Retinoic Acid Receptor-Related Orphan Receptor Î³t (RORÎ³t) Agonists

Perspective Cite This: J. Med. Chem. 2018, 61, 5794−5804

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Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Agonists as Potential Small Molecule Therapeutics for Cancer Immunotherapy Miniperspective Ruomeng Qiu and Yonghui Wang*

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Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, 201203 Shanghai, China ABSTRACT: The recent success of PD-1/PD-L1 antibodies for advanced cancer treatment has led to the conclusion that activating the immune system can be employed to fight cancer. These results also encourage the development of small molecule immunomodulators for cancer immunotherapy. RORγt is a key transcription factor mediating Th17 cell differentiation and IL-17 production, which is able to activate CD8+ T cells and elicit antitumor efficacy. Since RORγt agonists have been shown to increase basal activity of RORγt and promote Th17 cell differentiation, development of RORγt agonists could provide a unique approach to cancer immunotherapy. In this review, we summarize RORγt sterol and synthetic agonists, analyze the common ground of their mode of actions, and discuss the potential role of RORγt agonists as small molecule therapeutics for cancer immunotherapy. autoimmune diseases.20,21 Recent studies show that Th17 cells and IL-17 expression are broadly distributed in human tumors.22−25 It has been demonstrated that Th17 cells are actively involved in tumor immunology processes and have potential antitumor efficacy by eliciting remarkable activation of CD8+ T cells, which makes Th17 cells a potential source of targets for cancer immunotherapy.26,27 Retinoic acid receptor-related orphan receptor γt (RORγt) belongs to the nuclear receptor (NR) superfamily, which has provided a rich source of targets appropriate for small molecule therapeutic development of various human diseases.28 RORγt is specifically expressed by Th17 cells and is the key transcription factor to drive naive CD4+ T cells differentiated into Th17 cells.29 Since RORγt agonists have been shown to increase basal activity of RORγt and promote Th17 cell differentiation, development of RORγt agonists could provide a unique approach to cancer immunotherapy.30 Although there have been quite a few research articles in recent years on RORγt modulators, there are no reviews focusing exclusively on RORγt agonists. This review covers the reported RORγt sterol and synthetic agonists, their mode of actions (MOAs), and their potential role in cancer immunotherapy. Discovery progress from the first natural RORγ ligands to the most recently identified Th17 cell endogenous agonist of the orphan receptor RORγ has been summarized. Furthermore, we have elaborated on the published synthetic small molecule RORγt agonists, their structural insights, and the common ground of their MOAs. Finally, we

1. INTRODUCTION Cancer immunotherapy has generated inspiring clinical success recently after decades of research effort.1−3 Harnessing the human body’s own immune system, cancer immunotherapy has been widely recognized as a revolutionary approach to fight cancer, either as a standalone treatment or combined with surgery, radiotherapy, chemotherapy, and other established targeted therapy.4−7 In spite of contributions by other immune cell subtypes, CD8+ T cells have arisen as the prominent component in most antitumor immunity settings.8 However, one of the challenges that remain is tumor immune escape, as the activity of CD8+ T cells is down regulated in the tumor microenvironment.9,10 Multiple immunotherapeutic strategies including cytokines, vaccine, adoptive cell transfer, and checkpoint blockades are aimed at reactivating, improving, and maintaining responses by antitumor CD8+ T-cells.11−14 The recently approved antibody drugs, nivolumab and pembrolizumab as PD-1 inhibitors and atezolizumab, avelumab, and durvalumab as PD-L1 inhibitors,15 have demonstrated significant efficacy and are expected to be blockbusters in the future.16 Yet considering that small molecule therapeutics offer unique complementary advantages and are potentially synergistic with biologics, it is of great interest to develop alternative pathways available for small molecule intervention to boost the antitumor activity of CD8+ T cells.17,18 T helper 17 (Th17) cells, one of T-cell subsets differentiated from naive CD4+ T cells, are characterized by unique transcription factors and IL-17 production.19 Previously, Th17 cells have been well described as an essential inflammatory effector and shown to promote inflammation in various © 2018 American Chemical Society

Received: September 3, 2017 Published: February 7, 2018 5794

DOI: 10.1021/acs.jmedchem.7b01314 J. Med. Chem. 2018, 61, 5794−5804

Journal of Medicinal Chemistry

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discuss the potential of small molecule RORγt agonists as novel therapeutics for cancer immunotherapy. 1.1. NR Superfamily and MOA. The NR superfamily consists of 48 transcription factors in human, including the receptors for steroid hormones, lipophilic vitamins, thyroid hormone, and cholesterol metabolites.31 Approximately half of NRs are defined as orphan receptors since their ligands have not been identified. The NRs regulate a wide spectrum of physiological processes, and fairly all the NRs with identified ligands are developable targets for drug discovery.32 NR modulators have proven to be a rich source of pharmaceuticals, and in 2009 it was estimated that NR drugs accounted for approximately 13% of all pharmaceutical sales in the U.S. and an appreciable share of global sales.33 Nuclear receptors share a highly conserved domain that is divided into six subregions (regions A−F) based on sequence similarity. The structure consists of AF1 (activation function 1) region (A/B region), followed by zinc-finger DBD (DNA binding domain) (C region), hinge domain (D region), LBD (ligand binding domain) containing the AF2 (activation function 2) region (E region). Some receptors contain a region from LBD to the C-terminal, making up the F region. The ability of NR LBDs to trigger the transcriptional network is modulated by helix 12 at the C-terminal.34 Helix 12 is a crucial component of the NR LBDs because its ligand-induced reorientation contributes to the surfaces in a critical manner, recruiting or expelling coactivators/corepressors and regulating the level of transcriptional activity. Several cocrystal structures of NR LBDs have revealed that the interactions of ligands with helix 12 significantly determine AF2 stability. Besides, helix 12 has intermediary positions (rather than simply being active or inactive), suggesting that ligands can be designed to have different degrees of agonism/inverse agonism. Therefore, chemistry can generate not only receptor-specific ligands but also full or partial agonists/inverse agonists, leading to NRbased drug development. 1.2. RORγt. The retinoic acid receptor-related orphan receptor γ (RORγ or NR1F3) is a key NR of the ROR family, identified after RORα (RORa or NR1F1) and RORβ (RORb or NR1F2).35 RORα is most widely expressed in multiple organs, such as the liver, kidney, thymus, and brain.36,37 RORβ expression is restricted to the central nervous system.38 RORγ exists in two distinct isoforms, RORγ (RORγ1) and RORγt (RORγ2), and share the identical sequence through DBD to the LBD at the C terminus and only differ in their N-terminal sequences by only 24 amino acids.39,40 The mRNA for RORγ is widely observed in many tissues, but RORγt isoform is mainly expressed in the immune system, especially in thymus.41 RORγt is a key transcription factor for Th17 cell differentiation and IL-17 production.29 Other transcription factors, including STAT3, BATF, IRF4, RUNX1, and IκBζ, also play an important role in pharmacological processes of Th17 cell differentiation. In terms of small molecule drug discovery, RORγt contains a typical-NR ligand binding domain (LBD) that binds and interacts with small molecule ligands, making it an attractive therapeutic target.42

In 2010, Jin et al. reported four RORγ sterol agonists (Figure 1).43 These were the first discovered RORγ natural ligands that

Figure 1. First reported RORγ natural ligands as RORγ agonists.

led to the first determined RORγ structure. To explore cholesterol and its hydroxycholesterol derivatives’ role in RORγ, the interaction of RORγ with coactivator SRC1 (SRC1-2) either with or without cholesterol and hydroxycholesterols (HCs) was monitored in an AlphaScreen biochemical assay. RORα showed a high basal interaction with the SRC1-2 coactivator as a proof of concept. Ligands 1 (cholesterol), 2 (20α-hydroxycholesterol), 3 (22R-hydroxycholesterol), and 4 (25-hydroxycholesterol) also strongly enhanced the interaction of RORγ with coactivators instead of corepressors. Besides, the EC50 of the tested hydroxycholesterols were around 0.02−0.04 μM against RORγ, compared with the EC50 of 0.02 μM for cholesterol, which was previously identified as a natural ligand of RORα. These results suggested that endogenous hydroxycholesterols played an important role in modulating RORγdependent biological processes. Additionally, the crystal structures of 2, 3, and 4 with RORγ LBD were resolved (Figure 2). The cocrystal complex revealed that RORγ LBD contained 12 α-helices and two short β-strands that were folded into a typical three-layer helix sandwich. The coactivator (SRC2-2) adopted a two α-helix that directed hydrophobic side chains toward RORγ coactivator binding surface. The C-terminal AF2 was positioned in the active conformation, consistent with the agonist activity results of AlphaScreen assays. These structures revealed the binding modes of hydroxycholesterols with RORγ LBD and provided the typical coactivator-recruiting conformation of RORγ. In 2014, Soroosh et al. discovered a series of oxysterols as RORγt agonists (Figure 3).44 In a RORγ-LBD Gal4 reporter assay, several oxysterols showed significant agonism activity, with 5 (27-hydroxycholesterol), 7 (7β, 27-hydroxycholesterol), and 8 (7-keto, 27-hydroxycholesterol) having the best potency and efficacy. The direct binding of oxysterols with RORγ LBD was identified by a thermal shift assay and a 3H-labeled 25hydroxycholesterol binding assay, and promotion of coactivator recruitment and reduction of corepressor binding were observed. In murine and human primary cells, 7 and 6 (7α, 27-HC) promoted Th17 cell differentiation in a RORγtdependent manner. More importantly, these two oxysterols, 6 and 7, were found to be specifically produced by Th17 cells. In vivo administration of 7 observed enhanced IL-17 production in mice. In CYP27A1-deficient mice, which lacked an essential enzyme for generation of these oxysterols, a significant

2. RORγT AGONISTS AND THEIR MODE OF ACTIONS 2.1. Sterol RORγt Agonists. During exploration of RORγ endogenous ligands and their role in Th17 cell differentiation, a myriad of sterols as RORγ natural ligands have been discovered, most of which serve as RORγ agonists.43−46 5795



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Figure 2. (A) 4 with RORγ LBD. (B) overlay of 2 (gray), 3 (green), and 4 (blue). PDB codes are the following: 3KYT (RORγ/2), 3L0J (RORγ/3), and 3L0L (RORγ/4). Blue dashes represent hydrogen bond interactions.

were tested in a RORγ reporter assay in insect cells, and 46 of them were bioactive. Combined with overexpression, RNA interference, and genetic deletion of metabolic enzymes to investigate the RORγ-dependent transcriptional network, it was proven that the RORγ ligands could be cholesterol biosynthetic intermediates (CBIs) as lanosterol downstream and zymosterol upstream. Lipid molecules bound to RORγ were analyzed by LC/MS/MS, and they had consistent molecular weights with CBIs. In addition, CBIs stabilized the RORγ LBD and promoted coactivator recruitment, which was consistent with a previous study. This study suggested that possible endogenous ligands of RORγ were methylated CBIs or related metabolites, which is a major step toward the identification of RORγ endogenous ligands. Furthermore, the cocrystal complex of 9 (4α-carboxy-4βmethyl-zymosterol, 4ACD8) with RORγ was obtained (PDB code 4S14) (Figure 4). In the RORγ-9 cocrystal structure, helix 12 showed an agonism-like conformation, packed tightly to the LBD surface, and directly interacted with the coactivator. The cocrystal structure displayed a well-organized arrangement of 4ACD8 (9) in the RORγ LBD with the carboxy group of 9 interacting with RORγ residues (Q286, L287). These interactions ensured the universal binding affinity of CBIs to RORγ, similar to those of previously studied RORγ agonists. In 2015, Hu et al. confirmed that 11, a sterol produced after CYP51 demethylation, is an endogenous RORγ agonist (Figure 5).46 To identify whether precursors of cholesterol bind to RORγ and induce coactivator recruitment, a set of sterols was tested in RORγ coactivator recruitment profiling with ursolic

Figure 3. Oxysterol RORγt agonists.

reduction of IL-17 producing cells was observed, and it was phenotypically similar to RORγt knockout mice. These results suggested that naturally generated oxysterols acted as RORγ agonists, and the author speculates that oxysterols may potentially serve as RORγt endogenous ligands to drive IL-17 dependent immune responses. In 2015, Santori et al. showed that sterols were essential and sufficient to trigger the RORγ-dependent transcriptional network and that the RORγt ligand is a cholesterol biosynthetic intermediate in mammalian cells.45 To map the RORγ ligand biosynthetic pathway, 78 cholesterol biosynthetic intermediates

Figure 4. (A) Chemical structure of 4ACD8 (9); (B) RORγt-9 cocrystal structure; (C) key interactions of RORγ LBD with 9 (blue dashes represent hydrogen bond interactions). 5796



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possibility of developing synthetic small molecules to suppress or further activate ROR receptors. In 2012, GSK accidentally discovered a class of aryl amides as RORγ agonists while screening for RORγ small molecule inhibitors (Figure 7).48 The representative three aryl amide compounds (14−16) displayed their activities with an EC50 of ∼0.1 μM in IL-17 reporter assay. The three agonists also elevated thermal stability by ∼3 °C, indicating that these compounds directly bind to RORγ. Compound 15 promoted IL-17 production in a mouse Th17 cell differentiation assay with the maximum response of 220% at 3 μM, which further confirmed that RORγ activation and downstream biological effects were induced by these compounds. In 2014, Wang and colleagues at GSK discovered a novel series of tertiary amines ranging from RORγt agonist to inverse agonists (Figure 8).49 SAR explorations starting from inverse agonist 17 yielded a tertiary amine 18 as a potent RORγt agonist with an EC50 of 0.02 μM in a RORγ dual FRET assay. A cocrystal structure of 18 with the RORγ LBD was obtained (PDB code 4NIE), and the overall binding mode was essentially the same as previously predicted (Figure 9). Two hydrogen bond interactions of the sulfonyl group with Arg367 and the linker amide with a backbone carbonyl residue were observed. Furthermore, the sulfonylphenyl ring and the middle phenyl ring formed π−π stacking interactions with Phe377 and Phe378, respectively. Notably, the left-hand side (LHS) benzyl group occupied the hydrophobic pocket around Trp317 and Tyr502, the key site for stabilizing helix 12. Initiated from agonist 18, a series of tertiary amines with different sizes of 4-substituted moieties on LHS phenyl ring were designed, synthesized, and biologically evaluated in both a RORγ dual FRET assay and FRET assay. As the size of the substitution increased (compounds 18−20), maximum activation of the compounds decreased as indicated in a RORγ dual FRET assay. When the size of the LHS substituents was further enlarged, compound 21 did not affect the basal activity of RORγt and could be regarded as a neutral antagonist. Futhermore, compounds 22−24 could be considered inverse agonists according to the increased level of inhibition indicated in the RORγ FRET assay. The activity results together with the cocrystal structure revealed a typical RORγ mode of action. The strong association between substituent bulkiness and the inhibition level could be interpreted as differing sizes of LHS moieties push away helix 12 to different degrees, thus influencing the ability of coactivator and corepressor recruitment at different levels. Notably, in contrast to the stepwise inhibition variation, the potency was barely influenced by substituent size. Further evaluation of 23 was performed by peptide recruitment in a RORγ dual FRET assay. Consequently, neither coactivator nor corepressor was recruited by RORγ LBD. This was the first

Figure 5. Structures of 10 (zymosterol) and 11 (desmosterol).

acid added. Among these sterols, 10 and 11, two late-stage cholesterol precursors significantly promoted coactivator recruitment with EC50 values of 0.11 μM and 0.08 μM, respectively. In addition, 10 and 11 boosted IL-17 production in Th17 cell differentiation. Levels of selected sterols in Th17 cells were measured by LC/MS/MS. Only cholesterol and desmosterol were detectable among the tested sterols. Since cholesterol was unable to be generated by enzymes expressed in Th17 cells, it can be confirmed that desmosterol is an endogenous ligand in Th17 cells. 2.2. Synthetic RORγt Agonists. In 2010, Scripps Florida disclosed compound 13, the first published synthetic small molecule as a RORγ agonist (Figure 6).47 13 was discovered

Figure 6. Structures of 12 (T0901317) and 13 (SR1078).

from a series of compounds derived from 12, the previously identified inverse agonist of RORα/RORγ. By slightly changing from N-phenylbenzenesulfonamide to N-phenylbenzamide, 12 and 13 displayed opposite activity and different degrees of selectivity. 13 functioned as a RORα/γ agonist, while 12 functioned as a RORα/γ inverse agonist; in addition, 12 has promiscuous activity by modulating RORα, RORγ, LXR, and FXR, while 13 selectively modulated RORα/γ receptors, which indicated a high potential of 13 to be a chemical probe for RORα/γ function. In the AlphaScreen assay, 13 functioned as a ROR agonist on full-length RORα/γ. The agonism of 13 was further confirmed in HepG2 cells at 10 μM. The pharmacodynamics (PD) profile of 13 was also evaluated by ip administration at 10 mg/kg in mice, and increasing G6 Pase and FGF21 mRNA levels were detected in harvested mice livers, which was consistent with the fact that 13 functioned as a RORα/γ agonist. It is worth mentioning that 13 activates the receptor beyond the normal level, along with 12, indicating the

Figure 7. Aryl amides as RORγ agonists. 5797



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Figure 8. Discovery of tertiary amines as RORγt modulators.

Figure 9. (A) RORγ cocrystal structure with agonist 18; (B) key interactions of RORγ LBD with 18 (blue dashes represent hydrogen bond interactions, and pink dashes represent π−π stacking interactions).

Figure 10. Thiazole amides as RORγt modulators.

did not affect RORγt activity as seen in either FRET or dual FRET assays. However, the bulky group such as phenyl (27) showed decreased maximum inhibition in RORγ FRET assay. Furthermore, 27 behaved as a partial agonist, and it activated RORγt with a pEC50 of 5.5, achieving 150% maximum activation seen in RORγ dual FRET assay. When 5-substituted moieties were changed into benzyl (28) and phenoxy (29), the compounds became inverse agonists. These results indicated a correlation between RORγt partial agonism activity and the bulky phenyl moiety on this thiazole amide scaffold.

discovery that a certain RORγt inverse agonist did not recruit corepressors. These results need to be studied further. In 2015, Wang and colleagues reported a novel series of thiazole amides as RORγt modulators (Figure 10).50 Several partial agonists were identified, and the resolved cocrystal structure of 27 with the RORγ LBD provided structural insights into the mode of action. On the basis of the structure of HTS hit 17, a set of compounds with different 5-substitutions on the thiazole ring were evaluated in both RORγ FRET and dual FRET assays. Small alkyl moieties such as Me (25) and Et (26) 5798



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Figure 11. (A) Co-crystal structure of RORγ LBD with 27; (B) key interactions of RORγ LBD with 27 (blue dashes represent hydrogen bond interactions, and pink dashes represent π−π stacking interactions).

Figure 12. Sulfonamides as RORγt modulators.

Cocrystal structures of inverse agonist 31 (PDB code 4WQP) and agonist 33 (PDB code 4WPF) with the human RORγ LBD were solved (Figure 13). From an overlay of the

A cocrystal structure of RORγ LBD with 27 was obtained (PDB code 4XT9) and showed similar interactions to the previously reported tertiary amine RORγt agonist (Figure 11).50 The sulfonyl group formed hydrogen bonds with Arg367 and Leu287, and amide NH formed hydrogen bonds with the backbone carbonyl of Phe377, respectively (blue dash in Figure 11B). π−π stacking interactions between two phenyl rings of 27 and Phe377 and Phe388 were observed. The thiazole ring also had a π−π stacking interaction with Phe378 (pink dash in Figure 11B). The 5-thiazole phenyl group occupied the hydrophobic space near AF2 domain and thus stabilized the AF2 domain toward coactivator recruitment. The overlay of 27 with the full RORγt agonist 20-hydroxycholesterol showed that the 5-phenyl only partially occupied the hydrophobic pocket which made 27 a partial agonist. When a CH2 or O linker was added into the thiazole ring and 5-phenyl group, it pushed the phenyl group away from the hydrophobic site and thus may not be sufficient for AF2 domain stabilization, making 28 and 29 inverse agonists. Besides, addition of 2-methoxy group on the phenyl group led 30 to be a partial agonist because the 2methoxy moiety of 30 occupied more space in the hydrophobic site around the AF2 domain. It was demonstrated that different sizes of substituted moieties influenced the stability of helix 12, resulting in a functional switch only by minor structure changes. In 2014, Genentech reported divergent modes of action resulting from minor structural changes from phenylsulfonamides to benzylsulfonamides (Figure 12).51 In the process of optimization, parallel synthesis yielded the pair of benzylsulfonamide 32 and phenylsulfonamide 33, which was derived from inverse agonist 31. In a RORγ SRC1 recruitment assay, benzylsulfonamide 32 exhibited inverse agonist activity with an IC50 of 0.015 μM, while phenylsulfonamide 33 showed agonist activity with an EC50 of 0.25 μM.

Figure 13. Cocrystal structure inverse agonist 31 (purple) and agonist 33 (green) with RORγ LBD.

two complexes, it was found that the two ligands almost identically overlapped from the acetyl group through the middle phenyl ring. The compounds then oriented divergently. The terminal phenyl group of the agonist ligand 33 interacted with Trp317, His479, and Tyr502, stabilizing helix 12 in an agonist-like conformation, while the benzyl group of the inverse agonist 31 disrupted the His479-Tyr502 hydrogen bond near helix 12. This observation provided an explanation for 31 behaving in an inverse agonist manner. Moreover, in the inverse agonist 31 complex, the helix 11 C-terminal bent inward by 16°, while aromatic side chains rotated and the pocket apex was repacked in the absence of helix 12. Trp317 and Phe486 also 5799



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reorientated and the filled pockets formed following these changes. These switch-like motions explained how inverse agonist 31 interfered with helix 11 and helix 12, leading to inverse agonism activity. In 2016, a series of benzoxazepine benzamides as RORγt modulators were disclosed by AstraZeneca (Figure 14).52 The

Figure 16. Benzoxazinones as RORγt modulators.

their cocrystal structures with full-length or partial proteolyzed RORγ were solved (PDB codes 5IXK and 5IZ0, respectively) (Figure 17). The agonist-bound construct showed that the Figure 14. Benzoxazepine benzamides as RORγt modulators.

opposite behaviors induced by slightly modified compounds were also observed. In a cofactor recruitment assay, most compounds behaved as potent and selective RORγt inverse agonists, whereas five compounds behaved as RORγt agonists, promoting coactivator recruitment with efficacy up to 58% at a concentration of 1 μM. The soaked-in crystal structures of inverse agonist 34 and agonist 35 with RORγ-LBD were solved (PDB codes 5APJ and 5APH, respectively). However, the binding modes were similar and could not provide a solid explanation for the different mode of actions (Figure 15A). To

Figure 17. Overlay of inverse agonist 36 (BIO399, purple) and agonist 37 (BIO592, green).

agonist conformation was stabilized by His479-Tyr502 hydrogen bond, which was critical for agonist conformation. Next, cocrystallization with the inverse agonist 36 and RORγ518 was attempted but did not produce any crystals. By use of RORγ518 with proteolyzed AF2 helix, the inverse agonistbound construct was obtained. Together with the cocrystal structure of 37, it can be concluded that the agonist conformation of the cocrystal structure of 37 would be perturbed by Met358 and pushed by BIO399 into Phe506 of AF2 domain, suggesting that Met358 positioning is crucial for triggering RORγ inverse agonism. In 2016, Scripps reported 38, discovered from SAR exploration of the first identified RORγt agonist 13 (Figure 18).30 Using desmosterol as a control for agonism, the efficacy

Figure 15. (A) Superposition of soak-in crystal structures of 34 (purple) and 35 (green). (B) Superposition of cocrystal structure of 34 (red) and soak-in crystal structure of 35 (green).

identify the potential triggers for the inverse agonism of compound 34, cocrystallization with the RORγ LBD without a coactivator peptide was carried out (PDB code 5APK) (Figure 15B). In this way, a different binding mode of inverse agonist 34 was identified. The NH of the benzamide group formed a hydrogen bond interaction with His479, effectively disturbing the His479-Tyr502 agonist lock, which was a crucial interaction for maintaining the agonist conformation. Additionally, an induced fit binding mode was discovered, in which helix 11 residues formed a pocket around the halogen-substituted benzamide group. The induced-fit binding like mode of action has never been observed in previously reported inverse agonist cocrystal structures, and the detailed mechanism of achieving inverse agonism required further exploration. In 2016, Biogen reported benzoxazinone as RORγt modulators including an inverse agonist 36 and an agonist 37 (Figure 16).53 Having similar chemical scaffold, 36 was only three atoms larger compared to the benzo[1,4]oxazine-3-one ring of 37. To investigate how minor differences lead to inverse agonism, an X-ray crystallography study was carried out and

Figure 18. 13 and its analog 38 (SR0987).

of 13 as a RORγt agonist was determined in a Gal4 reporter assay. In the same system, 38 resulted in a further increase in the expression of IL-17. Unexpectedly, treatment with 38 also led to a decrease in the expression of PD-1 without impact on the expression of granzyme B (cytotoxicity marker). Together, these results suggested that treatment with 38 may enhance protective immunity by regulating the expression of IL-17 and PD-1 while maintaining the cytotoxic ability of these cells. And this hypothesis was also validated by a Th17 cell differentiation assay using Jurkat T cells. The discovery of dual function of 5800



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Additionally, obvious antitumor efficacy was observed at the compound concentration of 5 μM to 10 μM. Two patents were published in 2016 by Lycera which particularly provided dihydro-2H-benzo[b][1,4]oxazine sulfonamide and related compounds (e.g., 46 and 47) as follow-up to the 2015 patents (e.g., 44 and 45).58,59 Compared to WO2015131035 and WO2015171610, the exemplary compounds in WO2016201225 and WO2016179343 have similar core structures but with different left-side linkers or right-side substituents (Figure 21). Exemplary compounds were tested in a TR-FRET assay and a Gal4 RORγ assay, and the most potent compounds were less than 0.5 μM with the maximum response of greater than 200%. In June of 2015, Celgene paid Lycera $105 million to partner with Lycera’s anticancer T-cell technology, which was assumed by emerging evidence as orally available RORγ agonists.61 In January 2017, the RORγ agonist named LYC-55176 from Lycera entered into phase 2A trial (NCT02929862) and was orally dosed as a single agent for locally advanced or metastatic solid tumors.62 In addition to Lycera, several pharmaceutical companies have begun to initiate anticancer RORγ agonist projects (Table 1). Collectively, it can be concluded that synthetic RORγ agonists potentially serve as novel small molecule therapeutics for cancer immunotherapy.

anti-PD-1 and T cell activation of RORγt agonists may provide a unique approach to cancer immunotherapy. To study the mode of action in view of structural insights, hydrogen/deuterium exchange (HDX) mass spectrometry was applied with desmosterol, 13, and 38. When treated with either 13 or 38, protection to solvent exchange was also observed in helix 12 but was not observed with desmosterol. This phenomenon was consistent with the stabilized conformation of helix 12 in previously reported cocrystallization structures, and it provided an explanation for why 38 acted as a RORγt agonist. Doebelin et al. from the Scripps Institute reported a series of N-arylsulfonylindolines as RORγ agonists (Figure 19).54 The

Figure 19. N-Arylsulfonylindolines as RORγ agonists.

first RORγ agonist was accidentally discovered by Nmethylation of the amide linker of inverse agonist 39. Similar to previously reported mode of action studies, the linker played a major role in deciding specific pharmacology wherein a potent RORγ inverse agonist could be converted into a RORγ agonist, while the binding affinity was not much influenced. After SAR exploration of multiple linkers, ether (-CH2O-) was identified as preferred linker to exhibit RORγ agonism. Further optimization had minor effects on in vitro activity. HDX data were able to delineate the structural mechanism for the switch in pharmacology from an inverse agonist 39 to an agonist 40 through increased helix 12 stabilization, a phenomenon that was constantly observed in nuclear receptors. These RORγ agonists had poor metabolic stability probably caused by the high lipophilicity. Yet considering adequate plasma exposure following ip dosing, these compounds may be used as tools to investigate RORγ activation in animal tumor models. In 2015, Lycera disclosed three patents claiming RORγt agonists.55−57 The patents provided sulfonamido and related compounds, methods of boosting RORγ activity and/or enhancing the amount of IL-17 in a subject, and methods of treating various medical disorders especially aiming at various cancers. In patent WO2015131035 A1, three sulfonamido compounds (41−43) were listed as potent RORγt agonists (Figure 20) with average EC50 values under 0.5 μM in a TRFRET assay and a Gal4 reporter assay. Adoptive transfer of Tc17 to mice EG7 tumor model and Tc17/Th17/both to mice B16F10 tumor model was exemplified, demonstrating RORγt agonist suppressing tumor growth through T-cell activation.

3. DISCUSSION AND CONCLUSIONS The NR superfamily has been considered attractive targets involved in various human diseases. Since NRs contain a conserved ligand-binding domain, they are ideal receptors for development of small molecuar ligands. As a member of the NR superfamily, many RORγt small molecule modulators have been discovered, including endogenous sterol ligands and synthetic small molecule modulators. In recent years, substantial progress has been made in the discovery of RORγt agonists, even though some studies may not be focused on agonists at first. Sterols, as first identified RORγ ligands, led to determination of the RORγ structure and initiated progress toward the identification of endogenous RORγ agonists. Synthetic RORγt agonists have been unexpectedly discovered through agonist/inverse agonist conversion, and their crystallization structures revealed similar mode of actions. Stabilization of a hydrophobic pocket formed around Trp317 and Tyr502 near helix 12 is crucial in recruiting coactivators and functioning in an agonist-like manner. Minor structural changes to synthetic ligands, such as enlarging substituents or changing linkers, pushed the hydrophobic pocket and disrupted the His479-Tyr502 lock, resulting in a functional switch between agonist and inverse agonist. In general, together with discovered RORγt modulators, recent progress of RORγt structural insights has provided key principles guiding structurebased design for discovery of synthetic RORγt agonists.

Figure 20. Exemplary RORγt agonists in patent WO2015131035 A1. 5801



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Figure 21. Representative RORγt agonists in Lycera’s patents.

Table 1. Current Active Projects of Anticancer RORγt Agonistsa

a

drug name

active company

active indication

status

added date

RORγt agonist (cancer) RORγ agonists (oral, cancer) RORγ agonists (cancer) INV-71 LYC-55716

Nuevolution Celgene Corp Aurigene Innovimmune Lycera Corp

cancer cancer cancer cancer advanced solid tumor

discovery discovery discovery discovery phase 2A

Jan 24, 2017 Nov 08, 2016 Dec 18, 2015 Jan 20, 2017 Jan 05, 2017

Data retrieved from Cortellis, Thomson Reuters.60

Since Th17 cell differentiation and IL-17 have been demonstrated to elicit an antitumor effect by activating CD8+ T cells, RORγt, as the key lineage transcription factor to initiate the Th17 cell differentiation and IL-17 production, is considered a potential target for cancer immunotherapy. Moreover, several synthetic RORγt agonists have been shown to inhibit PD-1 expression on Th17 cells and regulate other pathways favorable to antitumor immunity. A recent paper reported that a RORγ antagonist has an antitumor effect on prostate cancer by acting on upstream of the androgen receptor (AR), yet this is resulted from a distinct mechanism.63 In theory, there is also the potential risk that RORγt agonists could cause autoimmune disorders, resulting from increased levels of IL-17. However, no experimental findings or clinical evidence has been reported for this side effect so far.64 Therefore, along with LYC-55716 entering into phase 2A study, the development of RORγt agonists may provide a promising approach toward novel small molecule therapeutics for cancer immunotherapy.

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discovery for autoimmune diseases and cancer immunotherapy. He has published more than 50 scientific papers and is co-inventor of 30 PCT international patents.

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ACKNOWLEDGMENTS We thank all the present and past members of the working group that were not actively involved in this publication for the help with some of the ideas and for the stimulating discussions: Ting Tang, Yafei Huang, Mingcheng Yu, Nannan Sun, Wei Fu. We thank Professor Di Zhu for helpful discussions. We are grateful to Tony Wang and Caitlin Wang for the proofreading of the manuscript. This work was supported by National Science Foundation of China (Grant 81573276) and Shanghai Biopharmaceutical Science and Technology Supporting Plan (Grant 15431900300).

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ABBREVIATIONS USED AF2, activation function 2; CBI, cholesterol biosynthetic intermediate; CYP51, cytochrome P450, family 51; FRET, fluorescence resonance energy transfer; HC, hydroxycholesterol; HDX, hydrogen/deuterium exchange; HTS, high throughput screening; IL-17, interleukin 17; LBD, ligand binding domain; LHS, left-hand side; MOA, mode of action; NR, nuclear receptor; PD, pharmacodynamics; PD-1, programmed death 1; PD-L1, programmed death ligand 1; RORγt, retinoic acid receptor-related orphan receptor γt; SAR, structure− activity relationship; SRC, steroid receptor coactivator; Th17 cell, T helper 17 cell

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 21 5198 0118. E-mail: [email protected]. cn. ORCID

Yonghui Wang: 0000-0002-0262-2431 Notes

The authors declare no competing financial interest.

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Biographies Ruomeng Qiu is a graduate student in medicinal chemistry under the supervision of Professor Yonghui Wang at Fudan University. She earned her B.S. degree from the School of Pharmacy at Fudan University in 2015. Her research interests include discovery of novel RORγt agonists for cancer immunotherapy and phenotypic drug discovery of small molecular IL-17 suppressors.

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