 2604

Probe Structure & Characteristics

CID/ML#Target NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]Fold SelectiveSecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID 4694339/ML209RORγ460[SID 99455330, AID 489037]RORγInactive up to 96 μM [SID 99455330, AID 489039]> 200Th17: 400 nM [SID 99455330, AID 492954]
VP16Inactive up to 96 μM [SID 99455330, AID 489036]> 200

Recommendations for Scientific Use of the Probe

The retinoic acid-related orphan receptor RORγt is required for the differentiation of thymocytes, lymphoid tissue inducer cells, and inflammatory T helper-expressing interleukin 17a (Th17) cells. The identification of small molecules modulating RORγt activity may provide a means to regulate Th17 mediated immune response. While small molecule antagonists of RORγt have been identified, these inhibitors are neither selective nor potent below micromolar concentrations. The diphenylpropanamide probe series presented here selectively inhibits RORγt-mediated activity at submicromolar concentrations in cell assays, including thymocytes. This probe will be a useful pharmacological tool to study cellular activities controlled by RORγt. Furthermore, this diphenylpropanamide series represents a lead scaffold to develop novel therapeutics for Th17-related autoimmune diseases.

1. Introduction

Retinoic acid-related orphan receptor gamma (RORγ) is a member of the nuclear hormone receptor superfamily. A single gene encodes for two isoforms, RORγ1 and RORγt, generated by alternative splicing that differ only in their amino termini1. While RORγ1 is widely expressed in many tissues, including liver, adipose, skeletal muscle and kidney, RORγt is expressed in CD4+ CD8+ thymocytes, lymphoid tissue inducer cells, and Th17 cells, which are T lymphocytes that populate intestinal lamina propria and produce interleukin 17a (IL-17)1. RORγt is necessary and sufficient for the differentiation of Th17 cells2, which comprise one of the distinct effector T cell lineages involved in the regulation of host defense, particularly against extra-cellular microorganisms at mucosal barriers3. Recent studies indicate that Th17 cells play key proinflammatory roles in a variety of autoimmune diseases and in cancer4. For instance, mice lacking IL-17 or the p19 subunit of IL-23, which is required for the expansion and/or function of Th17 cells in vivo, are resistant to experimentally-induced disease states, including autoimmune encephalomyelitis, collagen-induced arthritis, and inflammatory bowel disease57. Th17 cells, and the cytokines they produce, also appear to have essential functions in the pathogenesis of psoriasis8. Treatment of mice with neutralizing anti-IL-17 antibodies ameliorates autoimmune inflammation of the central nervous system, while transfer of cells producing IL-17 exacerbates the disease phenotype6.

Despite the potential of RORγt as a therapeutic target for these diseases, there are only a few known small molecule RORγt antagonists. As shown in Figure 1, all trans-retinoic acid (ATRA) and a synthetic retinoid, ALRT 1550, inhibit both RORγ and RORγ, but not RORγ transcriptional activity9. A recent patent application disclosed a series of benzo[1,4]diazepines, exemplified by LE 540 as a RORγt antagonist, but selectivity was not discussed10. Digoxin was recently reported as a selective RORγt inhibitor with an IC50 value of 1.98 μM11. In addition, a non-selective inhibitor for both RORγ and RORγt, SR1001, has been described12. The limitations of the RORγt inhibitors reported to date are potencies above one micromolar and lack of specificity.

Figure 1. Examples of RORγt antagonists.

Figure 1

Examples of RORγt antagonists.

The goal of this project was to identify a new structural series that selectively inhibits RORγt-mediated activity in cells with potency below one micromolar. Such a series could provide excellent opportunities not only for understanding regulatory mechanisms for RORγt, but also for developing therapeutic intervention for a number of diseases where RORγt activity is implicated.

2. Materials and Methods


All commercially available reagents and solvents were purchased and used without further purification. HPLC purification was performed using a Waters semi-preparative HPLC equipped with a Phenomenex Luna® C18 reverse phase (5 micron, 30 × 75 mm) column having a flow rate of 45 mL/min. The mobile phase was a mixture of acetonitrile and H2O, each containing 0.1% trifluoroacetic acid. During purification, a gradient of 30% to 80% acetonitrile over 8 minutes was used with fraction collection triggered by UV detection (220 nM). 1H spectra were recorded using an Inova 400 MHz spectrometer (Varian). LCMS was used to analyze samples’ purity. Method: Agilent 1200 series LC/MS equipped with a Zorbax™ Eclipse XDB-C18 reverse phase (5 micron, 4.6 × 150 mm) column having a flow rate of 1.1 mL/min. The mobile phase was a mixture of acetonitrile and H2O, each containing 0.05% trifluoroacetic acid. A gradient of 5% to 100% acetonitrile over 8 minutes was used during analytical analysis.

2.1. Assays

See Appendix 1 for a table of assay details and corresponding AIDs.

For screening and initial follow up, a cell-based reporter assay was used to detect RORγt-mediated activity. This assay, called RORγt, employed Drosophila Schneider cells that were stably transfected with two vectors: (1) a gene expressing a fusion of the Gal4 DNA binding domain and RORγt transactivation domain under the control of the metallothionine promoter; (2) a Photinus luciferase reporter regulated by the Gal4 binding site enhancer, UAS. The addition of copper to the medium induced expression of the Gal4-RORγt fusion, which subsequently induced expression of the UAS-luciferase reporter. Small molecule inhibitors of RORγt activity were detected by a decrease in luciferase reporter activity.

To identify non-specific and non-selective inhibitors, three other engineered Drosophila Schneider cell lines were used; these differed only in the Gal4-transactivation domain fusion. The first assay, called VP16, fused the Gal4 DNA binding domain to the transactivation domain of VP16, a virus-encoded protein that induces gene transcription and is unrelated to ROR family members. The VP16 assay was used as a counterscreen for RORγt to identify nonspecific inhibitors that target components common to both assays, such as the Gal4 DNA binding domain or the UAS luciferase reporter. The other two assays, termed RORα and DHR3, fused the Gal4 DNA binding domain to the transactivation domain of RORα or DHR3 (Drosophila hormone receptor 3). These assays were used in follow up studies to confirm the selectivity of RORγt inhibitors. The itemized protocol used for all four assays is shown in Table 1.

Table 1. Itemized protocol for RORγ, VP16 and RORγ reporter assays used in the qHTS and follow up studies.

Table 1

Itemized protocol for RORγ, VP16 and RORγ reporter assays used in the qHTS and follow up studies.

The activity and selectivity of compounds were tested in mouse T lymphocyte differentiation assays, which provide a more appropriate model to assess endogenous RORγt function. RORγt activity is required for T lymphocytes to differentiate into the Th17 lineage. In the Th17 assay, Th17 lymphocyte differentiation was tracked by the induction of a GFP reporter under the control of the endogenous IL-17a promoter using flow cytometry. Compounds that block RORγt activity prevent expression of the IL17a-GFP reporter. To confirm the selectivity of RORγt inhibitors, compounds were tested in a mouse Th1 lymphocyte differentiation assay. T lymphocytes differentiate into Th1 cells in the presence of IL-12 and IL-2. Th1 differentiation does not require RORγt, and thus selective RORγt inhibitors should not block this process. In this assay, Th1 differentiation was tracked by the induction of interferon gamma (IFNγ) expression as measured by an anti-IFNγ antibody using flow cytometry. Selective RORγt inhibitors do not block IFNγ expression in this assay.

In both Th17 and Th1 assays, T cells were derived from the lymph nodes and spleens of 6–8 week old IL17a-GFP mice (Biocytogen LLC). B220− cells were isolated on an autoMACS Pro with bead depletion of B220+ cells (Miltenyi). Naive CD4+ T cell were further purified as TCRb+CD8− DAPI− CD19− CD4+CD25− CD62L+CD44low/Int by cell sorting on a FACSAria (BD). Cells were cultivated in an incubator at 37 °C and 5% CO2 in RPMI 1640 medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated FBS (Hyclone), penicillin-streptomycin, 2 mM glutamine and 0.1 mM non-essential amino acids. Cells were seeded on day 0 at a density of 0.4 ×105 cells per ml in 96-well plates coated with anti-CD3e (5 μg/mL) and anti-CD28 (10 μg/mL). Cells were cultured for 4–5 days with cytokines to induce the differentiation of either Th17 (0.3 ng/mL TGFβ, 20 ng/mL IL-6, anti-IFNγ, and anti-IL-4) or Th1 (10 ng/mL IL-12, 100 U/ml IL-2, and anti-IL-4) lineages. On day 1, compounds dissolved in DMSO were added. On day 4 or 5, cells were stained with DAPI and PECy7-conjugated CD4 (BD Biosciences) by incubating on ice for 15 min, and Th17 differentiation was measured by GFP expression from the IL-17a locus. To detect Th1 differentiation, cells were incubated for 5 hrs with phorbol ester (50 ng/mL; Sigma), ionomycin (500 ng/mL; Sigma) and GolgiStop (BD). Then, cells were fixed, permeabilized for 30 min on ice, and stained for 30 min on ice in permeabilization buffer with Alexa647-conjugated anti-IL17 (eBioscience) and PE-conjugated anti-IFNγ (eBioscience). An LSR II (BD Biosciences) and FlowJo software (Tree Star) were used for flow cytometry.

2.2. Probe Chemical Characterization

ML209 was prepared using the reaction in Scheme 1:

Scheme 1. Synthesis of probe molecule.

Scheme 1

Synthesis of probe molecule. (i) TFA, 80 °C 6 h; (ii) Hunig base, 3,5-cis-dimethylpiperidine, 60 °C DMA, 16 h; (iii) chiral separation by HPLC

(−) 3-(Benzo[d][1,3]dioxol-5-yl)-1-cis-3,5-dimethylpiperidin-1-yl)-3-(2-hydroxy-4,6-dimethoxy phenyl)propan-1-one (5k(−)): 1H NMR (400 MHz, DMSO-d6, 60 °C) δ ppm 0.65–0.74 (m, 1H), 0.81 (d, 6H, J = 6.3 Hz), 1.20–1.38 (m, 2H), 1.64–1.74 (m, 1H), 1.80–1.94 (m, 1H), 2.34–2.50 (m, 1H), 2.92–3.20 (m, 2H), 3.67 (s, 3H), 3.69 (s, 3H), 3.72–3.90 (m, 1H), 4.28–4.38 (m, 1H), 4.88 (t, 1H, J = 7.2 Hz), 5.88–5.89 (m, 2H), 6.02–6.04 (m. 2H), 6.67–6.73 (m, 2H), 6.82 (s, 1H), 9.21 (s, 1H); LC/MS: Retention time t = 4.39 min; Purity: UV220 > 95%, UV254 > 95%; (Column: IA analytical, 0.46 cm × 25 cm; Run time: 15 min; Flow rate: 1 mL/min; Mobile phase; 60/40 EtOH/Hexane; Detectors: DAD (220 and 254 nm) and PDR chiral detector); [α]D23 = −129.1 (c 1.0, CHCl3); HRMS (ESI): m/z calcd for C25H32NO6+ 442.2224, found 442.2221; Solubility: (PBS, pH 7.4, 23 °C) = 23 μM.

MLS numbers for probe and analogs:

NCGC00238427-02MLS003221401SID 99455330CID 46943339ML209Analog
NCGC00188324-02MLS003221402SID 99455236CID 46943400Analog
NCGC00188327-02MLS003221403SID 99455239CID 16746402Analog
NCGC00189187-01MLS003221404SID 99455271CID 16746329Analog
NCGC00189189-01MLS003221405SID 99455273CID 46943317Analog
NCGC00238447-01MLS003221406SID 99455346CID 46943351Analog

2.3. Probe Preparation

The probe molecule and analogs were prepared from phenols, cinnamic acids, and amines. As shown in Scheme 2, substituted phenols 1 reacted with appropriate cinnamic acids 2 in TFA to give lactones 3, which converted to the desired amides 5 by reacting with appropriate amines 4.

Scheme 2. Synthesis of probe molecule and analogs.

Scheme 2

Synthesis of probe molecule and analogs. (i) TFA, 60–100 °C 3–16 h; (ii) method A; R3R4NH (4), 60–80 °C DMA or THF; method B: R3R4NH (4), Me3Al.

4-(Benzo[d][1,3]dioxol-5-yl)-5,7-dimethoxychroman-2-one (3a).

4-(Benzo[d][1,3]dioxol-5-yl)-5,7-dimethoxychroman-2-one (3a)

A mixture of 3,5-dimethylphenol (1a, 10.0 mmol, 1.54 g) and 3,4-(methylenedioxy)cinnamic acid (2a, 10.0 mmol, 1.92 g) in TFA (30 mL) was heated at 70 °C for 3 hrs. After removing TFA, the residue was purified by silica-gel column chromatography using hexane/ethyl acetate (7–60%) to give 3a (2.13 g, 65%) as a white powder. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.83 (dd, J=15.8, 1.6 Hz, 1 H), 3.20 (dd, J=15.9, 6.9 Hz, 1 H), 3.74 (s, 3 H), 3.79 (s, 3 H), 4.43 (d, J=5.9 Hz, 1 H), 5.96 (s, 2 H), 6.33 – 6.50 (m, 3 H), 6.66 (d, J=1.6 Hz, 1 H), 6.79 (d, J=8.0 Hz, 1 H); LC/MS: Retention time t = 5.78 min; Purity: UV220 > 98%, UV254 > 98%.

3-(Benzo[d][1,3]dioxol-5-yl)-1-cis-3,5-dimethylpiperidin-1-yl)-3-(2-hydroxy-4,6-dimethoxyphenyl)propan-1-one (5k):

3-(Benzo[d][1,3]dioxol-5-yl)-1-cis-3,5-dimethylpiperidin-1-yl)-3-(2-hydroxy-4,6-dimethoxyphenyl)propan-1-one (5k):

3,5-dimethylpiperidine monotartrate salt (2.01 g, 7.62 mmol, 2.0 equiv) and Hunig base (2.46 g, 19.0 mmol, 5.0 equiv) were added to a solution of 3a (1.251 g, 3.81 mmol, 1.0 equiv) in 10.0 mL of DMA. The reaction mixture was heated at 60 °C overnight. The solvent was removed and the residue was dissolved in 50 mL of DCM. The solution was washed with sat. NaHCO3 (30 mL ×3). The organic layer was dried over MgSO4, filtered and concentrated. The crude product was purified by silica gel column chromatography eluting with 7–60% hexanes/ethyl acetate to afford amide 5k (869 mg, 52%) as a solid. Chiral separation of 5k by HPLC (Column: IA Preparatory 5 cm × 50 cm; Run Time: 40 minutes; Flow Rate: 35 mL/min; Mobile Phase: 60/40 EtOH/Hexane; Detectors: DAD (220 and 254 nm)) gave 5k() and 5k(+). Stability profile over 48 h (PBS, pH 7.4, 23°C) is shown in Figure 2.

Figure 2. Stability of ML209 in PBS buffer (pH 7.4, 23°C) plotted as LC peak area vs time for a 48 hr period.

Figure 2

Stability of ML209 in PBS buffer (pH 7.4, 23°C) plotted as LC peak area vs time for a 48 hr period.

3. Results

3.1. Summary of Screening Results

The compound library was tested using quantitative high throughput screening (qHTS), a method where each compound is tested at multiple concentrations to generate a titration-response curve for each sample13. Approximately 310,000 samples were assayed at six concentrations in both the RORγt (AID 2551) and VP16 (AID 2546) assays. The screening data were normalized to control wells treated with DMSO only, or control inhibitor compound, to establish 100% and 0% activity, respectively. For control inhibitors, the RORγt assay used C16, a specific RORγt antagonist identified in a previous small molecule screen (Jun Huh and Dan Littman, personal communication), while the VP16 assay used AG-879, a tyrosine kinase inhibitor14,15 that blocks the gene reporter activities in both RORγt and VP16 cells. The RORγt qHTS totaled 1531 plates, of which 98 % passed quality control; these showed a 0.55 ± 0.17 Z′ score and 9 ± 7 signal to background ratio. The VP16 qHTS totaled 1437 plates, of which 98 % passed quality control; these showed a 0.78 ± 0.06 Z′ score and 27 ± 6 signal to background ratio.

Concentration response curves (CRCs) were fit and classified as described13. Briefly, CRCs are placed into four classes. Class 1 contains complete CRCs showing both upper and lower asymptotes and r2 values > 0.9. Class 2 contains incomplete CRCs having only one asymptote and shows r2 values > 0.9. Class 3 curves are of the lowest confidence because they are poorly fit or based on activity at a single concentration point. Class 1 and 2 curves are divided further into subclasses to indicate efficacies 80% or greater (Class 1.1 and 2.1) or between 30% and 80% (Class 1.2 and 2.2). Class 4 compounds are inactives having either no curve-fit or an efficacy below threshold activity (3 SD of the mean activity). While both activators and inhibitors were recovered from the qHTS, this report focuses on the identification and characterization of antagonists. The RORγt qHTS resulted in 14,585 inhibitors with curve fits of good quality (Class 1.1, 1.2 and 2.1) and 19,724 of lower quality (Class 2.2 and 3; Table 2). The VP16 qHTS yielded 9,825 inhibitors with curve fits of good quality and 23,572 of lower quality (Table 2).

Table 2. Activity profile of the ROR qHTS.

Table 2

Activity profile of the ROR qHTS.

To derive nascent structure-activity relationships of RORγt-specific actives from the screen, the following process was implemented: RORγt actives were defined as those samples displaying Class 1 or 2 curves with at least 60% efficacy; these actives were deemed RORγt-specific if their corresponding activity in the VP16 assay was inactive (Class 4 curve), or if active, had at least a 10-fold lower IC50 value. This process identified 1,256 RORγt-specific actives whose structures were then clustered using a custom fragment-based program to yield 2,442 structural series and 173 singletons. Because an active could be part of more than one series, the number of series was larger than the number of actives clustered. After clustering, structurally related compounds with inconclusive or no activity were added to each series. Each series contained at least three compounds, of which at least one was active.

To prioritize compounds for follow up studies, the series were examined for selectivity and enrichment of RORγt actives. Specifically, a series was considered selective for RORγt over VP16 if at least 50% of the actives in the series were selective for RORγt, and the fraction of RORγt selective actives was significantly larger than that of the VP16 actives (p < 0.2). RORγt selective series that were significantly enriched with RORγt actives (p < 0.2) and had a promiscuity score less than 0.5 were prioritized for confirmation studies. The promiscuity score is a measure of a compound’s activity in other assays (100+) and was calculated using the formula (2Nactive+Ninconclusive)/Ntested, where Nactive denotes the number of assays where a compound was active, Ninconclusive denotes the number of assays where a compound showed inconclusive activity and Ntested denotes the total number of assays in which a compound was tested. Confirmation studies of prioritized series were performed using primary assays RORγt (AID 2762, 489037) and VP16 (AID 2763), selectivity assays, VP16 (AID 489036), RORα (AID 489039), DHR3 (AID 489038) and Th1 (AID 492962) and secondary assay Th17 (AID 492954). These tests identified the diphenylpropanamide series as the most promising series for further optimization.

3.2. Dose Response Curves for Probe

Figure 3. Activity ML209 in the RORγt in Drosophila cell assay.

Figure 3Activity ML209 in the RORγt in Drosophila cell assay

3.3. Scaffold/Moiety Chemical Liabilities

ML209 contains a phenol moiety, which may be metabolized in vivo.

3.4. SAR Tables

Table 3Structure-activity relationship study of the diphenylpropanamide series

Image ml209u5.jpg
EntryCmpd. No.CIDSIDNCGC IDRaRbRcIC50 (μM) (ROR γ)IC50 (μM) (Th17)IC50 (μM) (ROR α)Solubility (μM)
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u9.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u10.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u11.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u12.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u13.jpg
Image ml209u7.jpg
Image ml209u8.jpg
16.3±1.7n.d.> 4020.6
Image ml209u14.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u15.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u16.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u17.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u18.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u18.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u18.jpg
Image ml209u7.jpg
Image ml209u8.jpg
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u19.jpg
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u20.jpg
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u21.jpg
3.7±1.20.8> 4027.4
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u22.jpg
Image ml209u6.jpg
Image ml209u7.jpg
Image ml209u23.jpg
Image ml209u6.jpg
Image ml209u8.jpg
Image ml209u6.jpg
Image ml209u24.jpg
Image ml209u8.jpg
Image ml209u6.jpg
Image ml209u25.jpg
Image ml209u8.jpg
Image ml209u6.jpg
Image ml209u26.jpg
Image ml209u8.jpg
Image ml209u6.jpg
Image ml209u27.jpg
Image ml209u8.jpg
Image ml209u18.jpg
Image ml209u27.jpg
Image ml209u8.jpg
0.73±0.090.4> 4044.7

All analogs were synthesized. n.d.= Not determined; in. = inconclusive; Solubility data were measured in PBS, pH 7.4 at 23 °C.

3.5. Cellular Activity

CID16746402 (5a), the compound identified from the screen, showed no cytotoxicity at concentrations up to 96 μM.

3.6. Profiling Assays

Profiling of the probe in a panel of 21 nuclear receptors for antagonistic activity showed that the probe had weak activities against 4 receptors, which are ERRα (IC50 = 14 μM), LXRα (IC50 = 10 μM), TRα (IC50 = 4.5 μM) and TRβ (IC50 = 13 μM), as shown in Table 4.

Table 4. Profiling results for ML209 in a panel of 21 nuclear receptors for antagonistic activity.

Table 4

Profiling results for ML209 in a panel of 21 nuclear receptors for antagonistic activity.

4. Discussion

CID16746402 (5a) was identified from the primary screen. It had an IC50 value of 5 μM and displayed good selectivity for RORγt versus VP16 and RORγ. More importantly, this compound inhibited Th17 cell differentiation and had no effect on Th1 cell differentiation. Thus, CID16746402 was selected as a starting point for a hit-to-probe optimization effort. As shown in Figure 4, three areas of the hit compound were explored for structure-activity relationship (SAR) study. All analogs were evaluated in the RORγt assay (AID 489037) and the SAR results are summarized as follows: (1) In area A, cis-3,5-dimethylpiperidine and cis-3,5-dimethylmorpholine are the best groups among the tested amines (Table 3, entries 1–11); (2) In area B, while electron denoting groups at the 4 position are preferable, methoxy group at the 2 position deteriorates the activity (Table 3, entries 1, 20–24); (3) In area C, the 6-methoxy group is important for the activity (Table 3, entries 1, 14–15) and the 2-hydroxy group can be replaced by a methoxy group without loss of activity (5avs5n). Chiral separation of the stereoisomers of 5k gave 5k(−) and 5k(+). Compound 5k(−), the stereoisomer with negative optical rotation, was >20-fold more active than the stereoisomer with positive optical rotation (5k(+)), suggesting that the chiral center affects the activity. Compound 5k(−) was not active against RORγ, VP16, and DHR3 at concentrations up to 96 μM, indicating it was a highly selective RORγ transcriptional inhibitor. Since RORγt is a key regulator for Th17 cell polarization, selected analogs were further evaluated for antagonistic activity in a Th17 differentiation assay (AID492954). In general, the activities observed in the Th17 differentiation assay are in good agreement with the activities in the RORγt assay. Most importantly, the probe compound, compound 5k(−), inhibited Th17 cell differentiation with an IC50 value of 400 nM (Table 3, entry 12) and it did not inhibit Th1 cell differentiation at 3 μM.

Figure 4. SAR summary.

Figure 4

SAR summary.

4.1. Comparison to existing art and how the new probe is an improvement

To the best of our knowledge, the probe molecule is the most potent and selective RORγt transcriptional antagonist. In addition, the probe can be easily prepared from commercially available reagents.

4.2. Mechanism of Action Studies

We have shown that the probe molecule selectively inhibited the RORγt transcriptional activity and had no effect on the transcriptional activity of VP16, DHR3 or the close related nuclear hormone receptor RORγ. We are planning to test the probe compound in a RORγt binding assay to examine whether the probe molecule targets RORγt directly.

4.3. Planned Future Studies

We will test the probe molecule in a RORγ t binding assay to determine whether the probe binds to the receptor. In addition, we will perform both in vitro and in vivo PK studies of the probe molecule. Finally, we are planning to test the probe compound in related animal disease models, including EAE, CIA and IBD.

5. References

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Reiner SL. Development in motion: helper T cells at work. Cell. 2007;129(1):33–6. [PubMed: 17418783]
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Appendix 1. Listing of AIDs and assay details

PubChem AIDTypeTargetConc. RangeSamples Tested
2551Primary qHTSRORγt46 μM to 3 nM305467
2546Primary qHTSVP1646 μM to 3 nM304088
2762ConfirmatoryRORγt46 μM to 0.26 nM250
2763ConfirmatoryVP1646 μM to 0.26 nM250
489037SecondaryRORγt92 μM to 0.52 nM185
492954OrthogonalTh1720 μM to 0.1 μM37
489039Anti-targetRORα92 μM to 0.52 nM185
489036Anti-targetVP1692 μM to 0.52 nM185
489038Anti-targetDHR392 μM to 0.52 nM185
492962Anti-targetTh130 μM to 3 μM3


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