Small molecule inhibitors of RORγt: Targeting Th17 cells and other applications

Abstract

Nuclear hormone receptors (NHRs) form a family of transcription factors that are composed of modular protein structures with DNA- and ligand-binding domains (DBDs and LBDs). The DBDs confer gene target site specificity, whereas LBDs serve as control switches for NHR function. For many NHRs, both endogenous and synthetic small molecule ligands bind to small pockets within the LBDs, resulting in conformational changes that regulate transcriptional activity. This property of NHRs has been exploited by the pharmaceutical industry for therapeutic targeting of a wide variety of diseases, ranging from inflammatory diseases and cancer to endocrine and metabolic diseases. Th17 cells are CD4⁺ T helper effector cells that express several pro-inflammatory cytokines, including interleukin-17A (IL-17), and the actions of these cells have been linked to multiple human autoimmune diseases. Our laboratory previously identified the NHR RORγt, an immune cell-specific isoform of RORγ (retinoic acid receptor-related orphan nuclear receptor gamma), as a key transcription factor for the development of Th17 cells both in human and mouse. Although endogenous ligands for RORγt have not yet been reported, it is thought that RORγt activity and Th17-cell development can be modulated with highly specific small molecules that bind to the RORγt LBD and displace its endogenous ligands. Recent studies from multiple groups have reported the activities of such inhibitors. In this mini review, we describe how RORγt inhibitors were identified and how they may contribute to our understanding about RORγt and its biology.

Th17 cells, autoimmune diseases, and RORγt

Recent studies have shown that Th17 cells have key pro-inflammatory roles in cancer and a variety of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), collagen-induced arthritis (CIA), inflammatory bowel disease (IBD), and graft versus host disease [1–5]. Genome-wide association studies in humans have linked genes involved in Th17-cell differentiation and function (e.g. IL23R) with susceptibility to Crohn's disease, arthritis, and psoriasis [6–8]. In both mouse and human, not only Th17 cells, but also other immune cells, e.g. cells with the γδ T cell receptor and various innate lymphoid cells (ILCs) that express IL-17 and/or IL-22, are distinguished by their expression of RORγt [9, 10]. RORγt is required for the induction of IL-17 transcription and for the manifestation of Th17-dependent autoimmune disease in mice. Multiple other transcription factors have been shown to be important for the development of Th17 cells: BATF, STAT3, IRF4, RUNX1, and IκBζ [11–15]. Unlike RORγt, these factors are not NHRs and thus do not contain ligand binding domains, which renders them less attractive targets for drug development. Another ligand-regulated transcription factor, AhR (aryl hydrocarbon receptor), which is not a member of the NHR family, also augments Th17-cell and ILC differentiation and may be a good target for drug discovery [16, 17]. However, its genetic ablation in mice has a relatively mild effect on Th17-cell differentiation, leaving RORγt the most prominent target yet for the development of small molecules to regulate the functions and development of Th17 cells.

Of note, the ROR subfamily has three homologues in mammals: RORα, RORβ, and RORγ. Among these, RORβ is not expressed in immune cells, but RORα expression is high in Th17 cells both in vitro and in vivo. Importantly, RORα plays partially redundant roles in mouse Th17-cell differentiation [18]. However, its function may be less important in human cells, because the inhibition of RORγt activity alone is sufficient to completely block human Th17 cell differentiation in vitro (Huh JR and Littman DR, unpublished results). Moreover, severe developmental defects observed in RORα genetic null mice suggests that targeting RORα may generate detrimental side effects [19]. Unlike RORγt, which appears to be solely expressed in lymphoid lineage cells of the immune system, RORγ exhibits broader mRNA expression at low to moderate levels in most tissues including brain, liver, muscle, and adipose tissues [20]. Both RORα and RORγ are regulated in a circadian manner in these tissues, and they are thought to have metabolic regulatory functions that may be redundant [21, 22].

Th17 cells and small molecule inhibitors

Unlike many other NHRs, RORγt expression is sufficient (without adding exogenous agonists) to induce transcriptional activation of a reporter construct in various types of cells, and this suggests that RORγt is either constitutively active or its activating ligands are ubiquitously present. Regardless of activating mechanism, this feature allowed a cell-based RORγt reporter screen to be devised to identify small molecules inhibiting its transcriptional activity by binding to the RORγt LBD. Multiple reporter cell lines with not only RORγt, but also its closely related proteins such as RORα (RORγt mammalian homologue) and DHR3 (RORγt Drosophila orthologue) were generated. Since all these proteins share a high degree of similarity in protein structure, including ligand-binding pockets, small molecules that inhibit RORγt reporter activity, but not others, must be specifically acting on RORγt. By performing a small-scale small molecule screen with these insect cell-based reporter systems, the cardiac glycoside digoxin was identified as a specific inhibitor of RORγt transcriptional activation (Figure 1) [23]. Digoxin inhibited murine Th17-cell differentiation without affecting other T-cell lineages.

Figure 1.

Chemical structures of RORγ/γt small molecule inhibitors. The listed compounds exhibit selective inhibitory effects on RORγt versus RORα.

A crystal structure of the RORγt LBD complexed with digoxin illustrated its mode of action (inhibiting co-activator binding) and confirmed it as a RORγt inhibitor [24]. At high concentrations (over 300 nM), digoxin is toxic for human cells, as it targets the sodium-potassium ATPase, but less-toxic derivatives, 20,22-dihydrodigoxin-21,23-diol (Dig(dhd)) and digoxin-21-salicylidene (Dig(sal)) were generated and shown to reduce induction of IL-17 in human CD4⁺ T cells [23]. In addition, using the same RORγt and control reporter systems, a small molecule library comprising more than 300,000 compounds was screened at the NIH Chemical Genomics Center (NCGC). From this screen and the follow-up analysis, a series of Diphenylpropanamide compounds as selective RORγt inhibitors, including a highly potent compound ML 209 (also known as compound 4n) with an IC50 (half maximal inhibitory concentration) of 60 nM, were identified (Figure 1) (manuscript in preparation).

By modifying the Liver X Receptor (LXR) ligand T0901317, Griffin and Burris initially identified a small molecule, SR1001, which inhibited both RORα and RORγt activities [25]. Further chemical modification led to the development of a RORγ/γt specific inhibitor, SR2211 (Figure 1), which suppressed IL-17 production in the mouse T-cell lymphoma EL-4 [26]. By carrying out small-scale small molecule screens with primary human Th17 or EL-4 cells, ursolic acid (Figure 1) and azole-type fungicides were identified as RORγt or RORα/RORγt inhibitors, respectively [27, 28]. Likewise, these compounds inhibited IL-17 production. Importantly, treatment of animals with digoxin, SR1001, or ursolic acid was shown to not only delay onset, but also to reduce severity of the mouse model of multiple sclerosis, experimental autoimmune encephalomyetis (EAE) [23, 25, 27]. In addition, a recent study showed that digoxin treatment led to a beneficial outcome in the rat model of collagen-induced arthritis [29]. Therefore, RORγt activity can be modulated with small molecule inhibitors both in vitro and in vivo. These inhibitors and their chemical derivatives can be used as chemical probes for development of RORγt-targeted therapeutic agents that attenuate inflammatory lymphocyte function and autoimmune disease.

Of note, a comparison of RORγ LBD crystal structures complexed with either 25-hydroxyl cholesterol, a surrogate ligand, or digoxin revealed that these two molecules bind to RORγt LBD in a flipped orientation despite their shared core sterol structure [24, 30]. In addition, RORγt inhibitors thus far identified exhibit diverse chemical structures, consistent with a ligand binding pocket that is relatively large and perhaps has flexibility to accommodate a wide range of molecules. Digoxin was shown to antagonize the co-activator interaction by disrupting the localization of the RORγt helix H12 in its active conformation [24]. Its mechanism to facilitate the interaction of RORγt LBD with co-repressor peptides, however, is currently unknown.

The aforementioned RORγt small molecule inhibitors exhibited specific effects on Th17-cell differentiation without affecting T-cell differentiation into other lineages. They also inhibited the transcriptional activity of RORγt without affecting that of other NHRs. These results suggested that RORγt inhibitors possess activity specific for RORγ/γt. However, strictly speaking, the data have not ruled out the possibility that additional targets of such compounds may contribute to the observed biological effects. One means of testing this possibility is to compare genome-wide gene expression patterns in RORγt sufficient (wild-type) and deficient (genetic null) cells with or without compounds. Indeed, digoxin treatment in wild-type or RORγt knock-out T cells, cultured under Th17-cell differentiation conditions, largely phenocopied the gene expression profile in sham-treated RORγt knock-out cells [23], suggesting that RORγt is the sole dominant target of digoxin, at least under these conditions (no major off-targets exist). This is therefore a straightforward approach to determine if a particular RORγt inhibitor has additional target genes in relevant cells and tissues. Whether or not other RORγt inhibitors are truly specific will require more rigorous tests comparing their effects in wild-type versus RORγt-deficient cells.

Due to the transcriptional initiation from distinct start sites, RORγt lacks 21 amino acids that are present at the N-terminus of RORγ. Since both proteins contain identical LBDs, small molecules that inhibit RORγt activity will also inhibit RORγ. Indeed, digoxin suppressed IL-17 production from cells in which either RORγ or RORγt were expressed ectopically [23]. It remains to be determined if the development of RORγt-specific inhibitors is feasible. It is not known if the protein structure of the RORγt DBD is different enough from that of the RORγ DBD to allow differential regulation. If so, then it may be possible to develop small molecule inhibitors that bind to the LBD and subsequently induce a conformational change of the RORγt DBD but not the RORγ DBD. Such a change would then result in inhibition of DNA binding of RORγt.

Digoxin's binding to the LBD has been shown to decrease the DNA binding of the full-length protein presumably through a conformational change. A small molecule screen to identify RORγt, but not RORγ, inhibitory compounds would be an interesting one to execute in the future.

Small molecule inhibitors as tools to study biology

Other than serving as chemical templates for the development of beneficial therapeutic reagents, RORγt inhibitors can be utilized as chemical tools to enhance the understanding of RORγt-related biology. For example, using these small molecule compounds, we demonstrated that RORγt is required for the maintenance of IL-17 expression in mouse and human effector T cells. Here, we illustrate some examples and a sampling of the numerous potential applications.

RORγt in differentiated Th17 cells

RORγt had been shown to be crucial for the differentiation of Th17 cells [31, 32]. However, it was not known if it also plays important roles for the function of differentiated Th17 cells. To investigate this question, mouse Th17 cells, derived either in vivo or in vitro, and human memory Th17 cells isolated from the peripheral blood of healthy donors were subjected to treatment with digoxin or its non-toxic derivatives. Compared with DMSO as a control, these treatments led to the reduction of IL-17 production in cells from both mouse and human [23]. In vitro differentiated Th17 cells expressing a myelin-specific TCR were also adoptively transferred into lymphopenic mice, followed by administration of DMSO or digoxin. Transfer of these T cells induced EAE in DMSO-treated recipient mice, but there was delayed onset and reduced severity of disease progression in digoxin-treated animals. These data demonstrated that sustained RORγt activity is required for the function of differentiated Th17 cells, and hence these cells can be targeted for therapeutic benefit.

Identification of RORγt downstream target genes in human cells

A full understanding of RORγt function requires the identification of its target genes. Unlike other model organisms, a genetic analysis allowing comparison between RORγt sufficient and deficient cells is not available for human cells (although some comparison can be made using RNA interference). However, RORγt small molecule inhibitors can be administered to human CD4⁺ T cells cultured under Th17 polarization conditions. Those genes up- or down- regulated in the presence of RORγt inhibitor are by definition RORγt target genes. Off-target effects of small molecule compounds can be minimized by using multiple inhibitors and by comparing their effects. For example, genes whose expression is modulated by several different RORγt inhibitors with distinct chemical structures are likely to be true targets, rather than off-targets. If combined with RORγt ChIP-Seq (chromatin immunoprecipitation followed by genome-wide sequencing) analysis, this approach can also lead to the identification of genes that are directly regulated by RORγt.

Identification of RORγt ligands that enhance its activity

As mentioned earlier, RORγt expression in many cell types exhibited strong transcriptional activation even in the absence of exogenous ligands. Unlike several small molecule inhibitors recently identified, no compound has been shown to enhance RORγt activity. It may be relatively difficult to identify compounds that further increase RORγt activity beyond a plateau of regulation by endogenous ligand. A potential way to get around this problem is to use a highly specific RORγt inhibitor, such as digoxin, in reporter cell systems to decrease innate RORγt activity. Small molecule screens then can be performed to identify compounds that revert activity back to the steady-state level observed in the absence of antagonist or inverse agonist, by presumably competing against it. Identifying agonistic compounds for RORγt would be useful not only to develop therapeutic means to fight certain bacterial or fungal infections and cancers by augmenting RORγt and Th17 cell activities, but also to gain insights into the structure of endogenous ligands, which may provide new tools for agonizing or antagonizing the activity of the NHR.

RORγt cistrome analysis/GRO (Global run-on) sequencing

RORγt inhibitors can be used to perform a kinetic analysis of RORγt function. For example, during Th17-cell differentiation, RORγt activity can be suppressed at various time points with the help of small molecule inhibitors. Subsequently, it would be possible to investigate not only their effects on the transcription of various target genes, but also on DNA occupancy of other key transcription factors in the Th17 program, such as STAT3, BATF, and IRF4. Genome-wide RORγt ChIP or GRO sequencing analyses can be performed in the presence or absence of RORγt inhibitors to dissect RORγt-dependent transcription programs in great detail. Of note, we previously showed that digoxin treatment led to the dissociation of RORγt protein from its DNA targets, suggesting that RORγt inhibitors may be helpful to investigate the mechanisms of the RORγt-dependent recruitment of other transcription factors to target genes. Unlike genetic analyses with RORγt-null or conditional mutants, chemical analyses with RORγt inhibitors will result in a relatively rapid inhibition of RORγt activity with intact (but inactive) protein expressed. This feature will allow kinetic analyses of RORγt function, and comparison with genetic data may uncover ligand-independent functions of RORγt, should they exist. It is possible, for example, that transcriptionally inactive inhibitor-bound RORγt may still contribute to the expression or inhibition of particular genes by interacting with other transcription factors or cofactors. The chemical tools will also allow for examination of heritable versus non-heritable functions of RORγt. While expression of some target genes, e.g. IL-17, appears to require continuous binding and function of RORγt, other targets may become independent of its activity through RORγt-dependent epigenetic modifications of chromatin. The identities of target genes with distinct dependency on RORγt may be best pursued by performing RORγt Chip and GRO sequencing analysis at different time points during Th17-cell differentiation with or without RORγt inhibitors.

RORγt proteomic analysis

RORγt inhibitors can also be added to cell lines or primary cells expressing tagged RORγt protein. RORγt activity-dependent protein binding partners can then be identified by mass spectrometry. For example, co-activators are likely to be identified as binding partners in the absence of RORγt inhibitors whereas co-repressors may be identified in the presence of inhibitors.

Th17 cells and natural inhibitors

Digoxin, a highly specific RORγt inhibitor, is a natural product originally isolated from a plant, foxglove or digitalis. Digoxin and other cardiac glycosides are best known for their inhibition of the function of the Na/K ATPase pump in certain mammals [33], including human (but not mouse), a function which we will not discuss further in this review. Digoxin may inhibit RORγt activity simply by chance. On the other hand, digoxin, at some point during the course of evolution, may have been consumed as a dietary source, transported to the gut luminal space, and subsequently may have been used as a dietary component regulating the function of Th17 cells and ILCs that are abundant in the mammalian gut mucosa. In addition, previous reports have suggested the existence of endogenous digoxin-like molecules produced by the adrenal gland [34, 35]. Curiously, a human commensal bacterium, Eubacterium lenta, has been reported to metabolize digoxin into 20,22-dihydrodigoxin [36, 37], which has less potent RORγt inhibitory activity (Huh JR and Littman DR, unpublished results). In this case, human commensal E. lenta may have evolved to modify food-derived (digoxin) or host-derived (endogenous digoxin-like molecules) [38, 39] small molecules and to regulate host immune responses by augmenting Th17-cell function.

Another possibility is the existence of endogenous small molecules or natural ligands that do not resemble digoxin, but that nonetheless inhibit RORγt function. These molecules may or may not possess chemical structures similar to those of the recently identified small molecule inhibitors. Since no endogenous ligands of RORγt have been identified, it is unclear at this point whether synthetic RORγt inhibitors or natural products like digoxin will give us any clues as to the chemical structures of natural ligands. RORγt natural ligands, agonistic or antagonistic, may be produced from cells that express RORγt, such as Th17 cells and ILCs, or from cells with which these lymphocytes interact, such as epithelial or dendritic cells. One particularly intriguing possibility is that natural inhibitors or activators of RORγt may be provided by gut-resident bacteria, either as direct products or by enzymatic modification of host metabolites. As a nuclear hormone receptor and a key transcription factor governing the differentiation program of crucial pro-inflammatory cells, RORγt may thus serve as a control center that can be modulated by gut-residing bacteria that are now being recognized for their ability to control host immune responses [40].

Concluding remarks

Through unbiased small molecule screens and through candidate small molecule approaches, multiple RORγt inhibitors have recently been identified. Not surprisingly, these inhibitors blocked the function and differentiation of pro-inflammatory Th17 cells. In coming years, more potent inhibitors will be surely developed. Since antibodies directed at both upstream (IL-12p40 subunit of IL-23) and downstream (IL-17) effectors of the Th17 pathway have been shown to be effective therapies for autoimmune disease [41–43], it will be important to learn if RORγt inhibitors will be efficacious in clinical applications. RORγt is important for thymocyte development in young animals and also for lymphoid organ development. Therefore, it will be crucial to assess long-term effects of RORγt inhibitors for the development or maintenance of such tissues. Unlike RORγt, which is mainly expressed in immune tissues, its isoform RORγ exhibits a broader expression pattern. It will also be crucial to investigate the effects of prolonged inhibition of RORγ activity if inhibitors only active for RORγt cannot be developed. As discussed, the development of highly specific and potent RORγt inhibitors will also lead us to the better understanding of how RORγt functions both in normal development and disease settings.