Unexpected Allosteric Network Contributes to LRH-1 Co-regulator Selectivity.

Phospholipids (PLs) are unusual signaling hormones sensed by the nuclear receptor liver receptor homolog-1 (LRH-1), which has evolved a novel allosteric pathway to support appropriate interaction with co-regulators depending on ligand status. LRH-1 plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of metabolic and neoplastic diseases. Although the prospect of modulating LRH-1 via small molecules is exciting, the molecular mechanism linking PL structure to transcriptional co-regulator preference is unknown. Previous studies showed that binding to an activating PL ligand, such as dilauroylphosphatidylcholine, favors LRH-1's interaction with transcriptional co-activators to up-regulate gene expression. Both crystallographic and solution-based structural studies showed that dilauroylphosphatidylcholine binding drives unanticipated structural fluctuations outside of the canonical activation surface in an alternate activation function (AF) region, encompassing the ß-sheet-H6 region of the protein. However, the mechanism by which dynamics in the alternate AF influences co-regulator selectivity remains elusive. Here, we pair x-ray crystallography with molecular modeling to identify an unexpected allosteric network that traverses the protein ligand binding pocket and links these two elements to dictate selectivity. We show that communication between the alternate AF region and classical AF2 is correlated with the strength of the co-regulator interaction. This work offers the first glimpse into the conformational dynamics that drive this unusual PL-mediated nuclear hormone receptor activation.

Designed Spiroketal Protein Modulation.

Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure-based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co-activator recruitment. We solved the crystal structure of the spiroketal-hRXRα-TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co-crystal structure, the first of a designed spiroketal-protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes.

Defining the communication between agonist and coactivator binding in the retinoid X receptor α ligand binding domain.

Retinoid X receptors (RXRs) are obligate partners for several other nuclear receptors, and they play a key role in several signaling processes. Despite being a promiscuous heterodimer partner, this nuclear receptor is a target of therapeutic intervention through activation using selective RXR agonists (rexinoids). Agonist binding to RXR initiates a large conformational change in the receptor that allows for coactivator recruitment to its surface and enhanced transcription. Here we reveal the structural and dynamical changes produced when a coactivator peptide binds to the human RXRα ligand binding domain containing two clinically relevant rexinoids, Targretin and 9-cis-UAB30. Our results show that the structural changes are very similar for each rexinoid and similar to those for the pan-agonist 9-cis-retinoic acid. The four structural changes involve key residues on helix 3, helix 4, and helix 11 that move from a solvent-exposed environment to one that interacts extensively with helix 12. Hydrogen-deuterium exchange mass spectrometry reveals that the dynamics of helices 3, 11, and 12 are significantly decreased when the two rexinoids are bound to the receptor. When the pan-agonist 9-cis-retinoic acid is bound to the receptor, only the dynamics of helices 3 and 11 are reduced. The four structural changes are conserved in all x-ray structures of the RXR ligand-binding domain in the presence of agonist and coactivator peptide. They serve as hallmarks for how RXR changes conformation and dynamics in the presence of agonist and coactivator to initiate signaling.

Mutation in KANK2, encoding a sequestering protein for steroid receptor coactivators, causes keratoderma and woolly hair.

BACKGROUND: The combination of palmoplantar keratoderma and woolly hair is uncommon and reported as part of Naxos and Carvajal syndromes, both caused by mutations in desmosomal proteins and associated with cardiomyopathy. We describe two large consanguineous families with autosomal-recessive palmoplantar keratoderma and woolly hair, without cardiomyopathy and with no mutations in any known culprit gene. The aim of this study was to find the mutated gene in these families. METHODS AND RESULTS: Using whole-exome sequencing, we identified a homozygous missense c.2009C>T mutation in KANK2 in the patients (p.Ala670Val). KANK2 encodes the steroid receptor coactivator (SRC)-interacting protein (SIP), an ankyrin repeat containing protein, which sequesters SRCs in the cytoplasm and controls transcription activation of steroid receptors, among others, also of the vitamin D receptor (VDR). The mutation in KANK2 is predicted to abolish the sequestering abilities of SIP. Indeed, vitamin D-induced transactivation was increased in patient's keratinocytes. Furthermore, SRC-2 and SRC-3, coactivators of VDR and important components of epidermal differentiation, are localised to the nucleus of epidermal basal cells in patients, in contrast to the cytoplasmic distribution in the heterozygous control. CONCLUSIONS: These findings provide evidence that keratoderma and woolly hair can be caused by a non-desmosomal mechanism and further underline the importance of VDR for normal hair and skin phenotypes.

Coactivator recruitment of AhR/ARNT1.

A common feature of nuclear receptors (NRs) is the transformation of external cell signals into specific transcriptions of the signal molecule. Signal molecules function as ligands for NRs and, after their uptake, activated NRs form homo- or heterodimers at promoter recognition sequences of the specific genes in the nucleus. Another common feature of NRs is their dependence on coactivators, which bridge the basic transcriptional machinery and other cofactors to the target genes, in order to initiate transcription and to unwind histone-bound DNA for exposing additional promoter recognition sites via their histone acetyltransferase (HAT) function. In this review, we focus on our recent findings related to the recruitment of steroid receptor coactivator 1 (SRC1/NCoA1) by the estrogen receptor-α (ERα) and by the arylhydrocarbon receptor/arylhydrocarbon receptor nuclear translocator 1 (AhR/ARNT1) complex. We also describe the extension of our previously published findings regarding the binding between ARNT1.1 exon16 and SRC1e exon 21, via in silico analyses of androgen receptor (AR) NH2-carboxyl-terminal interactions, the results of which were verified by in vitro experiments. Based on these data, we suggest a newly derived tentative binding site of nuclear coactivator 2/glucocorticoid receptor interacting protein-1/transcriptional intermediary factor 2 (NCOA-2/ GRIP-1/TIF-2) for ARNT1.1 exon 16. Furthermore, results obtained by immunoprecipitation have revealed a second leucine-rich binding site for hARNT1.1 exon 16 in SRC1e exon 21 (LSSTDLL). Finally, we discuss the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an endocrine disruptor for estrogen related transcription.

Peroxisome proliferation-activated receptor δ agonist GW0742 interacts weakly with multiple nuclear receptors, including the vitamin D receptor.

A high-throughput screening campaign was conducted to identify small molecules with the ability to inhibit the interaction between the vitamin D receptor (VDR) and steroid receptor coactivator 2. These inhibitors represent novel molecular probes for modulating gene regulation mediated by VDR. Peroxisome proliferator-activated receptor (PPAR) δ agonist GW0742 was among the identified VDR-coactivator inhibitors and has been characterized herein as a pan nuclear receptor antagonist at concentrations of > 12.1 µM. The highest antagonist activity for GW0742 was found for VDR and the androgen receptor. Surprisingly, GW0742 behaved as a PPAR agonist and antagonist, activating transcription at lower concentrations and inhibiting this effect at higher concentrations. A unique spectroscopic property of GW0742 was identified as well. In the presence of rhodamine-derived molecules, GW0742 increased the fluorescence intensity and level of fluorescence polarization at an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a dose-dependent manner. The GW0742-inhibited NR-coactivator binding resulted in a reduced level of expression of five different NR target genes in LNCaP cells in the presence of agonist. Especially VDR target genes CYP24A1, IGFBP-3, and TRPV6 were negatively regulated by GW0742. GW0742 is the first VDR ligand inhibitor lacking the secosteroid structure of VDR ligand antagonists. Nevertheless, the VDR-meditated downstream process of cell differentiation was antagonized by GW0742 in HL-60 cells that were pretreated with the endogenous VDR agonist 1,25-dihydroxyvitamin D3.

Binding of the N-terminal region of coactivator TIF2 to the intrinsically disordered AF1 domain of the glucocorticoid receptor is accompanied by conformational reorganizations.

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.

Expression, purification and crystallization of the ancestral androgen receptor-DHT complex.

Steroid receptors (SRs) are a closely related family of ligand-dependent nuclear receptors that mediate the transcription of genes critical for development, reproduction and immunity. SR dysregulation has been implicated in cancer, inflammatory diseases and metabolic disorders. SRs bind their cognate hormone ligand with exquisite specificity, offering a unique system to study the evolution of molecular recognition. The SR family evolved from an estrogen-sensitive ancestor and diverged to become sensitive to progestagens, corticoids and, most recently, androgens. To understand the structural mechanisms driving the evolution of androgen responsiveness, the ancestral androgen receptor (ancAR1) was crystallized in complex with 5α-dihydrotestosterone (DHT) and a fragment of the transcriptional mediator/intermediary factor 2 (Tif2). Crystals diffracted to 2.1 Šresolution and the resulting structure will permit a direct comparison with its progestagen-sensitive ancestor, ancestral steroid receptor 2 (AncSR2).

Structure, energetics, and dynamics of binding coactivator peptide to the human retinoid X receptor α ligand binding domain complex with 9-cis-retinoic acid.

Retinoid X receptors (RXRs) are ligand-dependent nuclear receptors, which are activated by the potent agonist 9-cis-retinoic acid (9cRA). 9cRA binds to the ligand binding domain (LBD) of RXRs and recruits coactivator proteins for gene transcription. Using isothermal titration calorimetry, the binding of a 13-mer coactivator peptide, GRIP-1, to the hRXRα-LBD homodimer complex containing 9cRA (hRXRα-LBD:9cRA:GRIP-1) is reported between 20 and 37 °C. ΔG is temperature independent (-8.5 kcal/mol), and GRIP-1 binding is driven by ΔH (-9.2 kcal/mol) at 25 °C. ΔC(p) is large and negative (-401 cal mol(-1) K(-1)). The crystal structure of hRXRα-LBD:9cRA:GRIP-1 is reported at 2.05 Å. When the structures of hRXRα-LBD:9cRA:GRIP-1 and hRXRα-LBD:9cRA ( 1FBY ) homodimers are compared, E453 and E456 on helix 12 bury and form ionic interactions with GRIP-1. R302 on helix 4 realigns to form new salt bridges to both E453 and E456. F277 (helix 3), F437 (helix 11), and F450 (helix 12) move toward the hydrophobic interior. The changes in the near-UV spectrum at 260 nm of the hRXRα-LBD:9cRA:GRIP-1 support this structural change. Helix 11 tilts toward helix 12 by ≈1 Å, modifying the ring conformation of 9cRA. Hydrogen-deuterium exchange mass spectroscopy indicates GRIP-1 binding to hRXRα-LBD:9cRA significantly decreases the exchange rates for peptides containing helices 3 (F277), 4 (R302), 11 (F437), and 12 (E453, E456). The structural changes and loss of dynamics of the GRIP-1-bound structure are used to interpret the energetics of coactivator peptide binding to the agonist-bound hRXRα-LBD.

Structural Insights into the Interaction of the Intrinsically Disordered Co-activator TIF2 with Retinoic Acid Receptor Heterodimer (RXR/RAR).

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

Glucocorticoid resistance conferring mutation in the C-terminus of GR alters the receptor conformational dynamics.

The glucocorticoid receptor is a key regulator of essential physiological processes, which under the control of the Hsp90 chaperone machinery, binds to steroid hormones and steroid-like molecules and in a rather complicated and elusive response, regulates a set of glucocorticoid responsive genes. We here examine a human glucocorticoid receptor variant, harboring a point mutation in the last C-terminal residues, L773P, that was associated to Primary Generalized Glucocorticoid Resistance, a condition originating from decreased affinity to hormone, impairing one or multiple aspects of GR action. Using in vitro and in silico methods, we assign the conformational consequences of this mutation to particular GR elements and report on the altered receptor properties regarding its binding to dexamethasone, a NCOA-2 coactivator-derived peptide, DNA, and importantly, its interaction with the chaperone machinery of Hsp90.

P160/SRC/NCoA coactivators form complexes via specific interaction of their PAS-B domain with the CID/AD1 domain.

Transcriptional activation involves the ordered recruitment of coactivators via direct interactions between distinct binding domains and recognition motifs. The p160/SRC/NCoA coactivator family comprises three members (NCoA-1, -2 and -3), which are organized in multiprotein coactivator complexes. We had identified the PAS-B domain of NCoA-1 as an LXXLL motif binding domain. Here we show that NCoA family members are able to interact with other full-length NCoA proteins via their PAS-B domain and they specifically interact with the CBP-interaction domain (CID/AD1) of NCoA-1. Peptide competition, binding experiments and mutagenesis of LXXLL motifs point at distinct binding motif specificities of the NCoA PAS-B domains. NMR studies of different NCoA-1-PAS-B/LXXLL peptide complexes revealed similar although not identical binding sites for the CID/AD1 and STAT6 transactivation domain LXXLL motifs. In mechanistic studies, we found that overexpression of the PAS-B domain is able to disturb the binding of NCoA-1 to CBP in cells and that a CID/AD1 peptide competes with STAT6 for NCoA-1 in vitro. Moreover, the expression of an endogenous androgen receptor target gene is affected by the overexpression of the NCoA-1 or NCoA-3 PAS-B domains. Our study discloses a new, complementary mechanism for the current model of coactivator recruitment to target gene promoters.

Orphan nuclear receptor TR2, a mediator of preadipocyte proliferation, is differentially regulated by RA through exchange of coactivator PCAF with corepressor RIP140 on a platform molecule GRIP1.

Orphan nuclear receptor TR2 is a preadipocyte proliferator. Knockdown of TR2 in 3T3-L1 preadipocytes reduced their proliferation efficiency, whereas specific elevation of TR2 in these cells facilitated their proliferation. All-trans retinoic acid (RA) stimulates cellular proliferation in 3T3-L1 preadipocytes by activating TR2 through an IR0-type RA response element, which further activates c-Myc expression. In post-differentiated adipocytes, RA becomes a repressive signal for TR2 and rapidly down-regulates its expression. The biphasic effect of RA on TR2 expression in 3T3-L1 is mediated by differential RA-dependent coregulator recruitment to the receptor/Glucocorticoid Receptor-Interacting Protein 1 (GRIP1) complex that binds IR0 on the TR2 promoter. RA induces the recruitment of histone acetyl transferase-containing/GRIP1/p300/CBP-associated factor (PCAF) complex to the TR2 promoter in undifferentiated cells, whereas it triggers recruitment of histone deacetylase-containing/GRIP1/receptor-interacting protein 140 (RIP140) complex in differentiated cells. GRIP1 directly interacts with RIP140 through its carboxyl terminal AD2 domain. GRIP1 interacts with PCAF and RIP140 directly and differentially, functioning as a platform molecule to mediate differential RA-induced coregulator recruitment to TR2 promoter target. This results in a biphasic effect of RA on the expression of TR2 in undifferentiated and differentiated cells, which is required for RA-stimulated preadipocyte proliferation.

X-ray crystal structure of the novel enhanced-affinity glucocorticoid agonist fluticasone furoate in the glucocorticoid receptor-ligand binding domain.

An X-ray crystal structure is reported for the novel enhanced-affinity glucocorticoid agonist fluticasone furoate (FF) in the ligand binding domain of the glucocorticoid receptor. Comparison of this structure with those of dexamethasone and fluticasone propionate shows the 17 alpha furoate ester to occupy more fully the lipophilic 17 alpha pocket on the receptor, which may account for the enhanced glucocorticoid receptor binding of FF.

Unstable Protein Purification Through the Formation of Stable Complexes.

Purification of proteins containing disordered regions and participating in transient complexes is often challenging because of the small amounts available after purification, their heterogeneity, instability, and/or poor solubility. To circumvent these difficulties, we set up a methodology that enables the production of stable complexes in large amounts for structural and functional studies. In this chapter, we describe the methodology used to establish the best cell culture conditions and buffer compositions to optimize soluble protein production and their stabilization through protein complex formation. Two examples of challenging protein families are described, namely, the human steroid nuclear receptors and the HIV-1 pre-integration complexes.

Recognition and accommodation at the androgen receptor coactivator binding interface.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Transcriptional intermediary factor 1alpha mediates physical interaction and functional synergy between the coactivator-associated arginine methyltransferase 1 and glucocorticoid receptor-interacting protein 1 nuclear receptor coactivators.

In previous studies transcriptional intermediary factor 1alpha (TIF1alpha) was identified as a direct binding partner and potential transcriptional coactivator for nuclear receptors (NRs) but its overexpression inhibited, rather than enhanced, transcriptional activation by NRs. Here we show that TIF1alpha bound to and enhanced the function of the C-terminal activation domain (AD) of coactivator associated arginine methyltransferase 1 (CARM1) and the N-terminal AD of glucocorticoid receptor-interacting protein 1 (GRIP1). Furthermore, although TIF1alpha had little or no NR coactivator activity by itself, it cooperated synergistically with GRIP1 and CARM1 to enhance NR-mediated transcription. Inhibition of endogenous TIF1alpha expression reduced transcriptional activation by the GRIP1 N-terminal domain but not by the CARM1 C-terminal domain, suggesting that TIF1alpha may be more important for mediating the activity of the former than the latter. Reduction of endogenous TIF1alpha levels also compromised the androgen-dependent induction of an endogenous target gene of the androgen receptor. Finally, TIF1alpha formed a ternary complex with the GRIP1 N-terminal and CARM1 C-terminal domains. Thus, we conclude that TIF1alpha cooperates with NR coactivators GRIP1 and CARM1 by forming a stable ternary complex with them and enhancing the AD function of one or both of them.

Synergistic Regulation of Coregulator/Nuclear Receptor Interaction by Ligand and DNA.

Nuclear receptor (NR) transcription factors bind various coreceptors, small-molecule ligands, DNA response element sequences, and transcriptional coregulator proteins to affect gene transcription. Small-molecule ligands and DNA are known to influence receptor structure, coregulator protein interaction, and function; however, little is known on the mechanism of synergy between ligand and DNA. Using quantitative biochemical, biophysical, and solution structural methods, including 13C-detected nuclear magnetic resonance and hydrogen/deuterium exchange (HDX) mass spectrometry, we show that ligand and DNA cooperatively recruit the intrinsically disordered steroid receptor coactivator-2 (SRC-2/TIF2/GRIP1/NCoA-2) receptor interaction domain to peroxisome proliferator-activated receptor gamma-retinoid X receptor alpha (PPARγ-RXRα) heterodimer and reveal the binding determinants of the complex. Our data reveal a thermodynamic mechanism by which DNA binding propagates a conformational change in PPARγ-RXRα, stabilizes the receptor ligand binding domain dimer interface, and impacts ligand potency and cooperativity in NR coactivator recruitment.

Characterization of Soft Amyloid Cores in Human Prion-Like Proteins.

Prion-like behaviour is attracting much attention due to the growing evidences that amyloid-like self-assembly may reach beyond neurodegeneration and be a conserved functional mechanism. The best characterized functional prions correspond to a subset of yeast proteins involved in translation or transcription. Their conformational promiscuity is encoded in Prion Forming Domains (PFDs), usually long and intrinsically disordered protein segments of low complexity. The compositional bias of these regions seems to be important for the transition between soluble and amyloid-like states. We have proposed that the presence of cryptic soft amyloid cores embedded in yeast PFDs can also be important for their assembly and demonstrated their existence and self-propagating abilities. Here, we used an orthogonal approach in the search of human domains that share yeast PFDs compositional bias and exhibit a predicted nucleating core, identifying 535 prion-like candidates. We selected seven proteins involved in transcriptional or translational regulation and associated to disease to characterize the properties of their amyloid cores. All of them self-assemble spontaneously into amyloid-like structures able to propagate their polymeric state. This provides support for the presence of short sequences able to trigger conformational conversion in prion-like human proteins, potentially regulating their functionality.

Steroid receptor coactivators present a unique opportunity for drug development in hormone-dependent cancers.

Steroid receptor coactivators (SRCs) are essential regulators of nuclear hormone receptor function. SRCs coactivate transcription mediated by hormone stimulation of nuclear receptors and other transcription factors and have essential functions in human physiology and health. The SRCs are over expressed in a number of cancers such as breast, prostate, endometrial and pancreatic cancers where they promote tumor growth, invasion, metastasis and chemo-resistance. With their multiple roles in cancer, the SRCs are promising targets for the development of small molecule agents that can interfere with their function. For instance, perturbing SRC function with small molecule inhibitors and stimulators has been shown to be effective in reducing tumor growth in vivo. These early studies demonstrate that targeting the SRCs might prove effective for cancer treatment and more effort should be made to realize the untapped potential of developing drugs designed to target these coactivators.