N-terminal Domain Inhibitor Research Articles

Inhibition of the angiotensin-converting enzyme N-terminal catalytic domain prevents endogenous opioid degradation in brain tissue.

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin peptides in the periphery, and can also regulate endogenous opioid signaling by degrading enkephalin peptides in the brain. ACE has two catalytic domains, located in the N-terminal or C-terminal region of the protein, but little is known about the roles that these two catalytic domains play in regulating endogenous opioid degradation in brain tissue. Using acute brain slice preparations from mice of both sexes, we developed methods to study degradation of Met-enkephalin-Arg-Phe (MERF) by ACE, and investigated the role of each ACE catalytic domain in MERF degradation. Using mutant mouse lines with functional inactivation of either the N-terminal domain or the C-terminal domain, we incubated acute brain slices with exogenous MERF at a saturating concentration. The degradation MERF to produce Met-enkephalin was only reduced by N-terminal domain inactivation. Additionally, application of a selective N-terminal domain inhibitor (RXP 407) reduced degradation of both exogenously applied and endogenously released MERF, without affecting degradation of endogenously released Met-enkephalin or Leu-enkephalin. Taken together, our results suggest that the ACE N-terminal domain is the primary site of MERF degradation in brain tissue, and that N-terminal domain inhibition is sufficient to reduce degradation of this specific endogenous opioid peptide. Our results have exciting implications for the development of novel pharmacotherapies that target the endogenous opioid system to treat psychiatric and neurological disorders.

Hsp90α promotes lipogenesis by stabilizing FASN and promoting FASN transcription via LXRα in hepatocellular carcinoma.

Excessive lipid accumulation promotes the occurrence and progression of hepatocellular carcinoma (HCC), accompanied by high levels of fatty acid synthetase (FASN) and more active lipogenesis. Heat shock protein 90 (Hsp90) acts as a chaperone to maintain the stability and activity of the client proteins. Studies have revealed that Hsp90 regulates the lipid metabolism of HCC, but the effect of Hsp90 on FASN still remains unknown. This study aims to discover the mechanism of Hsp90 inhibition on lipid accumulation and investigate the different effects of Hsp90 N-terminal domain inhibitor STA9090 and C-terminal domain inhibitor novobiocin (NB) on FASN protein stability and transcription pathway in HCC. We found that HCC cells tended to store lipids, which could be disrupted by Hsp90 inhibitors in vivo and in vitro. High levels of Hsp90α and FASN in tumor tissue had correlation with poor prognosis of HCC patients and Hsp90α interacted with FASN to maintain its protein stability. Furthermore, N-terminal domain of Hsp90α was essential for process of sterol regulatory element binding protein 1 (SREBP1) to activate FASN transcription and Hsp90α prevented proteasomal degradation of liver X receptor α (LXRα) to upregulate FASN transcription via LXRα/SREBP1 axis. Our data reveals that Hsp90α promotes lipid accumulation by increasing the protein stability and FASN mRNA transcription, and can be alleviated by Hsp90 inhibitors, which provides a theoretical basis for Hsp90-targeted therapy on lipid metabolism in HCC.

N-terminal domain of androgen receptor is a major therapeutic barrier and potential pharmacological target for treating castration resistant prostate cancer: a comprehensive review.

The incidence rate of prostate cancer (PCa) has risen by 3% per year from 2014 through 2019 in the United States. An estimated 34,700 people will die from PCa in 2023, corresponding to 95 deaths per day. Castration resistant prostate cancer (CRPC) is the leading cause of deaths among men with PCa. Androgen receptor (AR) plays a critical role in the development of CRPC. N-terminal domain (NTD) is the essential functional domain for AR transcriptional activation, in which modular activation function-1 (AF-1) is important for gene regulation and protein interactions. Over last 2decades drug discovery against NTD has attracted interest for CRPC treatment. However, NTD is an intrinsically disordered domain without stable three-dimensional structure, which has so far hampered the development of drugs targeting this highly dynamic structure. Employing high throughput cell-based assays, small-molecule NTD inhibitors exhibit a variety of unexpected properties, ranging from specific binding to NTD, blocking AR transactivation, and suppressing oncogenic proliferation, which prompts its evaluation in clinical trials. Furthermore, molecular dynamics simulations reveal that compounds can induce the formation of collapsed helical states. Nevertheless, our knowledge of NTD structure has been limited to the primary sequence of amino acid chain and a few secondary structure motif, acting as a barrier for computational and pharmaceutical analysis to decipher dynamic conformation and drug-target interaction. In this review, we provide an overview on the sequence-structure-function relationships of NTD, including the polymorphism of mono-amino acid repeats, functional elements for transcription regulation, and modeled tertiary structure of NTD. Moreover, we summarize the activities and therapeutic potential of current NTD-targeting inhibitors and outline different experimental methods contributing to screening novel compounds. Finally, we discuss current directions for structure-based drug design and potential breakthroughs for exploring pharmacological motifs and pockets in NTD, which could contribute to the discovery of new NTD inhibitors.

Updates in Precision Oncology in Prostate and Urothelial Cancer

Basic science, translational, and clinical genomic advances in prostate and urothelial cancer have therapeutic implications. Poly(ADP-ribose) polymerase (PARP) inhibitors target cancer cells with homologous recombination repair genomic deficiencies to induce synthetic lethality. PARP inhibitors are approved for BRCA-altered castration resistant prostate cancer treatment either as single agent or in combination with novel hormonal therapies. Another genomic target in prostate cancer is the androgen receptor, especially overcoming resistance via the AR-V7 splice variant and other mechanisms. Strategies for overcoming this resistance, including N-terminal domain inhibitors and PROTAC degraders, are under investigation. Immune checkpoint inhibition is a treatment option for metastatic castrate resistant prostate cancer for patients with mismatch repair deficiency, and neoantigen-based vaccines are under development to harness the immune system to fight prostate cancer. In urothelial cancer, FGFR3 is an important prognostic and predictive marker, often indicative of luminal tumors by gene expression profiling. Erdafitinib targets FGFR alterations and is approved for treatment of refractory metastatic urothelial cancer. Antibody-drug conjugates, including enfortumab vedotin and sacituzumab govitecan, are additional targeted approaches utilized to manage urothelial cancer.

SAT022 A Mechanism Of Resistance To Novel Inhibitors Of The N-Terminal Domain Of Androgen Receptor

Abstract Disclosure: J. Setiawan: None. N.R. Mawji: None. A.H. Tien: None. R.J. Andersen: Stock Owner; Self; ESSA Pharma. M.D. Sadar: Stock Owner; Self; ESSA Pharma. Background: Androgen receptor (AR) is a steroid hormone receptor and a major therapeutic target in prostate cancer. Antiandrogen therapies directed at the AR C-terminal ligand-binding domain (LBD) provide initial benefit to patients with advanced prostate cancer, but they eventually progress to lethal metastatic castration-resistant prostate cancer (CRPC). AR N-terminal domain (NTD) inhibitors are a novel class of antagonists that were developed to overcome resistance mechanisms related to the AR-LBD, including splice variants lacking the LBD (AR-V7). While mutations in the AR-LBD have been well-characterized, the impact of NTD mutations on AR transcriptional activity and drug efficacy remains unclear. The mutation W435L was discovered from clinical samples of prostate cancer from patients treated with antiandrogens. This mutation stabilizes interaction between the AR’s NTD and LBD (N/C interaction) to increase AR transcriptional activity. Tryptophan residues W435 and W397 in AR NTD are essential in the binding site for the AR-NTD inhibitor EPI-7386 that is currently in clinical trials. We hypothesize that tryptophan mutations within the EPI-7386 binding site of AR are a potential mechanism of resistance. Methods: Site-directed mutagenesis was used to create expression vectors with W435 and W397 point mutations in AR. Plasmids were transfected into AR-negative CV-1 cells to compare the transcriptional activity of wild-type (WT) versus mutant ARs. IC50s for each inhibitor were generated for androgen-induced reporter gene constructs. A mammalian two-hybrid system assay was used to assess the impact of inhibitors and AR mutations on N/C interaction. Results: The AR-W435L mutation led to higher IC50s to inhibit androgen-induced MMTV-luciferase activity by NTD inhibitors and antiandrogens. Differences in N/C interaction were observed between WT and mutant ARs treated with EPI-7386. Importantly, the ability of another EPI analog, EPI-7170, to decrease AR-N/C interaction was not significantly impacted by W435L. These data support that loss of efficacy with EPI-7386 is not across the entire class of EPI analogs and may be specific to EPI-7386. Conclusion: Tryptophan mutations in the EPI-7386 binding site are predictive of resistance to this specific antagonist and do not broadly apply to the entire class of EPI analogs. Key differences were revealed between AR-LBD and AR-NTD inhibitors that support the recruitment of different tau regions in the NTD. Presentation: Saturday, June 17, 2023

Novel breakthroughs in advanced prostate cancer management

ABSTRACT Introduction Although PSA screening has been rationalized, prostate cancer continues to have the highest incidence rate in 2021, and alone accounts for 26% of cancer diagnoses in men. A thorough review of the medical literature highlights a vast array of approved and investigational treatments for prostate cancer. Thus, selecting the best treatment option for the appropriate patient at the right time is crucial. Hence, biomarkers help in defining optimal patient stratification, revealing potential processes through which a drug might exert its impact and aid in the tailoring of treatments for efficient personalized medicine. Areas covered This article is a pragmatic review of novel prostate cancer therapies that can help guide clinicians in tackling prostate cancer with the latest treatments. Expert opinion Local radiotherapy has proven to be a game changer for low burden, de novo metastatic prostate cancer. Androgen deprivation therapy continues to be the ultimate treatment. Delaying resistance to these agents will undoubtedly be a breakthrough in the treatment of prostate cancer. When it comes to metastatic castrate-resistant disease, treatment options become narrower. PARP inhibitors and N-terminal domain inhibitors offer new hope and have a synergistic effect, with immunotherapy adding promising agents to the therapeutic arsenal.

Differential Gene Expression Profiles between N-Terminal Domain and Ligand-Binding Domain Inhibitors of Androgen Receptor Reveal Ralaniten Induction of Metallothionein by a Mechanism Dependent on MTF1.

Simple SummaryInhibition of the androgen receptor (AR) remains the mainstay treatment for prostate cancer. All current therapies involving AR inhibition either directly or indirectly target its ligand-binding domain (LBD). We have developed the first novel compounds which target the N-terminal domain, (NTD) a region which is essential for AR transcriptional activity. First-generation ralaniten (NCT02606123), and second-generation EPI-7386 (NCT04421222) remain the only AR-NTD inhibitors to progress to clinical trials. Here we aim to characterize differences between different classes of AR antagonists targeting the AR-LBD and the AR-NTD, as well as next generation AR-NTD inhibitors. An incidental finding was that ralaniten was uniquely associated with increased metallothionein expression which was independent of AR activity. Instead, expression of metallothionein genes was driven by MTF-1 indicating a potential off-target effect. Neither AR-LBD inhibitor enzalutamide nor second-generation AR-NTD inhibitor EPI-7170 had this effect. This work has important implications for the development of novel AR-NTD inhibitors.Hormonal therapies for prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD). Clinical development for inhibitors that bind to the N-terminal domain (NTD) of AR has yielded ralaniten and its analogues. Ralaniten acetate is well tolerated in patients at 3600 mgs/day. Clinical trials are ongoing with a second-generation analogue of ralaniten. Binding sites on different AR domains could result in differential effects on AR-regulated gene expression. Here, we provide the first comparison between AR-NTD inhibitors and AR-LBD inhibitors on androgen-regulated gene expression in prostate cancer cells using cDNA arrays, GSEA, and RT-PCR. LBD inhibitors and NTD inhibitors largely overlapped in the profile of androgen-induced genes that they each inhibited. However, androgen also represses gene expression by various mechanisms, many of which involve protein–protein interactions. De-repression of the transcriptome of androgen-repressed genes showed profound variance between these two classes of inhibitors. In addition, these studies revealed a unique and strong induction of expression of the metallothionein family of genes by ralaniten by a mechanism independent of AR and dependent on MTF1, thereby suggesting this may be an off-target. Due to the relatively high doses that may be encountered clinically with AR-NTD inhibitors, identification of off-targets may provide insight into potential adverse events, contraindications, or poor efficacy.

Drugging the Undruggable: Targeting the N-Terminal Domain of Nuclear Hormone Receptors.

This chapter focuses on the development of drugs targeting the N-terminal domain of nuclear hormone receptors, using progress with the androgen receptor as an example. Historically, development of therapies targeting nuclear hormone receptors has focused on the folded C-terminal ligand-binding domain. Therapies were traditionally not developed to target the intrinsically disordered N-terminal domain as it was considered "undruggable". Recent developments have now shown it is possible to direct therapies to the N-terminal domain. This chapter will provide an introduction of the structure and function of the domains of nuclear hormone receptors, followed by a discussion of the rationale supporting the development of N-terminal domain inhibitors. Chemistry and mechanisms of action of small molecule inhibitors will be described with emphasis on N-terminal domain inhibitors developed to the androgen receptor including those in clinical trials.

Abstract P192: Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract Background: Androgen receptor (AR) signaling is a main driver of prostate cancer progression and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops, and new methods of inhibiting the AR pathway are needed. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients (NCT04421222). Here we further characterized the binding to AR NTD and the mechanism of action of EPI-7386. Methods: Target engagement was measured by Cellular Thermal Shift Assay (CETSA) and two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. qPCR, NanoString, and RNA sequencing were used to explore the activity of EPI-7386 on the AR transcriptome. To determine the effect of EPI-7386 on AR genomic occupancy, Chromatin immunoprecipitation sequencing (ChIP-seq) was performed. Results: We confirmed target engagement of EPI-7386 with an LBD truncated AR variant by CETSA using a cell line which expresses only AR-V567es, suggesting the interaction of EPI-7386 with AR NTD. In the same cell line, AR antagonist enzalutamide that binds to AR LBD showed no target engagement with AR-V567es. Furthermore, 2D NMR study results demonstrate an interaction of EPI-7386 with amino acid residues located in the transcription activation unit 5 (Tau-5) region of the AR NTD, a region which has been described to be involved in interactions with transcriptional cofactors such as CBP/p300. EPI-7386 strongly impaired the transcriptional activity and gene expression driven exclusively by LBD truncated AR variants including AR-V567es and AR-V7 and decreased cell viability. EPI-7386 has been shown to suppress the AR regulated transcriptome and the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-regulated gene expression. The analysis of the AR cistrome by ChIP-seq showed that EPI-7386 displaces genome-wide androgen induced AR binding and the combination with enzalutamide completely abrogated AR binding. Conclusion: EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Berenger Biannic, Han-Jie Zhou, Ronan Le Moigne. Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P192.

A phase 1 study to assess the safety, pharmacokinetics, and anti-tumor activity of the androgen receptor n-terminal domain inhibitor epi-506 in patients with metastatic castration-resistant prostate cancer

EPI-506 is the first of a new class of drugs targeting the N-terminal domain (NTD) of the androgen receptor (AR), potentially overcoming known resistance mechanisms to androgen receptor pathway inhibitors (ARPIs) among men with metastatic castration resistant prostate cancer (mCRPC). Patients with mCRPC who had progressed on prior ARPI were enrolled in this phase 1 open-label, adaptive 3 + 3 dose escalation study. The primary outcome was safety and tolerability of oral EPI-506. Secondary objectives included determination of the maximal tolerated dose (MTD), pharmacokinetic profile, and antitumor efficacy. 28 mCRPC patients were enrolled into 7 dose cohorts of EPI-506 ranging from 80-3600mg given once daily and 1800mg given twice daily. Six DLTs occurred in 4 patients; Grade 4 elevated amylase; Grade 3 abdominal pain; Grade 3 elevated ALT and Grade 3 elevated AST; Grade 2 nausea and Grade 1 vomiting which resulted in study drug intake of < 75% of the expected dose during the DLT assessment period. The most common drug-related adverse events included diarrhea, nausea and fatigue. Six patients had a PSA decline not meeting PSA response criteria. The study was terminated prior to reaching the MTD due to poor oral bioavailability. This phase 1 trial established the safety of EPI-506 and provides proof of concept for targeting the AR NTD. Next generation compounds with improved bioavailability and potency are in clinical development.

Structure-Activity Relationships of Benzothiazole-Based Hsp90 C-Terminal-Domain Inhibitors.

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC50, with the most potent being compounds 5g and 9i (IC50, 2.8 ± 0.1, 3.9 ± 0.1 μM, respectively). Based on these results, a ligand-based structure–activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response.

Abstract 1209: Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor

Abstract The androgen receptor (AR) is a key driver in the growth of prostate cancer and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients. Here we report the results of a comprehensive in vitro characterization of its mechanism of action. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. Target engagement was measured by a Cellular Thermal Shift Assay (CETSA). Both Nanostring and RNAseq were used to explore the activity of EPI-7386 on the AR transcriptome. Chromatin immunoprecipitation (ChIP)-seq and ChIP-qPCR were carried out to determine the effect of EPI-7386 on AR genomic occupancy. EPI-7386 exhibited potent activity in inhibiting full-length AR (AR-FL) driven transcriptional activity and also strongly impaired the transcriptional activity and the viability of cellular models driven exclusively by truncated AR protein. Using CETSA, we confirmed that EPI-7386 induced a thermal shift of both AR-FL and AR-V7 (lacking the AR LBD) in LNCaP and LNCaP95, respectively, which is an indication of AR target engagement by EPI-7386. ChIP analyses allowed a deeper understanding of epigenetic and transcriptional regulation driven by EPI-7386. It showed EPI-7386 inhibits androgen-activated AR binding to target gene loci such as KLK3. Additionally, EPI-7386 suppresses AR-regulated target gene expression in a comparable manner as lutamides in three human prostate cancer cell lines, LNCaP, 22Rv1, and VCaP, with a few notable exceptions. As a consequence, the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-associated transcriptional activity in both LNCaP and VCaP cells. In AR-V7 driven cell lines, LNCaP95 and 22Rv1, EPI-7386 showed superior activity to enzalutamide in inhibiting AR-regulated genes expression. In conclusion, EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients. The Phase I dose escalation first in human clinical trial of EPI-7386 single agent (NCT04421222) is currently enrolling. Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Han-Jie Zhou, Ronan Le Moigne. Comprehensive in vitro characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1209.

Preclinical and clinical pharmacology of EPI-7386, an androgen receptor N-terminal domain inhibitor for castration-resistant prostate cancer.

119 Background: EPI-7386 is the newest of the “anitens”, a new class of compounds designed to inhibit androgen receptor activity by binding to the N-terminal domain (NTD) of the AR. Through this novel method of AR inhibition, anitens can block AR transcription even in the presence of AR ligand-binding domain (LBD) resistance mechanisms including point mutations and splice variants. Compared to the first generation aniten, EPI-506, which showed poor pharmacokinetic properties in patients, EPI-7386 is metabolically stable in vitro and in vivo. A Phase 1 clinical trial of EPI-7386 in metastatic castration-resistant prostate cancer patients failing standard of care therapies is ongoing and the pharmacokinetic properties of the drug in preclinical models as well as in the initial cohort of patients are presented. Methods: The metabolic stability of EPI-7386 was evaluated in vitro in mouse, rat, dog, monkey, and human hepatocytes. Projected PK parameters in humans were estimated from in vitro and in vivo clearance correlation (IVIVC). Induction of CYP isoforms was evaluated in human hepatocyte cultures. In patients, plasma concentrations of EPI-7386 were determined by LC-MS-MS, and 4-beta-hydroxycholesterol levels in plasma were followed over time as an indirect indicator of CYP3A induction. Results: In vitro hepatocyte studies demonstrated good metabolic stability for EPI-7386 with an in vitro half-life &gt; 360 min. In animal PK studies, the terminal half-life of EPI-7386 was approximately 5.8 hours in mouse, 4.9 hours in rat, 13.4 hours in dog and the plasma clearance was low across species. The oral bioavailability of EPI-7386 ranged from 33–112% in mouse to &gt; 100% in rat and dog. Using IVIVC, a predicted human clearance of 0.16–0.39 mL/min/kg was calculated for EPI-7386, which was in line with allometric scaling from animal PK parameters. Human PK profiles of different doses of EPI-7386 were simulated using predicted oral bioavailability, clearance, and volume of distribution. Cmax and AUC0–24h for the Phase 1 first-in-human study (NCT04421222) starting dose of 200 mg dose were predicted to be 6,915 ng/mL and 137,278 ng•h/mL respectively. A comparison between estimated PK parameters and actual values observed in the first patient cohort will be presented. Human hepatocyte CYP induction studies showed that EPI-7386 is not an inducer of CYP1A2 but may have the potential to induce CYP2B6 and CYP3A4. A comparison of 4-beta-hydroxy cholesterol levels measured during the phase 1 will be presented along with a comparison drawn from in vitro models. Conclusions: Pre-clinical characterization predicts that EPI-7386 has the appropriate PK and metabolic properties to afford exposure in patients at potentially efficacious levels following once-daily oral administration. PK measurements in the initial cohort of patients treated in the Phase 1 study will be presented. Clinical trial information: NCT04421222.

Computational guided identification of novel potent inhibitors of N-terminal domain of nucleocapsid protein of severe acute respiratory syndrome coronavirus 2

The Coronavirus Disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 is an exceptionally contagious disease that leads to global epidemics with elevated mortality and morbidity. There are currently no efficacious drugs targeting coronavirus disease 2019, therefore, it is an urgent requirement for the development of drugs to control this emerging disease. Owing to the importance of nucleocapsid protein, the present study focuses on targeting the N-terminal domain of nucleocapsid protein from severe acute respiratory syndrome coronavirus 2 to identify the potential compounds by computational approaches such as pharmacophore modeling, virtual screening, docking and molecular dynamics. We found three molecules (ZINC000257324845, ZINC000005169973 and ZINC000009913056), which adopted a similar conformation as guanosine monophosphate (GMP) within the N-terminal domain active site and exhibiting high binding affinity (>−8.0 kcalmol−1). All the identified compounds were stabilized by hydrogen bonding with Arg107, Tyr111 and Arg149 of N-terminal domain. Additionally, the aromatic ring of lead molecules formed π interactions with Tyr109 of N-terminal domain. Molecular dynamics and Molecular mechanic/Poisson–Boltzmann surface area results revealed that N-terminal domain – ligand(s) complexes are less dynamic and more stable than N-terminal domain – GMP complex. As the identified compounds share the same corresponding pharmacophore properties, therefore, the present results may serve as a potential lead for the development of inhibitors against severe acute respiratory syndrome coronavirus 2. Communicated by Ramaswamy H. Sarma

Isolation and characterization of castration-resistant prostate cancer LNCaP95 clones.

The androgen receptor (AR) is a validated therapeutic target for prostate cancer and has been a focus for drug development for more than six decades. Currently approved therapies that inhibit AR signaling, such as enzalutamide, rely solely on targeting the AR ligand-binding domain and, therefore, have limited efficacy on prostate cancer cells that express truncated, constitutively active AR splice variants (AR-Vs). The LNCaP95 cell line is a human prostate cancer cell line that expresses both functional full-length AR and AR-V7. LNCaP95 is a heterogeneous cell population that is resistant to enzalutamide, with its proliferation dependent on transcriptionally active AR-V7. The purpose of this study was to identify a LNCaP95 clone that would be useful for evaluating therapies for their effectiveness against enzalutamide-resistant prostate cancer cells. Seven clones from the LNCaP95 cell line were isolated and characterized using morphology, in vitro growth rate, and response to ralaniten (AR N-terminal domain inhibitor) and enzalutamide (antiandrogen). In vivo growth of the clones as subcutaneous xenografts was evaluated in castrated immunodeficient mice. All of the clones maintained the expression of full-length AR and AR-V7. Cell proliferation of the clones was insensitive to androgen and enzalutamide but importantly was inhibited by ralaniten, which is consistent with AR-Vs driving the proliferation of parental LNCaP95 cells. In castrated immunodeficient animals, the growth of subcutaneous xenografts of the D3 clone was the most reproducible compared to the parental cell line and other clones. These data support that the enzalutamide-resistant LNCaP95-D3 subline may be suitable as a xenograft tumor model for preclinical drug development with improved reproducibility.

545P Preclinical profile of EPI-7386, a second-generation N-terminal domain androgen receptor inhibitor for the treatment of prostate cancer

Disease progression in metastatic castration-resistant prostate cancer (mCRPC) patients largely remains androgen receptor (AR) dependent. Resistance inevitably arises to current anti-androgens through numerous ligand binding domain (LBD)-associated mechanisms. EPI-7386 is a second-generation aniten which inhibits AR transcriptional activity by binding to the AR N-terminal domain (NTD), thus bypassing these resistance mechanisms. A phase I trial in patients with anti-androgen resistant mCRPC is currently being initiated. The preclinical anti-tumor and safety profile of EPI-7386 will be presented. EPI-7386 potency and selectivity was assessed using reporter and viability assays in cellular models. Pharmacodynamic markers were measured by qPCR, NanoString and RNAseq. The pre-clinical safety profile was assessed both in in vitro assays and in rat and dog toxicology studies. EPI-7386 has an IC50 of 421 nM in LNCaP reporter assays, it inhibited both AR full-length and AR-V7 dependent gene expression and maintained antitumor activity in AR-V7-expressing tumor models, including in in vivo models resistant to enzalutamide (ENZ). Transcriptomic analysis of EPI-7386 revealed on-target anti-AR activity, with a profile overlapping but distinct from ENZ, while the combination of the two agents demonstrated broader and deeper AR pathway inhibition. IND-enabling studies in rats and dogs showed on-target activity with good tolerability at drug exposures several fold higher than those active in tumor xenograft models. PK simulations suggest predicted tolerated and active doses can be achieved during the phase I dose escalation. EPI-7386, a second-generation aniten, is a potent AR NTD inhibitor, with a favorable safety and ADME profile, good pharmaceutical characteristics, and a long predicted human half-life supporting once-daily oral dosing with tablets. In vivo, EPI-7386 demonstrated anti-tumor activity as a single agent in the setting of anti-androgen clinical resistance and enhanced efficacy in combination therapy. A phase I study is scheduled to begin Q2 2020.

In silico screening of hundred phytocompounds of ten medicinal plants as potential inhibitors of nucleocapsid phosphoprotein of COVID-19: an approach to prevent virus assembly

Currently, there is no specific treatment to cure COVID-19. Many medicinal plants have antiviral, antioxidant, antibacterial, antifungal, anticancer, wound healing etc. Therefore, the aim of the current study was to screen for potent inhibitors of N-terminal domain (NTD) of nucleocapsid phosphoprotein of SARS-CoV-2. The structure of NTD of RNA binding domain of nucleocapsid phosphoprotein of SARS coronavirus 2 was retrieved from the Protein Data Bank (PDB 6VYO) and the structures of 100 different phytocompounds were retrieved from Pubchem. The receptor protein and ligands were prepared using Schrodinger’s Protein Preparation Wizard. Molecular docking was done by using the Schrodinger’s maestro 12.0 software. Drug likeness and toxicity of active phytocompounds was predicted by using Swiss adme, admetSAR and protox II online servers. Molecular dynamic simulation of the best three protein- ligand complexes (alizarin, aloe-emodin and anthrarufin) was performed to study the interaction stability. We have identified three potential active sites (named as A, B, C) on receptor protein for efficient binding of the phytocompounds. We found that, among 100 phytocompounds, emodin, aloe-emodin, anthrarufin, alizarine, and dantron of Rheum emodi showed good binding affinity at all the three active sites of RNA binding domain of nucleocapsid phosphoprotein of COVID-19.The binding energies of emodin, aloe-emodin, anthrarufin, alizarine, and dantron were −8.299, −8.508, −8.456, −8.441, and −8.322 Kcal mol−1 respectively (site A), −7.714, −6.433, −6.354, −6.598, and −6.99 Kcal mol−1 respectively (site B), and −8.299, 8.508, 8.538, 8.841, and 8.322 Kcal mol−1 respectively (site C). All the active phytocompounds follows the drug likeness properties, non-carcinogenic, and non-toxic. Theses phytocompounds (alone or in combination) could be developed into effective therapy against COVID-19. From MD simulation data, we found that all three complexes of 6VYO with alizarin, aloe-emodin and anthrarufin were stable up to 50 ns. These phytocompounds can be tested further for in vitro or in vivo and used as a potential drug to cure SARS-CoV-2 infection. Communicated by Ramaswamy H. Sarma

Abstract 1953: Pre-clinical development of the second-generation N-terminal domain androgen receptor inhibitor, EPI-7386, for the treatment of prostate cancer

Abstract Background: The vast majority of metastatic castration-resistant prostate cancers (mCRPC) progress with rising prostate-specific antigen, underlying a persistent dependence on the androgen receptor (AR) pathway. Despite standard of care treatment targeting the AR axis, anti-androgen resistance inevitably arise and involve mechanisms including AR gene amplification, ligand-binding domain (LBD) mutations and expression of constitutively active AR splice variants lacking the LBD (e.g. AR-V7). Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its transcriptional activity even in the presence of LBD-driven resistance. EPI-7386 is a potent and stable second generation NTD inhibitor (Aniten) currently in preclinical development. The efficacy, safety profile and distinct mechanism of action of this molecule in LBD inhibitor resistant CRPC models will be presented. Methods: EPI-7386 potency was assessed using reporter cellular models, or viability assays in a variety of cell lines, expressing or not the AR. Pharmacodynamic markers of activity in AR full length or AR-V7 driven models was determined by qPCR or RNAseq. The stability and selectivity of the molecule were characterized with screening and functional assays, while safety was assessed in specific IND enabling studies. Results: EPI-7386 has an IC50 of 421 nM in LNCaP reporter assays, a similar range to the most active LBD inhibitors, but contrary to these drugs, antitumor activity was maintained in models of CRPC expressing AR-V7. As expected, no activity was observed in non-AR driven models. EPI-7386 can inhibit effectively the expression of AR full length driven genes (PSA, FKBP5 or STEAP4), but can also modulate the expression of genes driven by AR V7, including UBE2C or B4GALT1. Additionally, EPI-7386 also demonstrated remarkable activity in several models in vivo, including a CRPC patient derived xenograft (PDX) resistant to enzalutamide (ENZ). Interestingly, RNAseq analysis showed on target activity, but with a different transcriptomic profile than ENZ, which suggests that the combination of both LBD and NTD AR inhibition could provide more robust and thorough inhibition of the AR-pathway. The enhanced activity of such combination was confirmed in vivo in the CRPC model VCaP. IND-enabling studies demonstrated on target activity, and well-tolerated profile for EPI-7386, supporting an IND filing in 1Q 2020. Early clinical development plans including efficacy endpoints will be presented. Conclusions: The second generation aniten compound EPI-7386 is more active and metabolically stable than EPI-506. It has a favorable safety and ADME profile, with predicted long half-life in human, supporting once daily dose. In vivo, EPI-7386 demonstrated potential as a single agent in overcoming anti-androgen clinical resistance as well as in combination therapy. The clinical strategy supporting the development of this new generation of aniten will be discussed. Citation Format: Nan Hyung Hong, Ronan Le Moigne, C. Adriana Banuelos, Nasrin R. Mawji, Teresa Tam, Jun Wang, Raymond J. Andersen, Alessandra Cesano, Marianne D. Sadar, Han-Jie Zhou, Peter Virsik. Pre-clinical development of the second-generation N-terminal domain androgen receptor inhibitor, EPI-7386, for the treatment of prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1953.

IND candidate EPI-7386 as an N-terminal domain androgen receptor inhibitor in development for the treatment of prostate cancer.

142 Background: The androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease. Anti-androgen resistance mechanisms include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively active AR splice variants lacking the LBD (e.g. AR-V7). Selective inhibition of the N-terminal domain (NTD) of the AR can inhibit its’ transcriptional activity even in the presence of LBD-driven resistance. In a phase I trial, the first-generation AR NTD inhibitor (aniten) EPI-002, demonstrated minor PSA declines in mCRPC patients. The efficacy and safety profile of second generation aniten IND-candidate EPI-7386 will be presented. Methods: Novel aniten chemical structures were developed to increase molecule potency using a wide variety of CRPC models. Similarly, the stability and selectivity of the molecule were characterized with screening and functional assays. IND-enabling studies further support the compounds’ safety profile. Results: EPI-7386 demonstrated a 20-fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human hepatocytes and across animal species. In vitro proliferation assays demonstrated on-target activity across a panel of prostate cancer cell lines, with activity in AR-V7-driven cellular models. EPI-7386 was able to induce tumor regression in CRPC xenografts and show superiority to enzalutamide (ENZ) in ENZ resistant models. In addition, the combination of ENZ with EPI-7386 demonstrated a more robust antitumor response. IND-enabling studies demonstrate a safe and well-tolerated profile supporting an IND filing in 1Q 2020. Pharmacodynamic markers specific to anitens will be presented, in addition to early clinical development plans. Conclusions: The next generation aniten compound EPI-7386 is more active and metabolically stable than EPI-002. It demonstrated potential as a single agent in overcoming anti-androgen clinical resistance as well as in combination therapy in earlier stages of the disease. The clinical strategy supporting the development of this new generation of aniten will be discussed.

Abstract B117: Treatment of castrated resistant prostate cancer with EPI-7386, a second generation N-terminal domain androgen receptor inhibitor

Abstract Background: The androgen receptor (AR) pathway drives most metastatic castration-resistant prostate cancers (mCRPC) even in late stages of the disease and resistance to ligand binding domain (LBD)-linked therapies inevitably emerges. Mechanisms of resistance to anti-androgens include AR gene amplification, C-terminal ligand-binding domain (LBD) mutations and expression of constitutively-active truncated AR splice variants lacking the LBD (e.g. AR-V7). Inhibition of the N-terminal domain (NTD) of the AR can inhibit transcriptional activity even in the presence of LBD-driven resistance. A Phase I clinical trial of the first-generation AR NTD inhibitor, EPI-506, demonstrated minor PSA declines in anti-androgen refractory mCRPC patients, revealing the need for more potent and metabolically stable NTD inhibitors. EPI-7386 represents a second generation of NTD inhibitors (Anitens) that are more active and more metabolically stable than EPI-506. Methods: Chemical structure activity relationships were explored to increase molecule potency while maintaining target specificity using a wide variety of CRPC models in vivo and in vitro. The stability and selectivity of the molecules were characterized with a panel of selective screening and functional assays. The mechanism of action and pathway engagement markers were followed by qPCR and RNAseq. Results: EPI-7386 was chosen as the IND candidate. The molecule demonstrated a 20-fold improvement in AR-driven cellular potency compared to EPI-002, while being highly stable in human and animal hepatocytes. In vitro proliferation assays demonstrated on-target activity across a panel of prostate cancer cell lines, with activity demonstrated in AR-V7-driven cellular models. The antiproliferative effect correlated with the inhibitory effect on AR driven genes, including specifically AR-V7 driven genes such as UBE2C. EPI-7386 controlled tumor growth and induced tumor regressions in several CRPC xenografts, including AR-V7-driven 22Rv1 and enzalutamide resistant LNCaP95 models, as well as the VCaP model. Importantly, the combination of enzalutamide with EPI-7386 demonstrated a more robust and consistent PSA and antitumor response in the VCaP model. EPI-7386 was well tolerated in animal models, and therapeutic levels could be achieved without displaying signs of toxicity in a 14-day rat study. Pharmacodynamic markers will be presented, in addition to their incorporation in the future clinical plan. Conclusions: The next generation aniten compound EPI-7386 is more active and metabolically stable than EPI-506. It shows on-target activity in AR full length and AR-V7 driven models, and demonstrates a high therapeutic index in preclinical models. As a single agent, EPI-7386 may overcome anti-androgen clinical resistance in advanced mCRPC as well as potentially in combination therapy with anti-androgens in earlier stages of the disease. The clinical strategy supporting the development of this Aniten N-terminal domain inhibitor of AR will be discussed. Citation Format: Ronan Le Moigne, C. Adriana Banuelos, Nasrin R Mawji, Teresa Tam, Jun Wang, Kunzhong Jian, Raymond J Andersen, Alessandra Cesano, Marianne D Sadar, Han-Jie Zhou, Peter Virsik. Treatment of castrated resistant prostate cancer with EPI-7386, a second generation N-terminal domain androgen receptor inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B117. doi:10.1158/1535-7163.TARG-19-B117