Article

The Orphan Nuclear TLX Is a Receptor for Synthetic and Natural Retinoids

Graphical Abstract Authors Kristine Griffett, Gonzalo Bedia-Diaz, Lamees Hegazy, ..., Thomas Koelblen, McKenna L. Wilhelm, Thomas P. Burris

Correspondence [email protected]

In Brief TLX is an orphan that plays important roles in , vision, and cancer. Griffett et al. found that both natural and synthetic retinoids bind directly to TLX and regulate its transcriptional activity. Retinaldehyde, an important visual pigment, is the preferential natural retinoid ligand for TLX.

Highlights d Synthetic and natural retinoids bind directly to the TLX nuclear receptor d Synthetic agonists and inverse agonists of TLX were identified d TLX displayed a preference for retinaldehydes over other natural retinoids d Retinaldehyde regulated TLX target in a TLX- dependent manner in RPE cells

Griffett et al., 2020, Cell Chemical Biology 27, 1272–1284 October 15, 2020 ª 2020 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2020.07.013 ll ll

Article The Orphan Nuclear Receptor TLX Is a Receptor for Synthetic and Natural Retinoids

Kristine Griffett,1 Gonzalo Bedia-Diaz,1 Lamees Hegazy,1 Ian Mitchelle S. de Vera,2 Udayanga S. Wanninayake,2 Cyrielle Billon,1 Thomas Koelblen,1 McKenna L. Wilhelm,3 and Thomas P. Burris1,3,4,* 1Center for Clinical Pharmacology, Washington University School of Medicine and St. Louis College of Pharmacy, St. Louis, MO 63110, USA 2Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO 63104, USA 3Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63110, USA 4Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2020.07.013

SUMMARY

TLX is an orphan nuclear receptor that plays a critical role in both embryonic and adult neurogenesis, as well in the pathogenesis of glioblastomas. TLX functions predominately as a transcriptional repressor, but no nat- ural ligands or high-affinity synthetic ligands have been identified. Here, we describe the identification of nat- ural and synthetic retinoids as functional ligands for TLX. We identified potent synthetic retinoids that directly bind to TLX and either activate or inhibit its transcriptional repressor activity. Furthermore, we identified all- trans and 11-cis retinaldehyde (retinal), retinoids that play an essential role in the visual cycle, as the prefer- ential natural retinoids that bind to and modulate the function of TLX. Molecular dynamics simulations fol- lowed by mutational analysis provided insight into the molecular basis of retinoid binding to TLX. Our data support the validity of TLX as a target for development of therapeutics to treat cognitive disorders and/or glioblastomas.

INTRODUCTION TLX is expressed within the limited regions of the brain where adult neurogenesis occurs (the dentate gyrus of hippocampus The mammalian orphan nuclear receptor TLX (NR2E1) is homol- and the subventricular zone [SVZ] of the lateral ventricles) and is ogous to the Drosophila tailless (Monaghan et al., 1995; Yu essential for neurogenesis (Li et al., 2012; Liu et al., 2008, 2010; et al., 1994) and is predominantly expressed in the central ner- Shi et al., 2004). Importantly, the phenotype of the conditional vous system where it plays important roles in maintaining neural knockout of Tlx in the adult mouse brain was limited to impaired stem cell (NSC) self-renewal in both the developing and adult special learning and memory without the array of other deficits brain (Shi et al., 2004). TLX displays a predominantly nuclear noted in the earlier knockout models (Zhang et al., 2008). Further- cellular localization and functions predominately as a constitu- more, brain-specific overexpression of TLX using the nestin pro- tively active transcriptional repressor (Sun et al., 2007; Yo- moter led to enhanced spatial learning and memory function koyama et al., 2008), and no natural ligands have been identified. (Murai et al., 2014). TLX has also been implicated in retinal devel- TLX does not recruit the classic nuclear receptor corepressors opment and function, and Tlx null mice display vision deficits such as SMRT and NCoR but rather relies on recruitment of atro- (Young et al., 2002; Yu et al., 2000). TLX is expressed in the Muller€ phin, LSD1, and HDACs to drive repression (Sun et al., 2007; Yo- glial cells, as well as the retinal pigment epithelial cells of the retina koyama et al., 2008; Zhang et al., 2006). A number of TLX target (Dwyer et al., 2011; Zhang et al., 2006), two cell types that play crit- genes have been identified that play critical roles in TLX’s role ical roles in the cone and rod visual pigment cycles. in the regulation of stem cell proliferation and self-renewal, TLX has also been implicated in development of glioblastoma including Pten, Gfap, p21, and Pax2 (Li et al., 2008; Shi et al., multiforme (Liu et al., 2010; Park et al., 2010), which is the most 2004; Yu et al., 2000; Zhang et al., 2006). common type of brain tumor in adults with a short median sur- In the developing mouse brain, TLX is expressed in the ventric- vival time (Louis et al., 2007). TLX expression is elevated in ular and subventricular zones (Li et al., 2008), and mice with either glioblastoma stem cells (GSCs), and overexpression of TLX in spontaneous or genetically engineered deletion of Tlx exhibit drives a phenotype with similarity to GSCs (Park microcephaly and retinopathies and display deficits in cognitive et al., 2010). Knockdown of Tlx expression in GSCs reduced function and abnormally aggressive behavior (Monaghan et al., the growth and self-renewal of these cells in culture, and impor- 1997; Young et al., 2002; Zhang et al., 2008). The principal func- tantly, reduced TLX expression reduced tumor size and tion of TLX in the developing brain appears to be to maintain increased survival in GSC-initiated tumor orthotopic xenografts NSCs in an undifferentiated state (Li et al., 2008). In the adult brain, in mice (Cui et al., 2016).

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Figure 1. Broad-Based Screening of Synthetic Compounds as Potential Ligands for the Nuclear Receptor TLX (A) Gal4-TLX cotransfection assay performed in HEK293 cells at 10 mM compound. BMS453 and BMS493 are shown to be potential agonists while CD437 and CD1530 show inverse agonist activity (n = 4).

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The roles of TLX in neurogenesis, GSCs, and vision suggest up to 10 mM(Figure S1A). We performed a cotransfection assay that targeting this nuclear receptor may hold therapeutic value utilizing the full-length TLX along with a luciferase reporter con- in treating a range of disorders such as cognitive dysfunction, taining a TLX DNA response element from the human Pten including Alzheimer’s disease, glioblastomas, and retinopathies. gene, which is a well-characterized TLX response gene (Sun Importantly, overexpression of TLX in the brains of mice, which et al., 2007). Consistent with the chimeric receptor data, both leads to increased cognitive function, does not induce malig- BMS compounds displayed agonist activity with similar IC50 nancies, suggesting that such a barrier may not exist for target- values of 159 nM (BMS453) and 54 nM (BMS493) (Figure 1C right ing increased neurogenesis. Here, we describe the identification panel). The ccrp (1–3) compounds displayed no activity in the of retinoids, both synthetic and natural, that act as ligands of TLX full-length TLX cotransfection assay (Figure S1B). The com- (both agonists and antagonists). pounds that modulated TLX activity, either enhancing transcrip- tional repression or antagonizing repression, were all ligands for RESULTS the RAR (BMS493, BMS453, CD437, CD1530, Ch55, and AC93253) but not all RAR ligands modulated TLX activity. Impor- Identification of Synthetic Retinoids as TLX Agonists tantly, the RAR pharmacological profile of the compounds did and Inverse Agonists not correlate with the pharmacological activity at TLX. For In order to identify putative TLX ligands, we utilized a cell-based example, BMS453 and BMS493 are both very efficacious TLX cotransfection reporter assay system that used a chimeric re- agonists, but BMS453 is an RARb agonist while BMS493 is an ceptor where the ligand-binding domain (LBD) of human TLX RAR inverse agonist. These data suggest that a subset of RAR fused to the DNA-binding domain (DBD) of the yeast transcrip- ligands may also function as TLX ligands and that there may tion factor GAL4 and a GAL4-responsive luciferase reporter be a substantial degree of ‘‘shared chemical space’’ with regard into HEK293 cells (Bramlett et al., 2003; Griffett et al., 2013; to their ligands. We directly compared the activities of a subset of Thomas et al., 2003). The active compounds were very limited TLX modulators in terms of their differential activity at TLX, RAR, and restricted to a subset of synthetic retinoids that were origi- and RXR by performing a cotransfection assays using chimeric nally designed to target the retinoic acid receptors (RARs) and Gal4-LBD receptors (TLX, RARa/b/g, and RXRa/b/g)in the retinoid X receptors (RXR). A panel of synthetic retinoids as- HEK293 cells. Compounds were tested at two concentrations sessed for their ability to modulate TLX transcriptional activity in on TLX (0, 1, and 10 mM) and at 1 mM against the RARs and the cotransfection assay is shown in Figure 1A. Although most of RXRs. As shown in Figure 1D, the TLX inverse agonists the compounds were inactive, we clearly identified both agonists (BMS493 and BMS453) displayed substantial activity beginning (enhancing the basal repressor activity of TLX) and inverse ago- at 1 mM, consistent with the dose responses in Figure 1C. The nists (decreasing the basal repressor activity of TLX). The struc- agonists, CD437 and CD1530, were less potent requiring turally related compounds, BMS453 and BMS493, were the 10 mM to observe increased transcription in the Gal4-TLX assay. most efficacious agonists and were able to induce substantially To determine if these compounds were TLX ligands that enhanced repression by TLX (Figure 1A). Other compounds dis- directly bound to the LBD, we utilized three distinct biophysical played inverse agonist activity such as CD437 and CD1530, methods: differential scanning fluorimetry (DSF), fluorescence which are structurally distinct from the BMS compounds, but polarization (FP), and nuclear magnetic resonance (NMR). A highly related to one another (Figures 1A and 1D). The typical DSF assay assessing thermal stability of the TLX LBD in the pres- transcriptional repressor effect of GAL4-TLX is shown in Fig- ence or absence of putative ligands demonstrated that com- ure 1B where it reduces basal transcription of the luciferase re- pounds that altered the transcriptional activity (BMS453, porter by 60% and inclusion of BMS453 results in even greater BMS493, and CD1530) of TLX also functioned as direct ligands repression of transcription (in a TLX-dependent manner). The ef- since they dose dependently increased the Tm, hence the ther- fects of BMS453 and BMS493 are dose dependent in this assay mal stability, of the LBD (Figure 1E). BMS753, which did not alter with half maximal inhibitory concentration (IC50) of 367 nM and the transcriptional activity of TLX, did not bind to the TLX LBD as 125 nM, respectively (Figure 1C left panels). We also evaluated it did not alter the Tm (Figure 1D). The ccrp (1–3) compounds famprofazone (ccrp1), 1-(1,5-dimethylpyrazole-3-carbonyl)- were also inactive in the DSF assay (Figure S1C). We confirmed 4-(diphenylmethyl)piperazine (ccrp2), and the direct binding of these compounds to TLX using an FP (ccrp3), three structurally unrelated compounds identified in a ‘‘ligand sensing’’ assay where we assessed the ability of the broad screen for TLX ligands (Benod et al., 2014) in this assay TLX LBD to interact with the nuclear receptor interaction domain and observed no activity, even when tested at concentrations of the corepressor atrophin (Zhang et al., 2006). We

(B) Transfection of HEK293 cells with Gal4-UAS-luciferase with or without Gal4-DBD TLX LBD treated with either DMSO or BMS453 at 10 mM (n = 4). (C) Dose response of the agonist compounds using the Gal4-DBD TLX LBD (left) and full-length TLX (right) luciferase reporter system in HEK293 cells with agonist compounds (n = 4). (D) Pharmacological profile of the ligands in the TLX, RAR, and RXR receptors. Gal4-TLX is shown as a dose response of 0, 1, and 10 mM, while the Gal4-RAR and Gal4-RXR receptors were tested at 1 mM (n = 4). (E) Protein thermal melt assay utilizing human TLX LBD and several of the compounds that demonstrated agonist (BMS453 and BMS493), inverse agonist (CD1530), and no activity (BMS753) in the luciferase assays (n = 3).

(F) Co-regulator recruitment assay (fluorescence polarization) determining the Kd of the atrophin peptide with titrated human TLX LBD without ligand (n = 4). (G) Co-regulator recruitment assay titrating the ligands identified in the Gal4 screening to modulate activity of TLX (n = 4). 1 (H) H-NMR spectra of the methyl region of human TLX LBD DMSO-d6 control (blue) overlaid with spectra after adding 1.05:1 molar equivalence of synthetic ligands (red). Data are presented as mean ± SEM.

1274 Cell Chemical Biology 27, 1272–1284, October 15, 2020 ll Article observed saturable binding of the fluorescein isothiocyanate clear receptors displaying constitutive repressor activity, (FITC)-labeled atrophin nuclear receptor interaction fragment including DAX-1, COUP-TF2, TR4, PNR, and SHP (Kruse et al., to the unlabeled TLX LBD protein in this assay as shown in Fig- 2008; Sablin et al., 2008; Tan et al., 2013; Wang et al., 2017; ure 1F. Addition of increasing concentrations of TLX modulators Zhi et al., 2015). (BMS453, BMS493, CD1530, and CD437) reduced the interac- Since there is no X-ray crystal structure of ligand-bound TLX, tion of the TLX LBD with atrophin, consistent with these com- we used molecular dynamics simulations (MD) to further delin- pounds directly binding to TLX and altering its conformation (Fig- eate the mechanism of agonist ligand BMS493 binding to TLX ure 1G). BMS753, which is inactive in cell-based and the DSF based on the published structure of RARa in complex with assay was also inactive in the FP assay (Figure 1G). Unexpect- BMS493 (Durrant and McCammon, 2011; Karplus and McCam- edly, all compounds, whether agonists or antagonists, caused mon, 2002; le Maire et al., 2010; Liu et al., 2017). BMS493 is an a similar effect in atrophin binding (a decrease), indicating that RAR inverse agonist that strengthens corepressor interaction this assay may only be effective in detecting a ligand-induced with the RAR LBD, leading to enhanced gene silencing (le Maire conformational change but not predicting agonism versus et al., 2010). Overlay of the co-crystal structure of the RAR- antagonism. We performed 1H-NMR spectroscopy of the TLX a-LBD/BMS493/N-CoRNR1 structure on the apo TLX LBD LBD with and without putative ligands in order to examine the structure shows that both receptors have similar three-dimen- interaction in greater detail. In the spectra, we monitored the sional structures with helix 12 of TLX occupying the same posi- perturbation of side-chain methyl groups from hydrophobic res- tion of the corepressor N-CoRNR1, but the structures differ on idues (i.e., Leu, Ile, and/or Val) by BMS453, BMS493, and the position of helix 11, which is fully extended in the RARa struc- BMS753, represented by seven shielded resonances at 0.26, ture but bent inside the LBP of TLX (Figure S3). Absence of b 0.22, 0.18, 0.16, 0.07, À0.17, and À0.32 ppm. Solution NMR sheets in TLX suggests a larger solvent-exposed region of the peak lineshape analysis provides details on the dynamic pro- LBP relative to RARa, and structural comparison between TLX cesses, including conformational changes and binding events and RARa suggests that the A ring of BMS493 would bind inside (Kleckner and Foster, 2011). Protein methyl side-chain peak the hydrophobic region of the LBP and the carboxylic group perturbation analysis is a convenient approach to monitor ligand would extend toward the solvent-exposed region (Figure S3A). binding as these resonances are in shielded, less chaotic regions Furthermore, ligand binding to TLX would require displacement of the 1H-NMR spectra that circumvent overlap with buffer and of helix 11 to accommodate the ligand (Figure S3A). BMS493 be- ligand peaks. The spectra overlays between the control (no com- haves as an agonist of TLX, and we anticipated that agonist bind- pound; DMSO-d6 alone) and either BMS453 or BMS493 display ing would cause slight structural rearrangement of the native au- clear chemical shifts and alterations in peak intensity, whereas torepressor confirmation, which can be examined with MD in the the inactive control compound, BMS753, provides spectra that nanosecond timescale. The ligand was positioned in the LBP of are indistinguishable from the control spectra (Figure 1H). The TLX based on the binding mode of BMS493 in the LBP of RARa. resultant perturbation maps of the chemical shift in hertz (left) During the simulations, helix 11 was displaced from its original and peak intensity (right) changes (expressed as intensity ratio position (specifically residues F362 and F363) to accommodate with respect to DMSO-d6 control) after adding BMS453, the ligand (Figures 3A and 3B; Video S1). Trajectories of the re- BMS493, and BMS753 compounds (Figure S2A). The corre- ceptor bound ligand were stable all over the simulations (1 ms) sponding spectra overlays are shown in Figure 1H. Thus, data with no major structural changes. Ligand interactions with TLX from three independent biophysical methods indicate that TLX were mainly hydrophobic; the A ring of the ligand was involved is directly bound by these compounds and they act as direct li- in hydrophobic interaction with amino acid residues F363 and gands. In addition, FP shows the binding results in the altered F362, and the B, C, and D rings were involved in hydrophobic recruitment of a peptide derived from atrophin, likely as the result contacts with L192, L242, I230, L268, I235, and I353 (Figure 2B). of a ligand-induced conformational change. After simulations, the LBP was predicted to have a volume of 373.5 A˚ 3 using SiteMap (Gampe et al., 2000). In order to confirm Analysis of Structural Basis of BMS493 Activation of TLX the binding mode predicted from the simulations, we mutated TLX displays the canonical architecture of nuclear receptor LBD: amino acid residues that were expected to mediate TLX binding a three-layer a-helical sandwich composed of nine a helices (H3 to BMS493 within the LBP. Specifically, F363 and F362, which to H12). Helix H12 occupies the coactivator binding groove, sug- were predicted to make critical interactions with BMS493 (Fig- gesting an autorepressed conformation of the LBD (Zhi et al., ure 2B), were mutated to alanines, and a double F363A/F362A 2015). The ligand-binding pocket (LBP) of apo TLX is packed mutant was also constructed. The effect of the mutations was with hydrophobic residues, and space in the pocket is also assessed in a cotransfection assay identical to that performed limited by the kink at the C-terminal end of helix 11 into the space in Figure 1C. As shown in Figure 2C, mutation of either of these that corresponds to the LBP in ligand-regulated nuclear recep- residues (F363, F362) individually resulted in a partial loss of the tors (Figure S2B–S2D) (Zhi et al., 2015). The bend of helix 11 in- agonist activity (enhanced transcriptional repression), whereas side the LBP of TLX is caused by hydrophobic interactions be- the double mutant completely lost the ability to be agonized by tween amino acid residues spanning helices 3, 6, and 11; BMS493. These experimental data are consistent with the specifically residues V361, F362, F363, and K365 (Helix 11) are ligand-binding model predicted by MD simulation. involved in hydrophobic contacts with L242 (helix 6) and A189, C186, and E183 (helix 3), leading to a fully occupied LBP and Retinaldehydes Are TLX Ligands leaving no room for ligand binding (Figures S2C and S2D). The We were intrigued by the selectivity profile of TLX indicating that autorepressed conformation of TLX is common among the nu- RAR and TLX share some, but not all, synthetic ligands. We

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Figure 2. Molecular Dynamics (MD) Simulations Support Synthetic Retinoids Binding within the Human TLX LBD (A) Overlay of apo TLX (brown) and ligand-bound TLX after MD (brown). (B) The binding pose of BMS493 in TLX LBD. (C) Cotransfection assay with the mutated amino acids compared with the wild-type TLX with DMSO or 10 mM BMS493 treatment (left) and the percentage activity of BMS493 compared with DMSO control for each mutant LBD (right) (n = 4). Data are presented as mean ± SEM.

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Figure 3. Natural Retinoids Activate and Bind the TLX Receptor (A) Gal4-DBD TLX LBD screening of natural retinoids at 10 mM concentration to determine changes in TLX activity in HEK293 cells (n = 4). (B) Chemical structures of the retinoids that displayed some activity in the initial Gal4 screening.

(legend continued on next page) Cell Chemical Biology 27, 1272–1284, October 15, 2020 1277 ll Article hypothesized that TLX and RAR may also share natural ligands iologically relevant, at least in some tissues. As described in the and in order to investigate this, we performed a similar screen Discussion, levels of retinaldehydes in the retina, where TLX is as that shown in Figure 1A using natural retinoid ligands for highly expressed, are likely in the 20 mMrange.Althoughdefining RAR and RXR. As shown in Figure 3A, while most of the natural the potential physiological role of retinaldehydes as TLX ligands in retinoids were inactive at a concentration of 10 mM, several the retina is well outside the scope of this study that identified a showed the ability to reduce the transcriptional repressor effect range of retinoids as TLX ligands, we did examine the ability of of TLX. The retinaldehydes were prominent in the actives with ATRAL (as well as BMS493) to modulate transcription driven by all-trans retinaldehyde (all-trans retinal; ATRAL) with the greatest a TLX target gene of particular relevance to the retina. The efficacy followed by several of the cis enantiomers (9-cis,11- RLBP1 gene, which encodes the cellular retinaldehyde-binding cis, and 13-cis RAL) displaying 30%–50% of the efficacy of protein (CRALBP), was demonstrated to be dysregulated in the ATRAL. All-trans retinoic acid (ATRA) was also active with retina of Tlx null mice, suggesting that it may be a direct TLX target 25% of the efficacy of ATRAL, but its cis enantiomers, as well gene (Zhang et al., 2006). We created a reporter containing a as, all-trans retinol (ATROL) were inactive (structures shown in 625 bp fragment of the RLBP1 promoter (with two putative TLX Figure 3B). In order to characterize the potency of the retinoids response elements) upstream of luciferase and cotransfected it on TLX as well as their relative selectivity versus RAR, we per- into HEK293 cells along with a TLX expression vector as we formed dose responses in the Gal4-chimeric receptor cotransfec- had performed with the Pten-luc reporter described above. We tion assay with ATRAL, 11-cis RAL, and ATRA. As shown in Fig- noted that cotransfection of TLX resulted in substantial repression ure 3C, ATRAL was effective in both activating RARa and of luciferase expression relative to cotransfection with an empty relieving repression of TLX with similar potencies (TLX half expression vector (Figure 4A, left panel). Furthermore, when we maximal effective concentration [EC50] = 1.72 mMversusRARa performed cotransfections that included dose responses of either EC50 =2.48mM). We observed that 11-cis RAL was more potent ATRAL or BMS493, we observed dose-responsive inverse ago- targeting TLX versus RARa (0.223 mM versus 1.23mM); however, nism for ATRAL and agonism for BMS493 (Figure 4A, right panel). 11-cis RAL increased transcription to a greater level on RARa These data indicate that retinaldehyde can modulate TLX activity than did TLX (Figure 3C, top panel). It must be considered that in the context of the full-length receptor. RARa is activating transcription in response to 11-cis RAL versus We next sought to determine if ATRAL could regulate TLX target TLX is reducing its transcriptional repression activity in response genes in a cell line expressing TLX naturally. We utilized the human to 11-cis RAL, so direct comparisons of efficacy are not neces- retinal pigment epithelial cell line ARPE-19, which was initially sarily valid. ATRA, a high-affinity agonist for RAR, was very active characterized as expressing RLBP1 (Dunn et al., 1996). We at RARa (EC50 = 87 nM with substantial efficacy), whereas it was observed TLX mRNA expression by qPCR as well as protein much weaker at TLX (EC50 =1.83mM) (Figure 3C, right panel). We expression via western blot, which we were effectively able to sup- also determined that these retinoids functioned as direct ligands press by 75% by siRNA treatment (Figure 4B). ARPE-19 cells were using FP and NMR. ATRAL, ATRA, and 11-cis RAL all induced a treated with either 10 mM ATRAL or vehicle for 24 h (following treat- conformational change in the TLX LBD that caused reduced inter- ment with scrambled RNA or TLX siRNA), and the expression of action with the atrophin peptide in the FP assay, while the inactive TLX target genes was assessed by qPCR. We examined the retinoid, ATROL, had no effect on atrophin peptide binding (Fig- expression of RLBP1 as well as additional TLX target genes previ- ure 3D). Using 1H-NMR, the control TLX spectrum was identical ously identified, including SOX5 (SRY-related HMG-box 5), to that of ATROL-TLX (Figure 3E, top left panel) consistent with SOX11, SLC1a1 (Solute Carrier Family 1 Member 1), FEZF2 (FEZ the cell-based and FP data, indicating it did not bind to or alter Family 2), RGMA (Repulsive Guidance Molecule TLX activity. On the other hand, ATRAL, 11-cis RAL, and ATRA BMP Co-Receptor A), and NEUROG2 (Neurogenin2) (Schmouth all induced substantial changes in the spectra (Figure 3E). ATRA et al., 2015). Given TLX’s activity as a transcriptional repressor, induced substantial intensity decrease of the three rightmost loss of expression should lead to an increase in expression of peaks (60%–80% peak intensity decrease), and the perturbation the target genes and that is what we observed in every case (Fig- pattern induced by ATRA was similar to that of synthetic ligands ure 4C). The increase in expression of the target gene due to loss of BMS453 and BMS493. ATRAL also induced substantial peak TLX expression ranged from 3.33 for Rgma to 58.83 for Sox5.As shifting, specifically to the peak at À0.17 ppm (5.8 Hz). 11-cis illustrated in Figure 3, we characterized ATRAL as an inverse RAL also induced a modest peak decrease to the three most agonist able to relieve the constitutive repressor activity of TLX, shielded resonances (10%–15% suppression) (Figures 3Eand and indeed we observed that ATRAL treatment increased the S3B). Thus, our prediction that TLX may function as a receptor expression of all seven of these target genes (2.23 to 64.23)(Fig- for natural retinoids was confirmed, and it is clear that the ligand ure 4C). In order to confirm that this effect of ATRAL was mediated preference of TLX is distinct from that of RARs and RXRs. by TLX, we assessed the ability of ATRAL to relieve repression af- It did not escape our attention that, although the affinity of TLX ter siRNA knockdown of TLX expression, and in each case the ef- for ATRA was much lower than tissue levels of this retinoid, the af- fect of ATRAL was substantially reduced or eliminated (Figure 4C). finity of TLX for the retinaldehydes might be in a range that is phys- These data are consistent with ATRAL functioning as a TLX ligand.

(C) Dose-response profiles of select retinoids using Gal4-DBD TLX LBD or Gal4-DBD RARa-LBD analyzed to demonstrate differences in efficacy (top) and potency (bottom) (n = 4). (C) Co-regulator displacement assay of human TLX LBD with ATROL (negative control), ATRA, and the ATRALs (n = 4). 1 (E) H-NMR spectra of the methyl region of TLX LBD DMSO-d6 control (blue) overlaid with spectra after adding 1.05:1 molar equivalence of ligands (red). Data are presented as mean ± SEM.

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Figure 4. All-trans Retinaldehyde Regulates TLX Target Genes in a TLX-Dependent Manner (A) The RLBP1 luciferase construct was cotransfected with empty vector (ÀTLX) or full-length TLX (+TLX) in HEK293 cells to determine basal luciferase levels. A dose response with BMS493 and ATRAL was then performed in HEK293 cells cotransfected with full-length TLX and RLBP1-luc (n = 4). (B) Effect of scrambled RNA control or TLX-specific siRNA on Tlx expression as detected by qPCR in APRE-19 cells (left panel). Effect of scrambled RNA control or TLX-specific siRNA on TLX expression as detected by western blot in APRE-19 cells (right panel). (C) qPCR analysis of TLX target in response to ATRAL (10 mM) and siRNA mediated TLX knockdown (n = 4). Data are presented as mean ± SEM.

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Figure 5. TLX Is Predicted to Bind Natural Retinoids in a Different Conformation than the Synthetic Retinoids (A) Overlay of lowest energy structures of simulated TLX-ATRAL before (cyan) and after (magenta) the MD simulations. (B) Movement of helix 3 opens a space for the ATR aliphatic ring movement and interaction with helix 12. (C) Inward movement of helix 3 toward helix 6 is mediated and stabilized by hydrophobic contacts between amino acid residues Leu192, Met195, Ala189, and Ser188 (helix 3) with W233 on helix 5. (D) Cotransfection assay with a mutated amino acid compared with the wild-type TLX suggests that tryptophan 233 is necessary for ATRAL activity compared with DMSO on the wild-type receptor based on luciferase activity (n = 4). Data are presented as mean ± SEM.

Structural Basis for Retinaldehyde Modulation of TLX of ATRAL made hydrophobic interactions with amino acid res- Activity idues W222, M379, I372, and L375 (Figures 5A and 5B). The We modeled ATRAL in the LBP of TLX in a similar pose to bind- extended tetraenyl group of ATRAL was involved in hydropho- ing of retinoic acid in RXRa (PDB: 1G5Y) (Gampe et al., 2000) bic contacts with F226, A190, I358, and F362 (Figure 5C). (Figure 5A) (see STAR Methods for more detail). During the ATRAL binding caused considerable structural changes to TLX-ATRAL simulations (1 ms), the trimethyl cyclohexene group TLX; primarily inward movement of helix 3 and the T155-E179

1280 Cell Chemical Biology 27, 1272–1284, October 15, 2020 ll Article loop movement toward helix 6 (Figures 5A and 5C; Video S2). Our observation that a distinct subset of synthetic retinoids act These observed structural changes are consistent with as TLX ligands led us to examine the potential of natural retinoids ATRAL’s function as an inverse agonist. One would expect to function as TLX ligands. Indeed, we found that a subset of that inverse agonist binding would induce conformational naturally occurring retinoids function as TLX ligands. Interest- changes and destabilization of the native constitutive autore- ingly, TLX preferred retinaldehydes in general, whereas RARs pressor conformation of TLX, which is a typical behavior of and RXR preferred acids. Based on the TLX activity of ATRAL, many nuclear receptors (Keidel et al., 1994; Kojetin and Burris, one important tool that we developed was a homology model 2013; Paige et al., 1999). Movement of H3 is initiated by hydro- of TLX binding to ATRAL based on the crystal structure of the phobic contacts of W233 (H5) with amino acid residues Leu192, RXR-ATRA (Gampe et al., 2000). MD simulation predicted that Met195, Ala189 and Ser188 (H3) and Pro164 (loop T155-E179). a particular amino acid residue within the LBD of TLX (W233) Further inward movement of the lower half of helix 3 is induced would be important for ATRAL activity. This was indeed the by molecular interactions between amino acid residues Ala181, case, confirming the validity of the model, and that these small Leu177 (helix 3) and Val245 and Thr241 (helix 6) (Figure 5C). molecules function as TLX ligands. These results provide a foun- Movement of helix 3 toward helix 6 significantly decreased dation for the structure-based design of novel TLX modulators. the solvent-exposed accessible surface area of ATRAL Given the relatively low affinity of TLX for the retinaldehydes (22.5 A˚ 2 compared with 86.8 A2 for BMS493), which contributed (single digit micromolar range), we sought to determine if there to ATRAL stability and prevented its rapid dissociation from the might be physiological conditions in which such concentrations LBP. There may be other major structural changes of TLX that of retinaldehydes were maintained (in tissues where TLX is ex- occur upon ATRAL binding on the milliseconds to seconds pressed). This led us to consider the retina given the high concen- timescale and are not observed during current 1 mM simula- tration of retinaldehydes in this tissue (along with the high level of tions. In order to test the validity of this model, we targeted mu- expression of TLX) as well as its role in vision. Visual phototrans- tation of W233 of TLX (to alanine). Using a cotransfection assay duction begins with a photon striking the chromophore 11-cis identical to that described in Figure 1C, we observed that while RAL bound by an opsin in photoreceptor cells, leading to photo- WT TLX is very responsive to ATRAL treatment, the W233A isomerization of 11-cis-RAL to ATRAL. This leads to a conforma- mutant lost all responsiveness to ATRAL (Figure 5D). Interest- tional change in the opsin and initiation of the signal transduction ingly, although the inverse agonist activity of ATRAL was cascade that allows for visual perception. ATRAL is reduced to completely lost, BMS493 still retained its ability to enhance ATROL in the photoreceptors and then transported to retinal repression, albeit at a reduced level (Figure 5D). Thus, the MD pigment epithelium (RPE) or Muller€ glial cells (cells within the simulations predictions of the importance of W233 for ligand retina where TLX is specifically expressed) for enzymatic regener- activity were confirmed by mutational analysis and support ation of 11-cis RAL followed by transport back to the photorecep- the validity of the MD simulation-based model. tors. Given that each photon perceived by the retina induces isomerization of a molecule of 11-cis RAL that must be recycled, DISCUSSION the concentration of retinaldehydes must be quite extraordinary to maintain photoreceptivity. In humans, rods are the predomi- We found that TLX bound directly to both synthetic and natural nant photoreceptors (95% of photoreceptors), and an average retinoids and identified both agonists, which enhance the human retina contains 92 million rods (Curcio and Allen, repressor activity, and inverse agonists, which suppress the 1990). Assuming 4 3 107 molecules of rhodopsin per rod (Na- repressor activity of TLX. The synthetic retinoids that functioned thans, 1992) and an average volume of a human retina of as TLX agonists were particularly potent with EC50 values 0.25 mL (average retinal thickness 0.225 mm and area of ranging from 54 nM to 367 nM in cell-based assays. Compounds 1,100 mm2), the concentration of rhodopsin in the retina is esti- that functioned as inverse agonists were considerably less mated to be in the range of 24 mM. One would expect that 11-cis potent (EC50 values >5 mM). Direct binding of these compounds RAL and ATRAL levels would fluctuate in a similar concentration to the TLX LBD was confirmed with multiple biophysical tech- range as rhodopsin in order to maintain photoreceptor respon- niques. Using molecular modeling techniques, we were able to siveness. Thus, it is possible that TLX functions as a retinaldehyde predict the mechanism of binding of BMS493 and confirm the receptor in the retina. Consistent with this hypothesis is our obser- predictions via mutational analysis of the LBP. Previously, three vation that ATRAL regulated the expression of several TLX target compounds (non-retinoids_ ccrp 1, 2, and 3) were identified as genes in a TLX-dependent manner in a human retinal pigment synthetic TLX agonists with Kd values ranging from 650 nM to epithelial cell line (Figure 4). There is no evidence that such levels 27 mM, but no inverse agonists were identified (Benod et al., of retinaldehydes are achieved in other tissues where TLX is ex- 2014). None of these compounds were active in cell- or pressed, thus retinaldehyde regulation may not play a role in these biochemical-based assays in our hands. At this point, it is un- other regions. The constitutive repressor activity of TLX may be clear why these compounds are inactive in multiple assays sufficient to drive the physiological role of the receptor or, alterna- where we detect both inverse agonists and agonists. Our data tively, other natural ligands may exist. indicate that it is possible to identify high-affinity ligands that can either potentiate (agonists) or block (inverse agonists) the SIGNIFICANCE transcriptional repressor activity of TLX. This suggests that it may be possible to develop small-molecule therapeutics target- TLX is an orphan nuclear receptor that exhibits constitutive ing TLX that could be used to treat cognitive disorders (agonists), transcriptional repressor activity and plays a critical role in as well as glioblastomas (inverse agonists). maintenance of neuronal stem cells. Genetic data suggest

Cell Chemical Biology 27, 1272–1284, October 15, 2020 1281 ll Article that TLX is a key regulator of cognitive function. Genetic ACKNOWLEDGMENTS deletion of Tlx leads to cognitive impairment, while overex- pression of Tlx in the brain improves cognition. In addition, Human Retinal Pigment Epithelial Cells (ARPE-19) were kindly provided by Dr. Enrique Rodriguez-Boulan (Weill Cornell Medical College). TLX is commonly overexpressed in glioblastomas, and sup- pression of expression of this receptor is therapeutic in an- AUTHOR CONTRIBUTIONS imal models. Thus, there is clear therapeutic potential for small-molecule ligands that would regulate TLX activity. K.G. performed cell-based assays, mutagenesis, biochemical assays, assis- Here, we demonstrate that retinoids, both synthetic and nat- ted with NMR studies, contributed to the study design, and prepared the ural, serve as direct ligands of TLX and modulate its tran- manuscript; G.B.-D. performed cell-based assays and analysis, and assisted in a variety of molecular biology techniques; L.H. performed and analyzed mo- scriptional activity. TLX ligands share chemical space with lecular dynamics simulations; I.M.S.dV. performed NMR and biochemical as- both the retinoic acid receptors (RARs) and retinoid X recep- says and analysis; U.S.W. assisted with NMR studies; C.B. performed cotrans- tors (RXRs), but there are substantial differences in receptor fection assays with Gal4 plasmids and ccrp compounds; T.K. performed specificity for the various receptors (TLX versus RARs cloning and protein purification; M.L.W. performed cell-based assays; T.P.B. versus RXRs). A limited number of synthetic retinoids origi- conceived the project, directed the study, and prepared the manuscript. nally developed to target RARs function as high-potency DECLARATION OF INTERESTS TLX ligands (50 nM EC50), and both agonists and inverse agonists were identified. These data suggest that high-affin- Authors declare no competing interests. ity TLX-specific ligands can be designed that may hold utility in treatment of diseases such as cancer and cognitive Received: February 17, 2020 dysfunction, including Alzheimer’s disease. In terms of nat- Revised: May 8, 2020 ural retinoids, TLX preferred retinaldehydes, which may be Accepted: July 17, 2020 Published: August 6, 2020 important given that these retinoids play an important role in the retina as visual pigments. 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STAR+METHODS

KEY RESOURCE TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-TLX Abcam Cat# Ab86276: RRID: AB_1925258 Bacterial and Virus Strains BL(21) DE3 cells New England Cat# C2527 BioLabs Chemicals, Peptides, and Recombinant Proteins DMSO Sigma Cat# D8418 ATRAL Sigma Cat# R2500 ATROL Sigma Cat# R7632 ATRA Sigma Cat# R2625 9-Cis RA Sigma Cat# R4643 BMS453 Tocris Cat# 3409 BMS493 Tocris Cat# 3509 BMS753 Tocris Cat# 3505 TLX-LBD This Paper N/A ATRO peptide: HiLyte Fluor 488-PPYADTPALRQLSEYARPHVAFSP-NH2 Anaspec custom IPTG Invitrogen Cat# 15529019 Ni NTA Affinity Column GE Healthcare Cat# 17524701 HiLoad 16/60 Superdex 75pg GE Healthcare Cat# 28989333 Sypro Orange ThermoFisher Cat# S6650 RIPA Lysis Buffer Fisher Scientific Cat# 50-103-5431 Critical Commercial Assays Quickchange II XL Site Directed Mutagenesis Kit Agilent Cat# 200521 PrimePCR Array for NR2E1 Target Genes (Custom) 384-well array card Bio-Rad Cat# 10025214 with 48 unique assays; human; ABI Quantstudio-384 Deposited Data RARa-BMS493 Structure le Maire et al., 2010 PDB:3KMZ RXRa Structure Gampe, et al., 2000 PDBID:1G5Y Experimental Models: Cell Lines HEK293 Cells ATCC Cat# CRL-1573 ARPE Cells ATCC Cat# CRL-2302 Oligonucleotides F362A Forward 5’- GAAGAAGTGGCTTTCAAAAAAACCATCGGC-3’ Integrated DNA N/A Technology (IDT) F362A Reverse 5’- GCCGATGGTTTTTTTGAAAGCCACTTCTTC-3’ IDT N/A F363A Forward 5’- GAAGTGTTTGCCAAAAAAACCATCGGCAAT-3’ IDT N/A F363A Reverse 5’- ATTGCCGATGGTTTTTTTGGCAAACACTTC-3’ IDT N/A F362/363A Forward 5’- GAAGAAGTGGCTGCCAAAAAAACCATCGGC-3’ IDT N/A F362/363A Reverse 5’- GCCGATGGTTTTTTTGGCAGCCACTTCTTC-3’ IDT N/A W233A Forward 5’- ATAGCACAAGCGGCCATTCCGGTTGATGCT-3’ IDT N/A W233A Reverse 5’- AGCATCAACCGGAATGGCCGCTTGTGCTAT-3’ IDT N/A Recombinant DNA pGL4.73 Promega Cat# E6911 pET45b Novagen/EMD Cat# 71327 Millipore pGL4.35[luc2P/9XGal4UAS/Hygro] Promega Cat# E1370 (Continued on next page)

Cell Chemical Biology 27, 1272–1284.e1–e4, October 15, 2020 e1 ll Article

Continued REAGENT or RESOURCE SOURCE IDENTIFIER Gal4-DBD-TLX-LBD in pBIND-Zeo vector This paper N/A Full-length TLX in pcDNA3.1+ vector Invitrogen Cat# V79020 Geneious – sequence analysis Biomatters https://www.geneious.com Topspin 3.5 pl 7 Bruker N/A I-TASSER suite Zhang Lab - UMich https://zhanglab.ccmb.med.umich.edu/ I-TASSER/ AMBER UCSF https://ambermd.org UCSF Chimera UCSF https://www.cgl.ucsf.edu/chimera/ Maestro Schro¨ dinger https://www.schrodinger.com/maestro Software and Algorithms Graphpad Prism Graphpad https://www.graphpad.com Gen5 Biotek https://www.biotek.com/products/software- robotics-software/gen5-microplate-reader- and-imager-software/ Protein Thermal Shift Applied Biosystems 4466037 Quantstudio Applied Biosystems N/A PrimePCR Analysis software (GeneStudy_1.0.030.1023) Bio-Rad https://www.bio-rad.com/en-us/product/ primepcr-pcr-primers-assays-arrays?ID= M0HROA15 Geneious – sequence analysis Biomatters https://www.geneious.com Topspin 3.5 pl 7 Bruker N/A I-TASSER suite Zhang Lab - UMich https://zhanglab.ccmb.med.umich.edu/ I-TASSER/ AMBER UCSF https://ambermd.org UCSF Chimera UCSF https://www.cgl.ucsf.edu/chimera/ Maestro Schro¨ dinger https://www.schrodinger.com/maestro Other Mini-PROTEAN TGX gel Bio-Rad Cat# 4561083 PVDF TransBlot Turbo Pack Bio-Rad Cat# 1704156 iScript cDNA synthesis kit Bio-Rad Cat# 1708891 iTaq universal SYBR green Bio-Rad Cat# 1725124 siSPORT NeoFX Transfection reagent Invitrogen Cat# AM4511M onTargetplus Human NR2E1 siRNA SMARTPool Dharmacon Cat# L-003419-00-0010

RESOURCE AVAILABILITY

Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Thomas Burris ([email protected]).

Materials Availability Plasmids and materials generated from this study are available by request to the lead author.

Data and Code Availability This study did not generate datasets to be deposited.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Cell Lines HEK293 Human embryonic kidney cells; hypotriploid; female (fetal). Cells were purchased from ATCC and grown in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, GemBio) and 2.5mM L-Glutamine (Gibco). e2 Cell Chemical Biology 27, 1272–1284.e1–e4, October 15, 2020 ll Article

ARPE-19 Human retinal pigmented epithelium cells; normal; male (19 years of age). Cells were a gift from Rodriguez-Boulan lab but originally purchased from ATCC. Cells were grown in DMEM-F12 media (Gibco) supplemented with 10% FBS (GemBio) and 2.5mM L-Gluta- mine (Gibco). o Mammalian cell culture conditions: All mammalian cells were grown at at 37 C in a humidified incubator with 5% CO2. Media was changed daily and cells were grown to 70% confluency prior to splitting (every 2-3 days). Cell lines are authenticated by ATCC and by STR profiling. Bacterial Strain The Escherichia coli BL(21)DE3 cells were purchased from New England BioLabs. The cells were stored at -80oC upon receipt, and were thawed on ice just prior to the transformation procedure for protein expression.

METHOD DETAILS

Cotransfection Assays Assays were performed in HEK293 cells as previously described (Griffett et al., 2013; Savkur et al., 2003; Wu et al., 2003). For screening assays, a chimeric Gal4-DBD human TLX-LBD in the pBIND vector was cotransfected with pGL4.35[luc2P/9XGA- L4UAS/Hygro] vector (Promega), while dose response assays utilized the full-length TLX sequence in pcDNA3.1+ (Invitrogen) and the TLX response element cloned from the human pten promoter in pTAL-Luc (Promega). For the RLBP1 promoter luciferase re- porter, 4877-3496bp upstream from the ATG in exon 1 of RLBP1 was cloned into the pTAL-Luc vector with a 5’ Kpn1 site and 3’ Xho1 site. The final cloned sequence is 625bp long and contains two putative TLX response elements (TGACCT sequences). All co- transfections were normalized to renilla (pGL4.73; Dual-Glo luciferase reagent; Promega) and subsequently DMSO for analysis.

TLX Knock-down Studies ARPE cells were cultured in DMEM-F12 medium (Gibco) with 10% FBS (Gembio) and plated at 75,000 cells per well in 12-well plates (Corning). 24-hours after plating, cells were transfected with either siTLX or scrRNA at 30 nM final concentration (in the well) using siSPORT NeoFX transfection reagent (Invitrogen) per manufacturer’s protocol. Cells were treated with either DMSO or ATRAL (10 mM) 24-hours after the transfection. RNA was isolated 24-hours after the drug treatment and cDNA was synthesized for the QPCR assays using Bio-Rad iScript cDNA synthesis kit per manufacturer’s protocol. Expression was analyzed by QPCR using a custom array plate (Bio-Rad PrimePCR) per manufacturer’s protocol. n=4 was used for all cell-based assays and plotted as mean ± SEM. TLX protein was also assessed for knock-down by western blot. ARPE cells were grown in 10cm dishes and trans- fected as described above. 48-hours after the transfection, cells were collected and lysed in RIPA buffer supplemented with 1X pro- tease inhibitor cocktail, centriguged, and the soluble fraction was collected. The supernatant was run on a 4-20% TGX (Bio-Rad) gel and transferred to PVDF membrane. The membrane was blocked for 1 hour with 5% skim milk in TBST and incubated overnight at 4oC with TLX antibody (Abcam, 1:500) in blocking buffer. The membrane was washed three times in TBST followed by a 1 hour in- cubation with HRP-linked secondary antibody (Cell Signaling Technologies, 1:10,000), washed three more times, then Bio-Rad ECL detection reagent was added. The blot was imaged on an Invitrogen iBright Imager (ThermoFisher).

Protein Expression Human TLX LBD residues 179-385, subcloned in pET-45b plasmid (Novagen), was expressed in E.coli BL(21) DE3 cells in terrific broth as 3C protease-cleavable hexahistidine-tagged fusion proteins. The bacterial culture was grown at 37oC until we obtained an optical density of 1 at 600 nm. Protein expression was induced by adding 1 mM IPTG and was allowed to proceed for 14 hr at 22oC. Cells were harvested, lysed, filtered and applied to a Ni NTA affinity column (HisTrap HP, GE Healthcare Life Sciences); pro- teins in wash buffer (50 mM Tris, pH 8.0, 500 mM NaCl, 5 mM TCEP, 20 mM imidazole, 10% glycerol, 1 mM CHAPS) were eluted against a 500 mM imidazole gradient and subsequently purified by size exclusion chromatography (HiLoad 16/60 Superdex 75 pg, GE Healthcare Life Sciences). The final buffer consisted of 25 mM Tris, pH 8.0, 50 mM NaCl, 5 mM TCEP, 0.5 mM EDTA, and 1 mM CHAPS.

Protein Thermal Shift For DSF, 0.1 mg/ml TLX-LBD in 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT, 10% Glycerol, 1% CHAPS was mixed with 1X Sypro Orange (ThermoFisher) and either DMSO, 0.1 mM, 1 mM, or 10 mM of test compound and run in triplicate on a 384-well MicroAmp Optical Plate (ThermoFisher). No protein control and no ligand references were also run simultaneously in triplicate on the same plate. Thermal stability was performed on a Quantstudio 7 Flex Instrument (ThermoFisher) and protein melt shifts were quantified by Boltz- mann-derived Tm using the Protein Thermal Shift (ThermoFisher) software. This experiment was performed twice to validate replica- tion of the data.

Peptide Recruitment Assays For fluorescence polarization, atrophin peptide with an N-terminus fluorescein tag (HiLyte Fluor 488-PPYADTPALRQLSEYARPHVAFSP- NH2; Anaspec) was diluted to 1 mM from a 100 mM DMSO stock, in the presence of 6 mM TLX LBD and varying ligand concentrations ranging from 10 nM to 100 mM in assay buffer consisting of 20 mM Tris HCl, pH 8.5, 2 mM CHAPS, 150 mM NaCl, 10% glycerol and freshly

Cell Chemical Biology 27, 1272–1284.e1–e4, October 15, 2020 e3 ll Article added 5 mM DTT. Fluorescence polarization assay was performed on a 384-well Corning 3820 black opaque plate using a Biotek Syn- ergy Neo Alpha plate reader at excitation and emission wavelengths of 485 nm and 538 nm, respectively. Data were analyzed and plotted using GraphPad Prism as mean ± SEM. Peptide recruitment assays were performed four times by two researchers to increase scientific rigor and validate that the assay was replicated.

Nuclear Magnetic Resonance Studies

One hundred micromolar (100 mM) of TLX LBD with 10% D2O were mixed with either 0.5% DMSO-d6 or 1.05:1 molar equivalence of ligand and transferred to a Shigemi tube. 1H NMR data were acquired at 298 K on a 700 MHz Bruker Avance II HDTM NMR instrument equipped with a QCI cryoprobe. For each spectrum, 1024 transients were collected into 32,000 data points, with a spectral width of 16.7 kHz. Data were analyzed using Topspin 3.5 pl 7. NMR studies were performed three times with the individual assessing the spectra blinded to the compound used.

Molecular Dynamics Simulations The apo human TLX crystal structure of the ligand binding domain (LBD) (4XAJ) was used to set up the simulation system for BMS493(Zhi et al., 2015). A fused maltose-binding protein (MBP) tag at the N terminus of TLX and the corepressor atrophin were removed. All mutations and gaps were fixed and missing residues 155-183 were modelled using I-TASSER suite (Loop T155- E179)(Roy et al., 2010; Yang et al., 2015; Zhang, 2008). The compound BMS493 was placed in the orthosteric pocket with atomic coordinates extracted from the RARa-BMS493 complex X-ray structure (PDB: 3KMZ) after aligning the two receptor LBDs. Molecular Dynamics (MD) were performed for 1 ns with the AMBER 16 software(Salomon-Ferrer et al., 2013a). No major structural changes were observed in the TLX-BMS49 simulations. A snapshot from the TLX bound BMS493 simulations was used in modeling all trans retinal (ATRAL) after removing BMS493. We initially explored different binding poses of ATR based on different X-ray structures of RAR and RXR receptors bound with retinoid ligands as templates(Chandra et al., 2017; Chao et al., 2008; Sato et al., 2010). However, all the simulations lead to ligand dissociation from the pocket because in contrast to RAR and RXR, TLX lacks a b-sheet region that protects the ligand inside the pocket and prevent it from dissociation out of the pocket (Figure S2A). So we explored positioning the ATR deeper inside the hydrophobic pocket in a similar pose to the X-ray structure of RXRa bound with a non-activating retinoic acid isomer (PDBID:1G5Y)(Gampe et al., 2000). This pose yielded stable simulations over 1us with major structural changes in helix 3 (Fig- ure 4). Ligand parameters were assigned according to the general AMBER force field (GAFF) and the corresponding AM1BCC charges using Antechamber(Jakalian et al., 2000; Wang et al., 2004). The FF14SB forcefield parameters were used for all receptor residues(Ponder and Case, 2003). Tleap module was used to neutralize and solvate the complexes using an octahedral water box of TIP3P water molecules. MD Simulations were performed on GPUs in Amber16 using the CUDA version of Particle Mesh Ewald Mo- lecular Dynamics (PMEMD)(Salomon-Ferrer et al., 2013b). Each system was first energy minimized using the steepest descent and conjugate gradient methods. Both systems were then gradually heated with the Langevin thermostat to 300K over 30 PS at constant volume using 1fs time step. Iinitial velocities were sampled from the Boltzman distribution while keeping week restraints on the solute and the ligand. Each system was then equilibrated in the isothermalÀisobaric ensemble (NPT) for 10 ns. Production simulations were performed for 1ns. All production simulations were performed at 300 K, using constant pressure periodic boundary with an average pressure of 1 atm. Isotropic position scaling was used to maintain the pressure with a relaxation time of 2 ps. Non-bonded interac- tions were cut off at 10.0 A˚ , and long-range electrostatic interactions were computed using the particle mesh Ewald (PME). The SHAKE algorithm was used to keep bonds involving H atoms at their equilibrium length. 1fs time step was used for the integration of Newton’s equations. CPPTRAJ module was used for calculating the root mean square deviation (RMSD)(Roe and Cheatham, 2013). Pictures were generated using UCSF Chimera and Maestro(2019; Pettersen et al., 2004).

Site-Directed Mutagenesis Four amino acids in the TLX LBD were identified in the MDS that indicated potential binding importance for either the synthetic or natural ligands (F362; F363; F362/363; and W233). The full-length human TLX construct in pcDNA3.1 was used for mutagenesis and subsequent cotransfection assays. Mutagenesis was performed using the Agilent Quickchange II XL kit per manufacturer’s pro- tocol. Primers for mutagenesis were synthesized by IDT and sequence information is in the Key Resources Table. Resultant plasmid DNA was sequenced by Genewiz and used for cotransfection assays.

QUANTIFICATION AND STATISTICAL ANALYSIS

All data fitting and statistical analysis were performed using Graphpad Prism software version 8.0. Reported values, exact n values, and statistical significance are indicated in the Figure Legends or within the STAR Methods text. Statistical significance was defined as p > 0.05 and determined by two-tailed Student’s t tests.

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