The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

RORγT is one of two protein products of the RORC (RAR related orphan receptor C) gene (NR1F3, nuclear receptor subfamily 1 group F member 3), and the other product, RORγ, is 21 amino acids longer. These receptors belong to the nuclear receptor subfamily of retinoic acid receptor-related orphan receptors (RORs), which also includes RORα (NR1F1) and RORβ (NR1F2). All these proteins are transcription factors that modulate gene activation by binding coactivators in a ligand-dependent manner [23,24]. RORγ/RORγT have the typical domain structure characteristic of other nuclear receptors [25], consisting of an N-terminal A/B domain, a DNA-binding domain (DBD/C domain), a hinge region (domain D) and a C-terminal ligand binding domain (LBD/E domain). The RORγ/RORγT isoforms are generated by transcription of RORC from two different promoters [26][27][28]. As mentioned earlier, RORγT is exclusively expressed in Th17 cells, where it orchestrates the expression of several cytokines [19,29]. In contrast, RORγ is broadly expressed [30] and regulates the circadian rhythm and lipid/glucose metabolism [31][32][33].
Due to the involvement of RORγ/RORγT in the development of the immune system, the regulation of metabolism, and the pathogenesis of autoimmune diseases and increasing evidence for their involvement in cancer biology, they have become the putative targets for drug design [34]. Indeed, an increasing number of articles have reported new compounds that modulate the activity of these receptors in agonistic [35] and inverse agonistic manners [36], with potential for use in adoptive cell therapy (ACT) or the treatment of select autoimmune diseases.
In this work, we present the identification of the compound AZ5104 as a specific agonist for the RORγ isoform in HepG2 cells. We also report that this compound negatively influenced the expression of the RORγT isoform and, as a consequence, inhibited the expression of RORγT-dependent interleukins and the differentiation of Th17 lymphocytes. Thus, we provide a new promising compound for potential therapeutic applications to treat autoimmune diseases with the Th17 component. In addition, the results of our study may also help to explain the molecular mechanism of the more frequent S. aureus infections in cancer patients treated with EGFR (epidermal growth factor receptor) inhibitors.

Identification of AZ5104 as a RORγ Activator
To search for new substances that modulate RORγ activity, we screened the L1600 Kinase Inhibitor Library (TargetMol) with a previously validated [37] RORγ-HepG2 reporter cell line. We identified 13 compounds ( Figure S1 and Table S1) in the initial chemical library screening, and these compounds were further processed in a two-step analysis that included: determination of dose-response curves and the ability to induce expression of the RORγ-dependent G6PC gene [38,39]. Only AZ5104 (Figure 1), an irreversible EGFR inhibitor [40], passed validation (true positive) while other twelve compounds were considered as false positives, (the false positive rate (FPR) was 2% which is significantly lower than observed by the other authors [41]). We then analyzed AZ5104 cytotoxicity in HepG2 cells, and the analysis showed that this compound is well tolerated by these cells at concentrations as high as 5 µM (Figure 2A), while in Th17 cells, exerted much higher toxicity ( Figure 2B and Figure S2). The dose-effect curve indicated that 2 µM AZ5104 was optimal for the transactivation of RORγ in the reporter cells ( Figure 3A). Next, we examined how overexpression of human RORγ and RORγT and mouse Rorγ and Rorγt in the RORγ-HepG2 reporter cell line influence the response to AZ5104. As shown in Figure 3B, we observed synergistic effects of protein overexpression and the examined compound. Interestingly we did not observe similar effects of this compound towards RORα and RORβ nuclear receptors ( Figure S3). To show that the effects of the AZ5104 are at least in part mediated by the interactions of this compound with the ligand binding domain of RORγ, we used another reporter system in which the LBD domain of the RORγ receptor was fused to the GAL4 DNA-binding domain [42]. As expected, we observed a statistically significant induction of the reporter after treatment with AZ5104 ( Figure 3C). human RORγ and RORγT and mouse Rorγ and Rorγt in the RORγ-HepG2 reporter cell line influence the response to AZ5104. As shown in Figure 3B, we observed synergistic effects of protein overexpression and the examined compound. Interestingly we did not observe similar effects of this compound towards RORα and RORβ nuclear receptors ( Figure S3). To show that the effects of the AZ5104 are at least in part mediated by the interactions of this compound with the ligand binding domain of RORγ, we used another reporter system in which the LBD domain of the RORγ receptor was fused to the GAL4 DNA-binding domain [42]. As expected, we observed a statistically significant induction of the reporter after treatment with AZ5104 ( Figure 3C).    human RORγ and RORγT and mouse Rorγ and Rorγt in the RORγ-HepG2 reporter cell line influence the response to AZ5104. As shown in Figure 3B, we observed synergistic effects of protein overexpression and the examined compound. Interestingly we did not observe similar effects of this compound towards RORα and RORβ nuclear receptors ( Figure S3). To show that the effects of the AZ5104 are at least in part mediated by the interactions of this compound with the ligand binding domain of RORγ, we used another reporter system in which the LBD domain of the RORγ receptor was fused to the GAL4 DNA-binding domain [42]. As expected, we observed a statistically significant induction of the reporter after treatment with AZ5104 ( Figure 3C).   HepG2 cells treated with AZ5104 showed a small (1.6-fold) but statistically significant induction of RORγ mRNA ( Figure 4A); however, we did not observe similar results on protein level ( Figure 4C). Notably, in the samples treated with increasing concentrations of AZ5104, we observed a substantial dose-dependent increase in the mRNA ( Figure 4B) and protein ( Figure 4C) expression of G6PC, a gene known to be regulated by RORγ [38,39]. This was accompanied by the decrease in levels of glucose 6-phosphate ( Figure 4D), which is a substrate for G6Pase. It should be noted that AZ5104 has the ability to induce G6PC expression much more (20-to 50-fold induction depending on the experiment) than is apparent from its RORγ transactivatory function. This may suggest that AZ5104 also influences a signal transduction pathway leading to the induction of another RORγ-independent factor regulating G6PC expression, though the results from the use of siRNA methodology ( Figure S4) showed that the contribution of RORγ in the process is significant.   Mean ± SD, n = 6. Luciferase activity was standardized per corresponding secreted alkaline phosphatase activity, which was used as transfection efficiency control. Results were normalized to the activity of nontreated, pCMV-XL5-transfected cells. (C) AZ5104 induces the activity of the GAL4-DBD RORγ fusion protein in the HepG2-pGL4.35 reporter cell line. Mean ± SD, n = 6. * p < 0.05 compared with control treatment. Luciferase activity was standardized per corresponding secreted alkaline phosphatase activity, which was used as transfection efficiency control. Results were normalized to the activity of nontreated, pCMV-XL5-transfected cells.
HepG2 cells treated with AZ5104 showed a small (1.6-fold) but statistically significant induction of RORγ mRNA ( Figure 4A); however, we did not observe similar results on protein level ( Figure 4C). Notably, in the samples treated with increasing concentrations of AZ5104, we observed a substantial dose-dependent increase in the mRNA ( Figure 4B) and protein ( Figure 4C) expression of G6PC, a gene known to be regulated by RORγ [38,39]. This was accompanied by the decrease in levels of glucose 6-phosphate ( Figure 4D), which is a substrate for G6Pase. It should be noted that AZ5104 has the ability to induce G6PC expression much more (20-to 50-fold induction depending on the experiment) than is apparent from its RORγ transactivatory function. This may suggest that AZ5104 also influences a signal transduction pathway leading to the induction of another RORγ-independent factor regulating G6PC expression, though the results from the use of siRNA methodology ( Figure S4) showed that the contribution of RORγ in the process is significant.

Molecular Docking Analysis
In the current study, we considered 3 host domains of RORγ in the context of binding to AZ5104 within the molecular docking approach. The estimated free energies of binding are presented in Table 1, and all final structures are presented in Figure 5. In the current study, we considered 3 host domains of RORγ in the context of binding to AZ5104 within the molecular docking approach. The estimated free energies of binding are presented in Table 1, and all final structures are presented in Figure 5.  The host domain is represented as a white ribbon, and only the residues in close contact with the ligand are explicitly shown. The interactions with these explicitly named residues contribute to the overall binding. Due to the complicated nature of intermolecular interactions, it is not possible to precisely decompose interactions into each contribution from separate residues. However, we can assume that the origin of some fraction of the interaction energy is provided by the hydrogen bonds. Among the three considered ligand-host complexes, two (i.e., AZ5104 bound to the 3L0J and 3B0W_B domains) constitute explicit hydrogen bonds ( Figure 5A,B). For the 3B0W_B ( Figure 5C) system, such explicit hydrogen bonding was not observed. In the case of all considered domains, the compound generally tends to occupy the same binding pocket of the host. In the case of 3L0J and 3B0W_B, the ligand adopts a similar orientation where the indole part of the ligand is located deeper in the binding pocket. In the case of the 3B0W_A domain, a different orientation is preferred, but due to lower interaction energy in an overall picture, this orientation seems to be slightly less favorable. The 3B0W domain forms an explicit hydrogen bond with the AZ5104 molecule. Here, the amino group of the glutamic acid residue (GLU379) interacts with a carbonyl group of the ligand. In the case of 3L0J, the second domain forms hydrogen bonds with the ligand, and AZ5104 occupies almost the same space, but the hydrogen bond is between the carbonyl group of the phenylalanine residue (PHE377) and the amine group of AZ5104. The magnitude of an interaction of all the considered domains with the guest system is similar. Additionally, a slightly weaker interaction was observed for 3B0W_A and 3B0W_B compared to that for 3L0J. The molecular docking results described shortly above do not point out clearly the most favorable domain. From the point of view of the current study, all of them are equally likely to occur in the experiment. Only the further investigation carried out at the level of theory incorporating the quantum effects might provide additional insight and thus the final resolution.

AZ5104 Inhibits Th17-Specific Genes Expression
In the next series of experiments, we used human Th17 cells that exclusively express the shorter isoform of the RORC gene, RORγT. Both isoforms share the majority of the amino acid sequence, differing by only 21-amino acids in the N-terminal A/B domain. Their ligand binding domains are identical, which is why we expected that AZ5104 would activate RORγT-dependent transcription in Th17 cells as it did in HepG2 cells. However, when we analyzed the cytotoxicity of the compound in CD4+ cells differentiating into Th17 lymphocytes, we noticed that concentrations above 500 nM significantly decreased cell viability ( Figure 2B). Unexpectedly, analysis of RORγT expression using real-time RT-PCR revealed that AZ5104 decreased these expression levels in a dose-dependent manner. This decrease was associated with decreases in IL17A/IL17F expression, the expression of other Th17-related cytokines (Figure 6), e.g., IL9 [43], IL21 [15], IL22 [44] and CSF2 [45], and IL-17 secretion ( Figure S5). Such results were not observed for the FOXP3 and IL23A [46] genes ( Figure 6H,I). Further analysis indicated that AZ5104 decreased RORγT occupancy at the IL17A/F promoters (Figure 7). In another approach, we used fully differentiated Th17 to check whether shorter exposure of cells to the AZ5104 could have different effects. Analyzing cell viability, we observed that concentrations above 0.5 µM are also cytotoxic (above 20%) to these cells ( Figure S2), and expression of the selected Th17 genes is already decreased ( Figure S6). One explanation for these results is that in Th17 cells, AZ5104 is not able to act as an agonist of RORγT because the observed cytotoxicity prevents the compound from reaching an effective intracellular concentration. Alternatively, some putative compound-mediated signaling events do not allow AZ5104 agonist activity. In that respect, the dose-dependent inhibitory effects of AZ5104 on the expression of IL17A/IL17F and other Th17 cytokines are consistent with the hypothesis that this compound affects the signaling pathway regulating tissue-specific RORγT expression. Because AZ5104 was previously identified as an EGFR inhibitor [40], we decided to analyze the phosphorylation status of downstream elements of this signaling pathway. We observed a strong inhibition of phosphorylation levels of pSRC, pERK1/2, and pSTAT3 in CD4+ cells treated with AZ5104 while differentiating towards Th17 cells (Figure 8). Interestingly, AZ5104 had no effect on the SRC-ERK1/2-STAT3 pathway in the HepG2 cell line and, noteworthy, phosphorylation of STAT3 on Tyr705 was not visible in these cells ( Figure S7) which suggests tissue-specific differences in the signaling network [47]. noteworthy, phosphorylation of STAT3 on Tyr705 was not visible in these cells ( Figure S7) which suggests tissue-specific differences in the signaling network [47].   noteworthy, phosphorylation of STAT3 on Tyr705 was not visible in these cells ( Figure S7) which suggests tissue-specific differences in the signaling network [47].

Discussion
In the human genome, 48 genes encoding proteins belonging to the nuclear receptor family have been identified thus far [48]. These genes express a significantly higher number of proteins due to differential promoter usage and alternative splicing, creating isoforms with different tissue distributions and functions [26,49,50]. Nuclear receptors play important roles in development and homeostasis [51,52] and are molecular targets for various therapies, including anti-cancer therapies, e.g., tamoxifen targeting ERα [53], enzalutamide targeting AR [54], hormone replacement, e.g., benzothiophene raloxifene targeting ER [55], immune system modulation dexamethasone targeting GR [56] and metabolic diseases, e.g., clofibrate and gemfibrozil targeting PPARα and γ [57,58]. The pathogenic role of RORγT, which participates in the development of Th17 cells in the development of certain autoimmune diseases, has been recognized, and RORγT has become a potential target for therapy against these diseases. Thus, many researchers have focused on the search for substances with inverse agonist activity against RORγT [36]. Nevertheless, due to the ability of Th17 cells to increase the immune response, at least in some types of cancer [59], and their use in adoptive cell therapy [60][61][62], an increasing number of researchers have also recognized the therapeutic potential of RORγT agonists [35,37].
In this study, we screened a kinase inhibitor library using a validated reporter cell line [37] and identified AZ5104 (Figure 1) as a compound with RORγ agonist activity. AZ5104 was able to induce the transcriptional properties of RORγ (Figure 3), and induce the RORγ-dependent expression of the G6PC gene (Figure 4). Molecular docking experiments revealed that AZ5104 binds to the LBD of RORγ/RORγT with a significant docking score (Table 1, Figure 5). However, in Th17 cells, we were not able to reach similar concentrations of the compound due to high toxicity, and we observed different biological effects of AZ5104, including the inhibition of RORγT expression ( Figure 6A) and

Discussion
In the human genome, 48 genes encoding proteins belonging to the nuclear receptor family have been identified thus far [48]. These genes express a significantly higher number of proteins due to differential promoter usage and alternative splicing, creating isoforms with different tissue distributions and functions [26,49,50]. Nuclear receptors play important roles in development and homeostasis [51,52] and are molecular targets for various therapies, including anti-cancer therapies, e.g., tamoxifen targeting ERα [53], enzalutamide targeting AR [54], hormone replacement, e.g., benzothiophene raloxifene targeting ER [55], immune system modulation dexamethasone targeting GR [56] and metabolic diseases, e.g., clofibrate and gemfibrozil targeting PPARα and γ [57,58]. The pathogenic role of RORγT, which participates in the development of Th17 cells in the development of certain autoimmune diseases, has been recognized, and RORγT has become a potential target for therapy against these diseases. Thus, many researchers have focused on the search for substances with inverse agonist activity against RORγT [36]. Nevertheless, due to the ability of Th17 cells to increase the immune response, at least in some types of cancer [59], and their use in adoptive cell therapy [60][61][62], an increasing number of researchers have also recognized the therapeutic potential of RORγT agonists [35,37].
In this study, we screened a kinase inhibitor library using a validated reporter cell line [37] and identified AZ5104 (Figure 1) as a compound with RORγ agonist activity. AZ5104 was able to induce the transcriptional properties of RORγ (Figure 3), and induce the RORγ-dependent expression of the G6PC gene (Figure 4). Molecular docking experiments revealed that AZ5104 binds to the LBD of RORγ/RORγT with a significant docking score (Table 1, Figure 5). However, in Th17 cells, we were not able to reach similar concentrations of the compound due to high toxicity, and we observed different biological effects of AZ5104, including the inhibition of RORγT expression ( Figure 6A) and RORγT-dependent transcription ( Figure 6B-G). All these observations are consistent with the hypothesis that the observed effects of AZ5104 in Th17 cells are not mediated by binding of AZ5104 to the LBD of RORγT but rather by the ability of this compound to inhibit downstream elements of EGFR signaling. This is further supported by observations that AZ5104 inhibited pSRC, pERK1/2, and pSTAT3 in Th17 cells in a dose-dependent manner (Figure 8) but did not in HepG2 cells ( Figure S7). STAT3 plays a crucial role in the development of Th17 cells [11,12], and its activity (through Tyr705 phosphorylation) is regulated by EGFR, SRC and ERK [63][64][65][66]. In that respect, our results are in line with previous observations that AhR-dependent activation of SRC and STAT3 is linked to enhanced Th17 differentiation [67] and that inhibition of ERK leads to inhibition of Th17 development [68]. To our knowledge, our report is the first showing the potential involvement of EGFR signaling in Th17 differentiation, adding another element to the complex process regulating the development of these lymphocytes. Epithelial growth factor (EGF)-mediated activation of EGFR has already been implicated in disorders with increased immunoreactivity, including asthma [69], in which Th17 cells may play an important pathogenic role [70,71]. Interestingly, cancer patients treated with EGFR inhibitors are more susceptible to S. aureus [72,73], a pathogen that is neutralized by Th17 lymphocytes [2]. In light of our results, this might be due to a decrease in the number of Th17 cells.
AZ5104, which was synthesized as an irreversible inhibitor of EGFR [40], is a demethylated metabolite of AZD9291 (osimertinib) [74,75]. The latter compound was approved by the Food and Drug Administration and the European Medicines Agency to treat T790M EGFR-positive non-small cell lung cancer [76]. The concentration of AZD9291 in patient serum can reach up to 795 nM [77], while another study [78] reported an average concentration of 409 ± 152 nM which is higher or very close to the concentrations of AZ5104 used in the present study (assuming that both compounds have a similar pharmacokinetic profile).

Cell Culture and Reagents
The HepG2 cell line (human hepatocellular carcinoma) was purchased from ATCC (Manassas, VA, USA) and maintained under standard conditions in Dulbecco's Modified Eagle's Medium (DMEM) (PAN Biotech GmbH, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech GmbH) at 37 • C in an atmosphere of 5% CO2. The RORγ-HepG2 reporter cell line stably transfected with the reporter plasmid (RORE)6-tk-Luc [79] was described in our previous study [37] and was cultured in the same medium as the parental HepG2 cell line with the addition of 50 µg/mL hygromycin B (PAN Biotech GmbH). The HepG2-pGL4.35 reporter cell line was described in our previous study [80] and was maintained in DMEM (PAN Biotech GmbH) supplemented with 50 µg/mL hygromycin B (PAN Biotech GmbH). AZ5104 was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Screening of the Chemical Library
The L1600 Kinase Inhibitor Library was purchased from TargetMol (Boston, MA, USA). RORγ-HepG2 cells were seeded into 96-well white plates. The next day, they were incubated with 1 µM concentration of compounds from the tested library for 24 h. Following incubation, the cells were harvested and lysed, and luciferase activity in the cell lysates was determined using an Infinite ® 200 PRO (Tecan) with d-Luciferin (luciferase substrate) (Cayman Chemical). A 2-fold induction of transcriptional activity was used as the cut-off value for the selection of positive compounds in an initial screening.

Cell Viability
The cytotoxicity of AZ5104 in HepG2 cells was evaluated with the neutral red uptake assay [81]. The absorbance of each sample was determined spectrophotometrically at 550 nm using a Sunrise microplate reader (Tecan, Männedorf, Switzerland). The cytotoxicity of AZ5104 in Th17 cells was determined using the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol. The luminescence of each sample was determined with an Infinite ® 200 PRO (Tecan, Männedorf, Switzerland).

Vectors, Transfection and Luciferase Assay
The RORγ expression plasmid was described previously [37]. Human RORγT, RORα, mouse Rorγ and Rorγt, and control pCMV-XL5 vectors were purchased from OriGene Technologies (Rockville, MD, USA). The RORβ expression vector was described previously [82] and was a kind gift from Roland Schvle. The GAL4-DBD RORγ fusion construct was described previously [83] and was a kind gift from Patrick Griffin. RORγ-HepG2 cells were seeded into 96-well white plates. The next day, they were incubated with increasing concentrations of AZ5104 for 24 h. Following incubation, the cells were harvested and lysed, and luciferase activity in the cell lysates was determined using an Infinite ® 200 PRO (Tecan) with d-Luciferin (luciferase substrate) (Cayman Chemical). In experiments involving the expression vectors, the RORγ-HepG2 cells were cotransfected with the pCMV-SEAP vector (a kind gift from S. Schlatter, Zurich) as a control for transfection efficiency. Alkaline phosphatase activity in culture medium was determined spectrophotometrically at 405 nm.

Naive CD4+ T Cell Isolation and Differentiation into Th17 Cells
To obtain fully differentiated human Th17 lymphocytes, the protocol described previously by Wilson et al. [84] was used. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by centrifugation through Ficoll. Buffy coats from healthy, anonymous donors were bought as waste material from the Regional Center for Blood Donation and Blood Treatment (Łódź, Poland). The naive CD4+ fraction was isolated using CD4 M-pluriBeads ® anti-hu (pluriSelect Life Science, Leipzig, Germany). After isolation, cells were cultured for 5 days under Th17-polarizing conditions: Yssel's medium containing human AB serum, cytokines (50 ng/mL human IL-1β, 30 ng/mL human IL-6, 10 ng/mL human IL-23, and 10 ng/mL human TGF-β) and beads coated with anti-CD2, anti-CD3, and anti-CD28 antibodies (T cell activation/expansion kit from Miltenyi Biotec, Bergisch Gladbach, Germany). The cytokines were purchased from PeproTech (Rocky Hill, NJ, USA).

siRNA Methodology
To downregulate the expression of the RORC gene, the duplexes of Stealth siRNA against RORC (HSS109302) transcript were used (Invitrogen, Carlsbad, CA, USA). As a negative control, siRNA-A with a scrambled sequence was used (Santa Cruz, Dallas, TX, USA). HepG2 cells were nucleofected with each siRNA using Amaxa SF Cell Line for 4DNucleofector™ X Kit L (Lonza, Basel, Switzerland) according to the manufacturer's protocol.

Chromatin Immunoprecipitation (ChIP)
To perform ChIP, CD4+ cells were cultured in Th17 polarizing conditions in the presence of 0.4 µM AZ5104 for 5 days. The cells were fixed with formaldehyde to cross-link proteins with DNA, harvested, and lysed, and the DNA was subjected to sonication with a VCX-130 sonicator (Sonics & Materials Inc., Newtown, CT, USA). For chromatin immunoprecipitation, the EZ-Magna ChIP A/G Kit from EMD Millipore (Billerica, MA, USA) was applied. The following antibodies were used: normal mouse IgG (EMD Millipore) and anti-ROR gamma antibody [162C2a] (ab58670, Abcam, Cambridge, UK). The relative enrichment of the IL17A and IL17F promoters was analyzed with real-time PCR methodology using a SYBR Green I Master Mix on a LightCycler 96 from Roche. The reactions were run under the following conditions: 95 • C for 10 min, followed by 40 cycles of 95 • C for 20 s, 58 • C for 20 s, and 72 • C for 20 s. Primers complementary to IL17A and IL17F were described previously [80]: IL17A, 5 -GCAGCTCTGCTCAGCTTCTA-3 (forward) and 5 -GGGCTTTTCTCCTTCTGTGG-3 (reverse); and IL17F, 5 -CTCTGATTTGTGGGCAATGG-3 (forward) and 5 -CCGGAGTTACTGACGAATGC-3 (reverse). Soluble chromatin collected before immunoprecipitation was amplified as input control. To calculate the relative abundance of a specific promoter sequence enriched by protein-specific immunoprecipitation, we used the dCt method with the Ct obtained for input DNA as a reference value as follows: 1000 × 2 −dCt , where dCt = Ct sample − Ct input.

Glucose 6-Phosphate Assay
To determine the amount of glucose-6-phosphate, the high sensitivity glucose 6-phosphate assay (Sigma Aldrich) was used according to the manufacturer's instruction. HepG2 cells were treated with increasing concentrations of AZ5104 for 24 h. After that time, cells were harvested and homogenized. Cell lysates were deproteinized with an Amicon Ultra-0.5 Centrifugal Filter Unit (EMD Millipore) prior to addition to the reaction. Fluorescence intensity (excitation = 535 nm/ emission = 587 nm) was measured using Infinite ® 200 PRO (Tecan).

Detection and Quantification of IL-17 (ELISA)
Naive CD4+ cells were cultured under Th17 polarizing conditions in the presence of selected concentrations of AZ5104 for 5 days. Then, the culture supernatants were collected, and the concentration of IL-17 was analyzed by ELISA using the Quantikine Human IL-17 Immunoassay kit (R&D Systems). The absorbance of the samples at 405 nm was determined in a Sunrise microplate reader (Tecan).

Docking Simulations
All docking simulations were performed as previously described [37,80] with the following modifications: within each molecular docking simulation, the AZ5104 molecule was considered as a ligand. The initial molecular information was obtained from the Pubchem database [87] as a SMILES code. This molecular representation had been turned into the 3D geometry with the OpenEye Toolkit [88,89]. As host structures, the ligand binding domains of RORγ with the PDB IDs 3B0W [90] and 3L0J [91] were considered.

Statistics
Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. The results obtained from human donors were analyzed using the Friedman repeated measures ANOVA on ranks followed by Tukey's post hoc test. A p-value of 0.05 or lower was considered statistically significant.

Conclusions
AZ5104 and AZD9291 are potentially interesting structures in the context of developing novel treatments for autoimmune diseases, though one should remember that these compounds have a rather high rate of side effects [92][93][94] and thus, new, less toxic analogs are needed.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. signal transducer and activator of transcription 3 SRC SRC proto-oncogene, non-receptor tyrosine kinase t-bet cell-specific T-box transcription factor TGF-β transforming growth factor beta Th17

Abbreviations
T-helper 17 cells