Advances in the understanding of androgen receptor structure and function and in the development of next-generation AR-targeted therapeutics.

Androgen receptor (AR) and its ligand androgens are important for development and physiology of various tissues. AR and its ligands also play critical role in the development of various diseases, making it a valuable therapeutic target. AR ligands, both agonists and antagonists, are being widely used to treat pathological conditions, including prostate cancer and hypogonadism. Despite AR being studied widely over the last five decades, the last decade has seen striking advances in the knowledge on AR and discoveries that have the potential to translate to the clinic. This review provides an overview of the advances in AR biology, AR molecular mechanisms of action, and next generation molecules that are currently in development. Several of the areas described in the review are just unraveling and the next decade will bring more clarity on these developments that will put AR at the forefront of both basic biology and drug development.

A comprehensive gas chromatography electron ionization high resolution mass spectrometry study of a new steroidal selective androgen receptor modulator (SARM) compound S42.

The synthetic 20-keto-steroid S42 (1) demonstrated selective androgen receptor modulator (SARM) properties in preclinical studies and, consequently, received growing attention also in the context of sports drug testing programs. Fundamental understanding of the behavior of S42 (1) and of relevant derivatives in gas chromatography-electron ionization MS experiments at high resolution (GC-EI-HRMS) is indispensable to develop a reliable qualitative and quantitative doping control method for S42 (1) and its metabolites in body fluid matrices. We present important fundamental mechanistic data on the EI fragmentation behavior of S42 (1) and of silyl ether derivatives as well as of stable isotope-labelled reference material.

The disordering effect of SARMs on a biomembrane model.

From medicine to sport, selective androgen receptor modulators (SARMs) have represented promising applications. The ability of SARMs to selectively interact with the androgen receptor (AR) indicates that this kind of molecule can interfere with numerous physiological and pathological processes controlled by the AR regulatory mechanism. However, critical concerns in relation to safety and potential side effects of SARMs remain under discussion and investigation. SARMs, being hydrophobic/organic compounds, can be subjected to hydrophobic interactions. In this perspective, we hypothesize that SARMs interact with lipid membranes, producing significant physical and chemical changes that could be associated with several effects that SARMs represent in biological systems. In this context, the effect of SARMs on lipid membranes mediated by non-specific interactions is little explored. Here, we report significant information related to the changes that ostarine, ligandrol, andarine, and cardarine produce in the thermodynamic properties of a lipid biomembrane model. Physical changes and chemical interactions of the systems were investigated by differential scanning calorimetry (DSC), dynamic light scattering (DLS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and theoretical calculations implementing density functional theory (DFT). We demonstrate that ostarine, ligandrol, andarine, and cardarine can strongly interact with a lipid biomembrane model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and accordingly, these molecules can be incorporated into the polar/hydrophobic regions of the lipid bilayer. By employing theoretical calculations, we gained insights into the possible electrostatic interactions between SARMs and phospholipid molecules, enhancing our understanding of the driving forces behind the interactions of SARMs with lipid membranes. Overall, this investigation provides relevant knowledge related to the biophysical-chemical effects that SARMs produce in biomembrane models and could be of practical reference for promising applications of SARMs in medicine and sport.

Synthesis of curcumin derivatives targeting androgen receptor for castration-resistant prostate cancer therapy.

In this work, a series of curcumin derivatives (1a-1h, 2a-2g, and 3a-3c) were synthesized for the suppression of castration-resistant prostate cancer cells. All synthesized compounds were characterized by 1H NMR, 13C NMR, HRMS, and melting point. The in vitro cytotoxicity study shows that compounds 1a, 1e, 1f, 1h, 2g, 3a, and 3c display similar or enhanced cytotoxicity against 22Rv1 and C4-2 cells as compared to ASC-J9, other synthesized compounds display reduced cytotoxicity against 22Rv1 and C4-2 cells as compared to ASC-J9. Molecular docking simulation was performed to study the binding affinity and probable binding modes of the synthesized compounds with androgen receptor. The results show that all synthesized compounds exhibit higher cdocker interaction energies as compared to ASC-J9. Compounds 1h, 2g, and 3c not only show strong cytotoxicity against 22Rv1 and C4-2 cells but also exhibit high binding affinity with androgen receptor. In androgen receptor suppression study, compounds 1f and 2g show similar androgen receptor suppression effect as compared to ASC-J9 on C4-2 cells, compound 3c displays significantly enhanced AR suppression effect as compared to ASC-J9, 1f and 2g. Compounds 1a, 1e, 1f, 1h, 2g, 3a and 3c prepared in this work have significant potential for castration-resistant prostate cancer therapy.

Inhibitors of the transactivation domain of androgen receptor as a therapy for prostate cancer.

The androgen receptor (AR) is a modular transcription factor which functions as a master regulator of gene expression. AR protein is composed of three functional domains; the ligand-binding domain (LBD); DNA-binding domain (DBD); and the intrinsically disordered N-terminal transactivation domain (TAD). AR is transactivated upon binding to the male sex hormone testosterone and other androgens. While the AR may tolerate loss of its LBD, the TAD contains activation function-1 (AF-1) that is essential for all AR transcriptional activity. AR is frequently over-expressed in most prostate cancer. Currently, androgen deprivation therapy (ADT) in the form of surgical or chemical castration remains the standard of care for patients with high risk localized disease, advanced and metastatic disease, and those patients that experience biochemical relapse following definitive primary treatment. Patients with recurrent disease that receive ADT will ultimately progress to lethal metastatic castration-resistant prostate cancer. In addition to ADT not providing a cure, it is associated with numerous adverse effects including cardiovascular disease, osteoporosis and sexual dysfunction. Recently there has been a renewed interest in investigating the possibility of using antiandrogens which competitively bind the AR-LBD without ADT for patients with hormone sensitive, non-metastatic prostate cancer. Here we describe a class of compounds termed AR transactivation domain inhibitors (ARTADI) and their mechanism of action. These compounds bind to the AR-TAD to inhibit AR transcriptional activity in the absence and presence of androgens. Thus these inhibitors may have utility in preventing prostate cancer growth in the non-castrate setting.

A Top-Down Design Approach for Generating a Peptide PROTAC Drug Targeting Androgen Receptor for Androgenetic Alopecia Therapy.

While large-scale artificial intelligence (AI) models for protein structure prediction and design are advancing rapidly, the translation of deep learning models for practical macromolecular drug development remains limited. This investigation aims to bridge this gap by combining cutting-edge methodologies to create a novel peptide-based PROTAC drug development paradigm. Using ProteinMPNN and RFdiffusion, we identified binding peptides for androgen receptor (AR) and Von Hippel-Lindau (VHL), followed by computational modeling with Alphafold2-multimer and ZDOCK to predict spatial interrelationships. Experimental validation confirmed the designed peptide's binding ability to AR and VHL. Transdermal microneedle patching technology was seamlessly integrated for the peptide PROTAC drug delivery in androgenic alopecia treatment. In summary, our approach provides a generic method for generating peptide PROTACs and offers a practical application for designing potential therapeutic drugs for androgenetic alopecia. This showcases the potential of interdisciplinary approaches in advancing drug development and personalized medicine.

Structural mechanism underlying variations in DNA binding by the androgen receptor.

The androgen receptor (AR) is a steroid activated transcription factor which recognizes DNA motifs resembling inverted repeats of a conserved 5'-AGAACA-3'-like hexanucleotides separated by a three-nucleotide spacer from a similar, but less conserved hexanucleotide. Here, we report the structures of the human AR DNA binding domain (DBD) bound to two natural AREs (C3 and MTV) in head-to-head dimer conformations, diffracting at 2.05 Šand 2.25 Å, respectively. These structures help to explain the impact of androgen insensitivity mutations on the structure integrity, DNA binding and DBD dimerization. The binding affinity of the AR DBD to different DNA motifs were measured by the BioLayer Interferometry (BLI) and further validated by Molecular Dynamics (MD) simulations. This shows that the high binding affinity of the first DBD to the upstream 5'-AGAACA-3' motif induces the cooperative binding of the second DBD to the second hexanucleotide. Our data indicate identical interaction of the DBDs to the upstream hexanucleotides, while forming an induced closer contact of the second DBD on the non-canonical hexanucleotides. The variation in binding between the DBD monomers are the result of differences in DNA occupancy, protein-protein interactions, DNA binding affinity, and DNA binding energy profiles. We propose this has functional consequences.

The design, synthesis and bioactivity evaluation of novel androgen receptor degraders based on hydrophobic tagging.

Prostate Cancer (PCa) easily progress to metastatic Castration-Resistant Prostate Cancer (mCRPC) that remains a significant cause of cancer-related death. Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Proteolysis-targeting chimaera (PROTAC) technology based on Hydrophobic Tagging (HyT) represents an intriguing strategy to regulate the function of therapeutically androgen receptor proteins. In the present study, we have designed, synthesized, and evaluated a series of PROTAC-HyT AR degraders using AR antagonists, RU59063, which were connected with adamantane-based hydrophobic moieties by different alkyl chains. Compound D-4-6 exhibited significant AR protein degradation activity, with a degradation rate of 57 % at 5 µM and nearly 90 % at 20 µM in 24 h, and inhibited the proliferation of LNCaP cells significantly with an IC50 value of 4.77 ± 0.26 µM in a time-concentration-dependent manner. In conclusion, the present study lays the foundation for the development of a completely new class of therapeutic agents for the treatment of mCRPC, and further design and synthesis of AR-targeting degraders are currently in progress for better degradation rate.

Discovery of Novel Anti-Resistance AR Antagonists Guided by Funnel Metadynamics Simulation.

Androgen receptor (AR) antagonists are widely used for the treatment of prostate cancer (PCa), but their therapeutic efficacy is usually compromised by the rapid emergence of drug resistance. However, the lack of the detailed interaction between AR and its antagonists poses a major obstacle to the design of novel AR antagonists. Here, funnel metadynamics is employed to elucidate the inherent regulation mechanisms of three AR antagonists (hydroxyflutamide, enzalutamide, and darolutamide) on AR. For the first time it is observed that the binding of antagonists significantly disturbed the C-terminus of AR helix-11, thereby disrupting the specific internal hydrophobic contacts of AR-LBD and correspondingly the communication between AR ligand binding pocket (AR-LBP), activation function 2 (AF2), and binding function 3 (BF3). The subsequent bioassays verified the necessity of the hydrophobic contacts for AR function. Furthermore, it is found that darolutamide, a newly approved AR antagonist capable of fighting almost all reported drug resistant AR mutants, can induce antagonistic binding structure. Subsequently, docking-based virtual screening toward the dominant binding conformation of AR for darolutamide is conducted, and three novel AR antagonists with favorable binding affinity and strong capability to combat drug resistance are identified by in vitro bioassays. This work provides a novel rational strategy for the development of anti-resistant AR antagonists.

The RNA secondary structure of androgen receptor-FL and V7 transcripts reveals novel regulatory regions.

The androgen receptor (AR) is a ligand-dependent nuclear transcription factor belonging to the steroid hormone nuclear receptor family. Due to its roles in regulating cell proliferation and differentiation, AR is tightly regulated to maintain proper levels of itself and the many genes it controls. AR dysregulation is a driver of many human diseases including prostate cancer. Though this dysregulation often occurs at the RNA level, there are many unknowns surrounding post-transcriptional regulation of AR mRNA, particularly the role that RNA secondary structure plays. Thus, a comprehensive analysis of AR transcript secondary structure is needed. We address this through the computational and experimental analyses of two key isoforms, full length (AR-FL) and truncated (AR-V7). Here, a combination of in-cell RNA secondary structure probing experiments (targeted DMS-MaPseq) and computational predictions were used to characterize the static structural landscape and conformational dynamics of both isoforms. Additionally, in-cell assays were used to identify functionally relevant structures in the 5' and 3' UTRs of AR-FL. A notable example is a conserved stem loop structure in the 5'UTR of AR-FL that can bind to Poly(RC) Binding Protein 2 (PCBP2). Taken together, our results reveal novel features that regulate AR expression.

Structural insights into the mechanism of resistance to bicalutamide by the clinical mutations in androgen receptor in chemo-treatment resistant prostate cancer.

Despite advanced diagnosis and detection technologies, prostate cancer (PCa) is the most prevalent neoplasms in males. Dysregulation of the androgen receptor (AR) is centrally involved in the tumorigenesis of PCa cells. Acquisition of drug resistance due to modifications in AR leads to therapeutic failure and relapse in PCa. An overhaul of comprehensive catalogues of cancer-causing mutations and their juxta positioning on 3D protein can help in guiding the exploration of small drug molecules. Among several well-studied PCa-specific mutations, T877A, T877S and H874Y are the most common substitutions in the ligand-binding domain (LBD) of the AR. In this study, we combined structure as well as dynamics-based in silico approaches to infer the mechanistic effect of amino acid substitutions on the structural stability of LBD. Molecular dynamics simulations allowed us to unveil a possible drug resistance mechanism that acts through structural alteration and changes in the molecular motions of LBD. Our findings suggest that the resistance to bicalutamide is partially due to increased flexibility in the H12 helix, which disturbs the compactness, thereby reducing the affinity for bicalutamide. In conclusion, the current study helps in understanding the structural changes caused by mutations and could assist in the drug development process.Communicated by Ramaswamy H. Sarma.

Synthesis, molecular docking and biological evaluation of 1,2,4-oxadiazole based novel non-steroidal derivatives against prostate cancer.

Prostate cancer is one of the most prevalent cancers in men leading to second most death causing cancer in men. Despite the availability of multiple treatment still the prevalence is high for prostate cancer. Steroidal antagonists associated with poor bioavailability, side effects while non-steroidal antagonists show serious side effects like gynecomastia. Therefore, there is a need of potential candidate for the treatment of prostate cancer with better bioavailability, good therapeutic effect and minimal side effects. In the same context, we have designed the series, SP1-SP25 based 3-phenyl-5-styryl-1,2,4-oxadiazole as the core structure. We successfully synthesized all 25 molecules in this series and characterized them using 1H, 13C NMR, and mass spectroscopy. Subsequently, we conducted MTT assays using PC-3 cells and observed that all the compounds exhibited a dose-dependent decrease in cell viability. Notably, compounds SP04, SP16, and SP19 demonstrated a significant decrease in cell viability and exhibited potent activity compared to the other synthesized molecules and standard drug bicalutamide. Among them, SP04 emerged as the one of the most potent compounds with an IC50 value of 238.13 nM and an 89.99 % inhibition of PC-3 cells, compared to synthesized molecules and standard drug bicalutamide. Furthermore, we conducted ROS assays and androgen receptor inhibition assays using the potent compound SP04 and bicalutamide. The results indicated that SP04 increased ROS production and decreased androgen receptor expression dose-dependent manner. Additionally, we conducted a docking study to analyse the interaction patterns within the active site of the androgen receptor. ADMET analysis revealed that all the compounds exhibited favorable physicochemical properties and manageable toxicity profiles.

Rational optimization of a transcription factor activation domain inhibitor.

Transcription factors are among the most attractive therapeutic targets but are considered largely 'undruggable' in part due to the intrinsically disordered nature of their activation domains. Here we show that the aromatic character of the activation domain of the androgen receptor, a therapeutic target for castration-resistant prostate cancer, is key for its activity as transcription factor, allowing it to translocate to the nucleus and partition into transcriptional condensates upon activation by androgens. On the basis of our understanding of the interactions stabilizing such condensates and of the structure that the domain adopts upon condensation, we optimized the structure of a small-molecule inhibitor previously identified by phenotypic screening. The optimized compounds had more affinity for their target, inhibited androgen-receptor-dependent transcriptional programs, and had an antitumorigenic effect in models of castration-resistant prostate cancer in cells and in vivo. These results suggest that it is possible to rationally optimize, and potentially even to design, small molecules that target the activation domains of oncogenic transcription factors.

The triazole fungicide metconazole inhibits the homodimerization of human androgen receptors to suppress androgen-induced transcriptional activation.

We assessed the mechanism of human androgen receptor-mediated endocrine-disrupting effect by a triazole fungicide, metconazole in this study. The internationally validated stably transfected transactivation (STTA) in vitro assay, which was established for determination of a human androgen receptor (AR) agonist/antagonist by using 22Rv1/MMTV_GR-KO cell line, alongside an in vitro reporter-gene assay to confirm AR homodimerization was used. The STTA in vitro assay results showed that metconazole is a true AR antagonist. Furthermore, the results from the in vitro reporter-gene assay and western blotting showed that metconazole blocks the nuclear transfer of cytoplasmic AR proteins by suppressing the homodimerization of AR. These results suggest that metconazole can be considered to have an AR-mediated endocrine-disrupting effect. Additionally, the evidence from this study might help identify the endocrine-disrupting mechanism of triazole fungicides containing a phenyl ring.

A hotspot for posttranslational modifications on the androgen receptor dimer interface drives pathology and anti-androgen resistance.

Mutations of the androgen receptor (AR) associated with prostate cancer and androgen insensitivity syndrome may profoundly influence its structure, protein interaction network, and binding to chromatin, resulting in altered transcription signatures and drug responses. Current structural information fails to explain the effect of pathological mutations on AR structure-function relationship. Here, we have thoroughly studied the effects of selected mutations that span the complete dimer interface of AR ligand-binding domain (AR-LBD) using x-ray crystallography in combination with in vitro, in silico, and cell-based assays. We show that these variants alter AR-dependent transcription and responses to anti-androgens by inducing a previously undescribed allosteric switch in the AR-LBD that increases exposure of a major methylation target, Arg761. We also corroborate the relevance of residues Arg761 and Tyr764 for AR dimerization and function. Together, our results reveal allosteric coupling of AR dimerization and posttranslational modifications as a disease mechanism with implications for precision medicine.

A partially open conformation of an androgen receptor ligand-binding domain with drug-resistance mutations.

Mutations in the androgen receptor (AR) ligand-binding domain (LBD) can cause resistance to drugs used to treat prostate cancer. Commonly found mutations include L702H, W742C, H875Y, F877L and T878A, while the F877L mutation can convert second-generation antagonists such as enzalutamide and apalutamide into agonists. However, pruxelutamide, another second-generation AR antagonist, has no agonist activity with the F877L and F877L/T878A mutants and instead maintains its inhibitory activity against them. Here, it is shown that the quadruple mutation L702H/H875Y/F877L/T878A increases the soluble expression of AR LBD in complex with pruxelutamide in Escherichia coli. The crystal structure of the quadruple mutant in complex with the agonist dihydrotestosterone (DHT) reveals a partially open conformation of the AR LBD due to conformational changes in the loop connecting helices H11 and H12 (the H11-H12 loop) and Leu881. This partially open conformation creates a larger ligand-binding site for AR. Additional structural studies suggest that both the L702H and F877L mutations are important for conformational changes. This structural variability in the AR LBD could affect ligand binding as well as the resistance to antagonists.

Synthesis of hydrocortisone esters targeting androgen and glucocorticoid receptors in prostate cancer in vitro.

Androgen and glucocorticoid receptors have been recently described as key players in processes related to prostate cancer and mainly androgen receptor's inactivation was shown as an effective way for the prostate cancer treatment. Unfortunately, androgen deprivation therapy usually loses its effectivity and the disease frequently progresses into castration-resistant prostate cancer with poor prognosis. The role of the glucocorticoid receptor is associated with the mechanism of resistance; therefore, pharmacological targeting of glucocorticoid receptor in combination with antiandrogen treatment was shown as an alternative approach in the prostate cancer treatment. We introduce here the synthesis of novel 17α- and/or 21-ester or carbamate derivatives of hydrocortisone and evaluation of their biological activity towards androgen and glucocorticoid receptors in different prostate cancer cell lines. A 17α-butyryloxy-21-(alkyl)carbamoyloxy derivative 14 was found to diminish the transcriptional activity of both receptors (in single-digit micromolar range), with comparable potency to enzalutamide towards the androgen receptor, but weaker potency compared to mifepristone towards the glucocorticoid receptor. Lead compound inhibited proliferation and the formation of cell colonies in both androgen and glucocortiocid receptors-positive prostate cancer cell lines in low micromolar concentrations. Candidate compound 14 showed to interact with both receptors in cells and inhibited the translocation of receptors to nucleus and their activation phoshorylation. Moreover, binding to receptor's ligand binding domains was assessed by molecular modelling. Lead compound also induced the accumulation of cells in G1 phase and its combination with enzalutamide was shown to be more effective than enzalutamide alone.

Study of novel androgen receptor V770 variant in androgen insensitivity syndrome patients reveals the transitional state of the androgen receptor ligand binding domain homodimer.

We report the discovery of the androgen receptor missense mutation V770D, that was found in two sisters suffering from complete androgen insensitivity. Experimental validation of AR V770 variants demonstrated that AR V770D was transcriptionally inactive due to the inability to dimerize and a reduced ligand binding affinity. The more conservative AR V770A variant showed a dimerization defect at low levels of DHT with a partial recovery of the transcriptional activity and of the receptor's ability to dimerize when increasing the DHT levels. With V770 located outside of the proposed LBD dimerization interface of the AR LBD homodimer crystal structure, the effects of the V770A mutation on AR dimerization were unexpected. We therefore explored whether the AR LBD dimerization interface would be better described by an alternative dimerization mode based on available human homodimeric LBD crystal structures of other nuclear receptors. Superposition of the monomeric AR LBD in the homodimeric crystal structures of GR, PR, ER, CAR, TRß, and HNF-4α showed that the GR-like LBD dimer model was energetically the most stable. Moreover, V770 was a key energy residue in the GR-like LBD dimer while it was not involved in the stabilization of the AR LBD homodimer according to the crystal structure. Additionally, the observation that 4 AIS mutations impacted the stability of the AR LBD dimer while 16 mutations affected the GR-like LBD dimer, suggested that the AR LBD dimer crystal is a snapshot of a breathing AR LBD homodimer that can transition into the GR-like LBD dimer model.

Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.