Article The Orphan Nuclear Receptor 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 nuclear receptor that plays important roles in neurogenesis, 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 genes 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 gene 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 astrocytes 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). 1272 Cell Chemical Biology 27, 1272–1284, October 15, 2020 ª 2020 Elsevier Ltd. ll Article 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). (legend continued on next page) Cell Chemical Biology 27, 1272–1284, October 15, 2020 1273 ll Article 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).