AR Ligands Research Articles

Safety and clinical activity of BMS-986365 (CC-94676), a dual androgen receptor ligand-directed degrader and antagonist, in heavily pretreated patients with metastatic castration-resistant prostate cancer

Metastatic castration-resistant prostate cancer (mCRPC) that progresses on androgen receptor pathway inhibitors (ARPIs) may continue to be driven by AR signaling. BMS-986365 is an orally administered ligand-directed degrader targeting the AR via a first-in-class dual mechanism of AR degradation and antagonism. CC-94676-PCA-001 (NCT04428788) is a phase I multicenter study of BMS-986365 in patients with progressive mCRPC. Patients who progressed on androgen deprivation therapy, one or more ARPIs, and taxane chemotherapy (unless declined/ineligible) were enrolled. The study included dose escalation (part A) and expansion (part B) of BMS-986365 up to 900 mg twice daily. Primary objectives were safety and tolerability, and to define maximum tolerated dose and/or recommended phase II dose. Key secondary endpoints included decline in prostate-specific antigen ≥50% (PSA50) and radiographic progression-free survival (rPFS). Parts A and B enrolled 27 and 68 patients, respectively. In part B, the median number of prior therapies was 4 (range 2-11). The most common treatment-related adverse events were asymptomatic prolonged corrected QT interval (47%) and bradycardia (34%). Part A maximum tolerated dose was not reached and recommended phase II dose selection is ongoing. Across part B three highest doses (400-900 mg twice daily, n= 60), PSA50 was 32% (n= 19), including 50% (n= 10/20) at 900 mg; median rPFS (95% confidence interval) was 6.3 months (5.3-12.6 months), including 8.3 months (3.8-16.6 months) at 900 mg; and rPFS was longer in patients without versus with prior chemotherapy: 16.5 months (5.5 months-not evaluable) versus 5.5 months (2.7-8.3 months), respectively. Efficacy was observed in patients with mCRPC with AR ligand binding domain (LBD) WT or with AR LBD mutations. BMS-986365 was well tolerated, with a manageable safety profile, and demonstrated activity in heavily pretreated patients with mCRPC with potentially higher benefit in chemotherapy-naive patients. These data show the potential of BMS-986365 to overcome resistance to current ARPIs, regardless of AR LBD mutation status.

In silico study of androgen receptor N-terminal domain and exploration of its modulators

The androgen receptor (AR, Uniprot: P10275) signaling plays a key role in the progression of prostate cancer, various AR-related ligands have been reported to treat prostate cancer. However, some resistance mechanisms limited the treating effect of these ligands. Since DBD binding or the allosteric binding sites in LBD of AR may allow the circumvention of some drug resistance mechanisms, anti-resistance is expected especially through the NTD (N-terminal domain) targeting. What’s more, studies have shown that compounds including EPI-001 and its derivatives which bind to the Tau-5 region on NTD could be promising molecules for AR-based therapeutics. Herein, we employed aMD (accelerated molecular dynamics) simulation to fold Tau-5 unit proteins into native structure correctly. Subsequently, based on the predicted structural features of Tau-5, the virtual screening was conducted to discover new compounds targeting AR-NTD. We picked up 8 compounds (according to their docking scores and partly similar structural consists as known AR ligands) and analyzed their interaction with Tau-5, compared with the positive control EPI-001, four of the pick-up compounds showed better glide scores. Interestingly, although compound 8 had a lower docking score, it consisted of a similar component as the ligand EIQPN and the amide derivatives, this predicts that compound 8 has also the potential to be modified into an excellent AR-NTD binding molecule. These 8 compounds were all commercially available and could be tested to check whether there was a hit compound to bind the AR-NTD and to regulate its bio-activities. Together, this study described an in silico VLS approach to discover AR-NTD ligands and provided more choices for developing AR-targeted therapies. Communicated by Ramaswamy H. Sarma

Mechanistic details for oxidative addition of PhICl2 to gold(I) complexes.

This study demonstrates that the oxidative addition of hypervalent iodine PhICl2 to Au(I) complexes LAuAr follows an unprecedented stepwise mechanism. The energy of the Au(I) dx2-y2 orbital determines the ease of the oxidative addition reaction; the fewer electron-withdrawing substituents present on the Ar ligand, the higher the energy of the dx2-y2 orbital and the easier the oxidative addition.

In Silico Identification of a β2 Adrenergic Receptor Allosteric Site that Selectively Augments Canonical β2AR‐Gs Signaling and Function

Objective & HypothesisActivation of β2‐adrenergic receptors (β2ARs) leads to airway smooth muscle (ASM) relaxation and bronchodilation. While this facilitates β2ARs agonists (β‐agonists) as the front‐line treatments for asthma and related obstructive airway diseases, their therapeutic efficacy of β‐agonists is limited by agonist‐induced β2AR desensitization and activation of non‐canonical β2AR signaling involving β‐arrestin. Accordingly, we undertook the identification of an allosteric site on β2AR and potential ligands, and ascertain their effects in‐vitro. We hypothesize that activation of specific allosteric site on β2AR could selectively modulate the activity of β‐agonists to overcome these limitations.MethodsWe employed the site identification by ligand competitive saturation (SILCS) computational method to map the entire 3D structure of an in silico‐generated β2AR intermediate conformation. Further, in‐silico database was screened to identify potential ligands using SILCS‐PHARM approach. Levels of cyclic‐AMP were used to assess activation of β2AR‐Gs activation using ELISA. Activation of downstream effector of cAMP, protein kinase A (PKA) was assessed by western blotting and luciferase assay. β‐arrestin recruitment to β2AR was assessed using Bioluminescence Resonance Energy Transfer (BRET) assay. ELISA and immunofluorescence were used to assess cell surface expression. Mutagenesis analysis was used to investigate the potential binding site for the allosteric modulators. Lastly, functional effects of the allosteric modulators were determined by assessing relaxation of human ASM cells embedded in 3D‐collagen gels and murine airways.ResultsDatabase screening using SILCS identified not only putative allosteric site but also potential drug‐like compounds that can bind to the site. Experimental assays in HEK293 cells and human ASM cells identified positive and negative allosteric modulators (PAM and NAM respectively) of β2AR as assessed by generation of cAMP. Co‐stimulation of cells with PAM or NAM modulated β‐agonist‐induced PKA activation. Allosteric modulators had no effect on β‐agonist‐induced recruitment of β‐arrestin to β2AR or β‐agonist‐induced loss of cell surface expression in HEK293 cells expressing β2AR. Mutagenesis analysis of β2AR confirmed the SILCS identified allosteric modulator binding site that comprises of F282, Y219, and R131. Finally, functional studies revealed augmentation of β‐agonist‐induced relaxation of contracted human ASM cells and bronchodilation of contracted murine airways by PAM.ConclusionThese findings identify a novel allosteric binding site on the β2AR, whose activation selectively augments β‐agonist‐induced Gs signaling and increases relaxation of ASM cells, the principal therapeutic effect of β‐agonists in obstructive lung disease such as asthma and COPD.

In Vitro characterization of the endocrine disrupting effects of per- and poly-fluoroalkyl substances (PFASs) on the human androgen receptor

Per- and poly-fluoroalkyl substances (PFASs) are used extensively in a broad range of industrial applications and consumer products. While a few legacy PFASs have been voluntarily phased out, over 5000 PFASs have been produced as replacements for their predecessors. The potential endocrine disrupting hazards of most emerging PFASs have not been comprehensively investigated. In silico molecular docking to the human androgen receptor (hAR) combined with machine learning techniques were previously applied to 5206 PFASs and predicted 23 PFASs bind the hAR. Herein, the in silico results were validated in vitro for the five candidate AR ligands that were commercially available. Three manufactured PFASs namely (9-(nonafluorobutyl)− 2,3,6,7-tetrahydro-1 H,5 H,11 H-pyrano[2,3-f]pyrido[3,2,1-ij]quinolin-11-one (NON), 2-(heptafluoropropyl)− 3-phenylquinoxaline (HEP), and 2,2,3,3,4,4,5,5,5-nonafluoro-N-(4-nitrophenyl)pentanamide (NNN) elicited significant antiandrogenic effects at relatively low concentrations. We further investigated the mechanism of AR inhibition and found that all three PFASs inhibited AR transactivation induced by testosterone through a competitive binding mechanism. We then examined the antiandrogenic effects of these PFASs on AR expression and its responsive genes. Consistently, these PFASs significantly decreased the expression of PSA and FKBP5 and increased the expression of AR, similar to the effects elicited by a known competitive AR inhibitor, hydroxyflutamide. This suggests they are competitive antagonists of AR activity and western blot analysis revealed these PFASs decreased intracellular AR protein in androgen sensitive human prostate cancer cells. Hence, the findings presented here corroborate our published in silico approach and indicate these emerging PFASs may adversely affect the human endocrine system.

Cell immunocapture microfluidic chip based on high-affinity recombinant protein binders

Cell immunocapture microfluidic devices represent a rapidly developing field with many potential applications in medical diagnostics. The core of such approach lies in the cell binding to antibody coated surfaces through their surface receptors. Here we show, that the small recombinant protein binders (PBs) can be used for this purpose as well, with the advantage of their constructional flexibility, possibility of fusion with range of tags and cheap mass production. For this purpose, two different PBs derived from Albumin Binding Domain (ABDwt) of streptococcal protein G, so called REX and ARS ligands with proved high affinity and selectivity to the human interleukin-23 (IL-23R) and IL-17 receptor A were used. Four PBs variants recognizing two different epitopes on two different receptors and two PBs variants binding to the same epitope on one receptor but having different peptide spacer with Avitag sequence necessary for their immobilization on sensor surface were tested for cell-capture efficiency. The glass microfluidic Y-system with planar immunocapture channel working in so-called stop-flow dynamic regime was designed. Up to 60–74% immunocapture efficiency of model THP-1 cells on REX/ARS surfaces and practically no cell binding on control ABDwt surfaces was achieved. Moreover, the specific immunocapture of THP-1 cells from mixture with IL-17RA negative DU-145 cells was demonstrated. We discuss the role of the epitope, affinity and immobilization spacer of PBs as well as the influence of stop-flow dynamic regime on the effectivity of THP-1 cell immunocapture. Results can be further exploited in design of microfluidic devices for rare cells immunocapture.

Genomic profiling and mutation burden of Chinese prostate patients with microsatellite instability.

e17531 Background: Prostate cancer with microsatellite instability-high (MSI-H) is identified as a distinct genotype that potentially benefit from immune checkpoint inhibitors. But the incidence and genomic profiling of microsatellite instability haven’t been well characterized in Chinese prostate cancer patients. Methods: Tumor tissue and matched blood sample from 241 Chinese prostate cancer patients were analyzed with targeted next-generation sequencing covering whole exon of 381 or 90 genes and 100 microsatellite loci. Tumor mutation burden (TMB), defined by non-silent somatic mutation counts in coding region per megabase (Mb), was calculated in tumors sequenced by 381-gene panel with coding region of 1.12Mb. Results: 7 patients (2.90%) were identified as MSI-H in overall population. Somatic or germline alterations of mismatch repair genes were detected in 5 of the 7 patients, including 2 MSH2 germline mutations, 2 MSH6 somatic mutations, 1 MSH2 somatic mutation, 1 PMS2 somatic mutation and 1 MLH3 somatic mutation. In MSI-H tumors, alterations in KMT2C (6/7), EGFR (5/7), KMT2D (5/7), JAK1 (5/7), PTEN (4/7), TP53 (3/7), ZFHX3 (3/7) and SPOP (3/7) were common. AR ligand binding domain was mutated in 3 of 7 patients, while no AR amplification was detected in MSI-H tumors. In 121 tumors sequenced by 381-gene panel, median TMB was 3.23 mutations/Mb. Median TMB was significantly higher in MSI-H tumors compared with MSS tumors (median TMB, 13.71 mutations/Mb versus 2.93 mutations/Mb, P < 0.001). Median counts of single nucleotide variants (SNVs) and insertion-deletions (Indels) were 13 and 10 in MSI-H tumors that were also higher than that of MSS tumors with median SNVs and Indels of 2 (P = 0.004) and 1 (P < 0.001), respectively. Conclusions: 2.9% patients were identified as MSI-H in Chinese prostate patients. MSI-H was associated with higher TMB, SNVs and Indels. Targeted next-generation sequencing is a practical tool for prostate cancer genotyping and treatment decision-making.

Ionization-Induced π → H Site Switching in Resorcinol–Arn (n = 1 and 2) Clusters Probed by Infrared Spectroscopy

Infrared (IR) spectra of resorcinol (Rs)-Arn clusters (n = 1 and 2) have been measured in the neutral and cationic ground states (S0 and D0) by IR dip and resonance-enhanced multiphoton ionization (REMPI)-IR spectroscopy. The OH stretching vibrations in S0 keep their frequency regardless of the number of Ar atoms and the conformation of the OH groups in Rs (rotamers RsI and RsII), demonstrating that the Ar atoms are attached to the aromatic π-ring (π-bound structure) in S0. In the D0 state, the IR spectra of Rs+-Arn reflect the difference in the Rs conformations (RsI+ and RsII+). For n = 1, the IR spectra of both rotamers are almost the same as those of the corresponding monomer cations, indicating that Ar ligands essentially remain π-bonded after ionization. In contrast, the IR spectra of Rs+-Ar2 show hydrogen-bonded and free OH stretching vibrations, demonstrating that for a significant fraction of the clusters, the Ar atoms migrate from the π-bound site to the OH groups. The ionization-induced π → H migration yields are not unity for both rotamers RsI+-Ar2 and RsII+-Ar2. This result is in sharp contrast to phenol+-Ar2, in which one of the Ar atoms migrates to the OH site with 100% yield. The mechanism leading to the nonunity yield in Rs+-Ar2 is discussed in terms of the number of OH binding sites and Franck-Condon factors. The ionization excess energy dependence of the IR spectra of Rs+-Ar2 and its Rs+-Ar fragments is discussed in terms of the Ar binding energies estimated from the photoionization and photodissociation efficiency spectra.

Abstract 4397: Gli Activation by Steroid Receptors in Prostate and Breast Cancer Cells

Abstract Background: The nuclear steroid receptor superfamily encompasses a group of proteins best known for their functions as primary transcription factors that are conditionally active when bound to a ligand. Here, we show that prominent members of this family (androgen receptor [AR], estrogen receptor [ER-α] and glucocorticoid receptor [GR]) have a secondary function of coordinately activating the Gli family of transcription factors. Previously, we found that liganded AR recognizes and binds to Gli2/Gli3 proteins at their Protein Processing Domains. This binding prevents their degradation and stabilizes them in their high molecular weight, transcriptionally-active forms. This interaction bypasses the need for signaling through Hedgehog/Smoothened to stabilize Gli. To determine whether other steroid receptors have this activity, we tested the ability of human ER-α and GR to bind to Gli3 and whether they increased Gli activity in exogenous (293FT) and endogenous breast and prostate cancer cell systems. Methods: Human AR, ER-α or GR expression vectors were co-transfected into 293FT cells along with a myc-tagged Gli3. Protein extracts were tested for co-immunoprecipitation of AR-/ER-/GR-Gli3 complexes. 293FT cells were co-transfected with AR, ER, or GR along with a Gli-reporter vector. Luciferase activity was measured in transfected cells upon treatment with vehicle, R1881 (AR ligand), estradiol (E2-ER ligand); or dexamethasone (dex-GR ligand). LNCaP (express AR), MCF7 (express ER) or LNCaP-AI cells (express GR) were transfected with reporter in the presence or absence of R1881, E2, dex or vehicle and luciferase activity was measured. AR-/ER-Gli3 complexes were detected by in situ proximity ligation assays in prostate cancer or breast cancer cells. The effect of AR or ER-α siRNA knockdown in LNCaP or MCF7 cells on Gli3 protein stability was measured on western blot. Results: Pulldowns of AR, ER or GR co-immunoprecipitates with Gli3. Transfection with AR or ER increased Gli reporter activity in the presence of vehicle but was further increased by R1881 or E2 treatment. Gli reporter activity was unchanged by transfection with GR in the presence of vehicle but the presence of 5 or 10nM of dex tripled this activity. Gli-luciferase activity was significantly increased in R1881-treated LNCaP cells, E2-treated MCF7 cells and in dex-treated LNCaP-AI cells. PLA detected the presence of AR-Gli3 and ER-Gli3 complexes in nuclei of LNCaP and MCF7 cells. AR and ER-α knockdown destabilized Gli3 protein in LNCaP and MCF7 cells. Conclusion: Collectively our results established a secondary function (Gli activation) shared by an important evolutionary spectrum of human steroid receptors (AR, ER, and GR). As Gli is oncogenic and regulates the expression of many growth-related genes, our observations may explain the pro-oncogenic effects of steroid receptors in steroid-dependent tumour systems. Citation Format: Shabnam Massah, Na Li, Sarah Truong, Jane Foo, Gail Prins, Ralph Buttyan. Gli Activation by Steroid Receptors in Prostate and Breast Cancer Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4397.

Synthesis, biological evaluation and molecular modelling studies of 1,3,7,8-tetrasubstituted xanthines as potent and selective A2A AR ligands with in vivo efficacy against animal model of Parkinson’s disease

In the present study, an attempt has been made to develop a new series of 1,3,7,8-tetrasubstituted xanthine based potent and selective AR ligands for the treatment of Parkinson's disease. Antagonistic interactions between dopamine and A2A adenosine receptors serve as the basis for the development of AR antagonists as potential drug candidates for PD. All the synthesized compounds have been evaluated for their affinity toward AR subtypes using in vitro radioligand binding assays. 1,3-Dipropylxanthine 7a with a methyl substituent at N-7 position represents the most potent compound of the series and displayed highest affinity (A2A, Ki = 0.108 µM), however incorporation of a propargyl group at 7-positon of the xanthine nucleus seems to be the most appropriate substitution to improve selectivity towards the A2A subtype along with reasonable potency. Antiparkinsonian activity has been evaluated using perphenazine induced catatonia in rats. Most of the synthesized xanthines significantly lowered the catatonic score as compared to control and displayed antiparkinsonian effects comparable to standard drug. All the synthesized compounds were subjected to grid-based molecular docking studies to understand the key structural requirements for the development of new molecules well-endowed with intrinsic efficacy and selectivity as adenosine receptor ligands. In silico studies carried out on newly synthesized xanthines provided further support to the pharmacological results.

Unraveling the protonation site of oxazole and solvation with hydrophobic ligands by infrared photodissociation spectroscopy.

Protonation and solvation of heterocyclic aromatic building blocks control the structure and function of many biological macromolecules. Herein the infrared photodissociation (IRPD) spectra of protonated oxazole (H+Ox) microsolvated by nonpolar and quadrupolar ligands, H+Ox-Ln with L = Ar (n = 1-2) and L = N2 (n = 1-4), are analyzed by density functional theory calculations at the dispersion-corrected B3LYP-D3/aug-cc-pVTZ level to determine the preferred protonation and ligand binding sites. Cold H+Ox-Ln clusters are generated in an electron impact cluster ion source. Protonation of Ox occurs exclusively at the N atom of the heterocyclic ring, in agreement with the thermochemical predictions. The analysis of the systematic shifts of the NH stretch frequency in the IRPD spectra of H+Ox-Ln provides a clear picture of the sequential cluster growth and the type and strength of various competing ligand binding motifs. The most stable structures observed for the H+Ox-L dimers (n = 1) exhibit a linear NHL hydrogen bond (H-bond), while π-bonded isomers with L attached to the aromatic ring are local minima on the potential and thus occur at a lower abundance. From the spectra of the H+Ox-L(π) isomers, the free NH frequency of bare H+Ox is extrapolated as νNH = 3444 ± 3 cm-1. The observed H+Ox-L2 clusters with L = N2 feature both bifurcated NHL2 (2H isomer) and linear NHL H-bonding motifs (H/π isomer), while for L = Ar only the linear H-bond is observed. No H+Ox-L2(2π) isomers are detected, confirming that H-bonding to the NH group is more stable than π-bonding to the ring. The most stable H+Ox-(N2)n clusters with n = 3-4 have 2H/(n - 2)π structures, in which the stable 2H core ion is further solvated by (n - 2) π-bonded ligands. Upon N-protonation, the aromatic C-H bonds of the Ox ring get slightly stronger, as revealed by higher CH stretch frequencies and strongly increased IR intensities.

Ligand Structure Determines Nanoparticles' Atomic Structure, Metal-Ligand Interface and Properties.

The nature of the ligands dictates the composition, molecular formulae, atomic structure and the physical properties of thiolate protected gold nanomolecules, Aun(SR)m. In this review, we describe the ligand effect for three classes of thiols namely, aliphatic, AL or aliphatic-like, aromatic, AR, or bulky, BU thiol ligands. The ligand effect is demonstrated using three experimental setups namely: (1) The nanomolecule series obtained by direct synthesis using AL, AR, and BU ligands; (2) Molecular conversion and interconversion between Au38(S-AL)24, Au36(S-AR)24, and Au30(S-BU)18 nanomolecules; and (3) Synthesis of Au38, Au36, and Au30 nanomolecules from one precursor Aun(S-glutathione)m upon reacting with AL, AR, and BU ligands. These nanomolecules possess unique geometric core structure, metal-ligand staple interface, optical and electrochemical properties. The results unequivocally demonstrate that the ligand structure determines the nanomolecules' atomic structure, metal-ligand interface and properties. The direct synthesis approach reveals that AL, AR, and BU ligands form nanomolecules with unique atomic structure and composition. Similarly, the nature of the ligand plays a pivotal role and has a significant impact on the passivated systems such as metal nanoparticles, quantum dots, magnetic nanoparticles and self-assembled monolayers (SAMs). Computational analysis demonstrates and predicts the thermodynamic stability of gold nanomolecules and the importance of ligand-ligand interactions that clearly stands out as a determining factor, especially for species with AL ligands such as Au38(S-AL)24.

Abstract 3739: Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML)

Abstract Despite the impressive success of molecular targeted therapy in chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKI) such as imatinib (IM), a proportion of patients exhibit suboptimal response and intolerance to the drug. While the mechanism by which resistance or intolerance develops is not completely clear, it is possible that several secondary modifiers could determine disease progression and drug response in CML. Nuclear Hormone Receptors-NHRs are attractive but less explored therapeutic targets in myeloid leukemia. The impressive success of treatment with ATRA, a RAR ligand, in the treatment of APL has not been translated to other myeloid leukemia subtypes. Recently, Prost et al., 2015 showed that Peroxisome proliferator-activated receptor-γ agonist Pioglitazone eliminates the residual CML stem cell pool and these patients remained in complete molecular response even after withdrawal of IM. The role of other NHRs in CML TKI resistance has not been explored extensively. Based on the in-vitro drug sensitivity data, CML cell lines KU812, EM2 were grouped as sensitive (IC50-0.15 & 2.29uM) and Lama84, KCL22 were grouped as resistant (IC50-54.45 & 66uM) to IM. The NHRs and coregulators that are differentially expressed between IM sensitive and resistant cell lines were identified using NHR RT2 PCR profiler array (SAbiosciences). Based on +2 fold cutoff, the differentially expressed NHRs between test and control group were identified. Eight NHRs (AHR, AR, ESR1, ESRRG, PPARγ, RXRA, RXRB & THRA) were upregulated in the sensitive compared to the resistant cell lines, which was further validated by qRT-PCR. To understand the effect of these NHRs in sensitizing resistant CML cells to Imatinib, KCL22 and Lama84 cells were pretreated with NHR ligands, followed by increasing concentrations of Imatinib (5µM-100µM). Ligands of RXR (9-cis retinoic acid), AR (testosterone) and ESRRG1 (GSK4716) significantly improved IM sensitivity by increasing the RNA expression of IM influx transporter hOCT1, which was also reported by Wang et al., 2012; Austin et al., 2014 using ligands for RXR, PXR and PPARγ. Pretreatment with these ligands did not have significant effect on the mRNA expression of BCR-ABL, but there was increased inhibition of the BCR-ABL downstream targets such as pCRKL, pSTAT5 and pAKT at the protein level in combination with IM. The AR, RXR & ESRRG ligands significantly decreased the IC50 to IM (p<0.0001) in primary CML cells (n=22) in combination with IM. We also observed significant sensitization of primary samples (n=7) to IM when used in combination with clinically available RXR and AR ligands isotretinoin (p=0.0011) & stanozolol (p=0.0054). Our finding suggests that NHR ligands, especially RXR, AR & ESRRG when used in combination with IM could overcome resistance. Further studies are warranted to confirm the mechanistic effects of these ligands in modulating drug resistance in CML. Citation Format: Bharathi M. Rajamani, Sreeja Karathedath, Raveen Stephen Illangeswaran, Esther Sathya Bama Benjamin, Vikram Mathews, Aby Abraham, Poonkuzhali Balasubramanian. Nuclear hormone receptors modulate imatinib resistance in chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3739.

Abstract 5900: Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells

Abstract Background and objective: Androgen deprivation therapy (ADT) is among the most effective of all cancer therapies for prostate cancer; however, resistance to therapy leading to castrate resistant prostate cancer (CRPC) is inevitable in most patients. Recently we demonstrated that CYP3A5 expressed in prostate cancer cells facilitates the nuclear translocation of AR, directly impacting the transcriptional regulation of AR-dependent genes promoting prostate cancer cell growth. Our preliminary data show that CYP3A5 is a part of the HSP90-AR complex, known to regulate AR nuclear localization and activation. Additionally, we observed that CYP3A5 inhibition also blocks growth of prostate cancer cells lacking AR ligand binding site (22RV1). This observation suggested presence of an alternative mechanism of regulation of prostate cell growth by CYP3A5, independent of its AR activation. We hypothesize that CYP3A5 inhibition can also sensitize prostate cancer cells to therapy by downregulating expression of drug resistance genes independent of its AR regulation. Our aim is to decipher the role of CYP3A5 in therapeutic resistance, by screening for CYP3A5 regulated cancer drug resistance genes and to further understand the underlying mechanism of this regulation. Method: We used cancer drug resistance PCR array after CYP3A5 siRNA knockdown to identify CYP3A5 regulated genes involved in therapeutic resistance. Further we evaluated the role of CYP3A5 in inducing synthetic lethality in combination with DNA damage repair inhibiting drugs by imagining the γ-H2AX foci. Results: CYP3A5 siRNA knockdown downregulates 11 genes involved in cancer drug resistance: TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1 with a p≤0.005 value. Of the list of downregulated genes, the three genes TOP2A, BRCA2 and BRCA1 have the most fold change and their loss is known to impair homologous recombination (HR), leading to synthetic lethality. HR defects in CRPC, sensitizes these tumors to PARP and topoisomerase inhibitors. We tested the effect of CYP3A5 inhibition in inducing HR defects by analyzing γ-H2AX foci counts in combination with topoisomerase inhibitor etoposide. Increased number of γ-H2AX foci suggest that CYP3A5 siRNA may induce synthetic lethality in prostate cancer cells when used in combination with DNA damage repair inhibiting drugs by impairing HR and downregulating TOP2A and BRCA1/2 expression. Conclusions: CYP3A5 inhibition downregulates DNA damage repair genes (TOP2A and BRCA1/2) and cell cycle progression genes (CCNE1, CDK 2/4) and impairs HR. CYP3A5 inhibition can sensitize CRPC to PARP and topoisomerase inhibitors as it induces synthetic lethality by downregulating TOP2A and BRCA1/2 expression. CYP3A5 inhibition can be potentially exploited therapeutically in combination with PARP inhibitors as it functionally impairs HR and causes synthetic lethality. Citation Format: Priyatham Gorjala, Ranjana Mitra, Oscar Goodman. Inhibition of CYP3A5 down-regulates cancer drug resistance genes and induces synthetic lethality in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5900.

An open-label phase 1/2a study to evaluate the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of TRC253, an androgen receptor antagonist, in patients with metastatic castration-resistant prostate cancer (mCRPC).

TPS403 Background: Resistance to AR-targeted therapy is a challenge in the treatment of mCRPC. Single amino acid mutations of the AR ligand binding domain may mediate resistance to second generation AR inhibitors, including enzalutamide (Rathkopf, Annals of Oncology 2017). Development of potent antagonists of wild-type (WT) AR as well as mutated AR is a priority. TRC253 is an orally available, high-affinity, small molecule antagonist of AR with inhibitory activity against WT AR as well as mutated variants. TRC253 blocks AR nuclear translocation and AR binding to DNA and antagonizes transcription. TRC253 does not have agonist activity toward WT or mutated ARs. Methods: This phase 1/2a study of TRC253 in patients with mCRPC will be conducted in two parts: dose-escalation (part 1) and dose-expansion (part 2). Objectives include assessment of safety, selection of a phase 2 dose, and to evaluate PSA response at week 12. Secondary objectives include the evaluation of the extent of receptor occupancy (FDHT PET) and anti-tumor effects of TRC253. Toxicity and efficacy assessments will be determined using NCI-CTCAE and PCWG3 criteria. Dose escalation will begin with single-patient cohorts. Cohorts will be expanded to 3 patients when pre-defined grades of drug-related toxicity occur. TRC253 doses in part 1 are 40 mg, 80 mg, 160 mg, 240 mg, 320 mg, and 400 mg. The DLT evaluation period will be the first 28 days of continuous daily dosing. Six patients have been enrolled to Part 1 to date. Part 2 will consist of two cohorts of up to 30 patients each. Cohort 1 (AR F876L mutation positive) and cohort 2 (AR F876L mutation negative) will receive TRC253 at the RP2D. Circulating tumor DNA will be analyzed in plasma samples to test for AR mutations. In Part 1 patients must have received ≥ 2 prior therapies. Part 2 patients must have demonstrated acquired resistance to enzalutamide or apalutamide. Descriptive statistics will be used to summarize patient characteristics, safety, efficacy, PK, and immunologic biomarkers. Clinical trial information: NCT02987829.

Detection of HRD gene mutations and copy number changes in cfDNA from prostate cancer patients.

e16546 Background: Recent genomic surveys of prostate cancer have identified somatic mutations in metastatic castration-resistant prostate cancer (mCRPC). In this study, we examined mCRPC patients for AR aberrations and mutations in the HRD (homologous recombination DNA-repair) pathway which may confer platinum or PARP inhibitor sensitivity. Methods: A novel targeted-hybrid-capture NGS assay capable of identifying deleterious mutations, copy number amplification and gene copy loss, was applied to circulating, cell-free DNA (cfDNA) extracted from plasma samples from 20 mCRPC patients. Samples were collected between 3/2010 – 10/2015 and stored at -20C. The gene panel used in the assay included AR and several genes in the HRD pathway — ATM, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK2, FANCA, HDAC2, NBN, PALB2, and RAD51. Sequencing libraries created with the cfDNA extracted from 2.8-4 mLs of plasma had an average unique read coverage depth of 2282 genome equivalents (range 445 – 5136, median 2181). Results: Somatic variations were observed in 17 of the 20 samples analyzed. Copy number variation (CNV) was observed in 11 samples. AR amplification, linked to resistance to abiraterone and enzalutamide, was observed in 30% (6) of samples. Canonical AR ligand binding domain mutations, such as T787A and L702H were also detected. ATM, BRCA1, BRCA2, HDAC2, and FANCA gene deletions were also detected, as well as frameshift, nonsense, and other deleterious mutations in HRD genes. Significant CNV in multiple genes was observed in at least 5 patient samples. Mutations were detected across the entire collection date range, speaking to the robustness of cfDNA. Conclusions: Non-invasive tumor mCRPC genotyping appears to be feasible. The ability to detect HRD gene alterations suggests cfDNA testing may be suitable for the detection of HRD pathway defects and overall genome instability.

Ionization spectroscopies and theoretical calculations of cis and trans 3-fluoro-N-methylaniline-Arn(n = 1,2) van der Waals clusters: Structures and binding energies

The ab initio and dispersion-corrected density functional theory (DFT) calculations of the van der Waals (vdW) clusters of cis and trans 3-fluoro-N-methylaniline-Arn (3FNMA-Arn) (n=1,2) predict that, for cis and trans 3FNMA-Ar1 clusters, the π-bound geometry with the Ar atom sitting over the ring is the global minimum in both neutral and cationic states, while for cis and trans 3FNMA-Ar2 clusters, the [π|π]-bound sandwich structure with two Ar ligands occupying nearly equivalent π-bound positions above and below the ring plane is the global minimum. The vibronic spectra of cis and trans 3FNMA-Ar1 clusters in the S1 state were recorded by using one-color and two-color resonant two-photon ionization (R2PI) techniques, the comparison of which yields an estimate of the binding energy of cluster in the S1 state. It is found that the linear correlation between the redshift of the S1←S0 electronic transition energy (E1) of cluster and the E1 of the monomer also holds for the Ar clusters of hetero-di-substituted aromatics. By recording the photoionization efficiency (PIE) curves and mass-analyzed threshold ionization (MATI) dissociation spectra of cis and trans 3FNMA-Ar1 clusters, the ionization energies (IE) and binding energies of clusters in the D0 state are obtained. The MATI spectra of the cis and trans 3FNMA-Ar1 cations exhibit significant progressions of the vdW bending mode (bx), indicating the structural changes of the clusters in the bending coordinate upon ionization. The combination of the three kinds of spectroscopic methods provides the binding energies of cis and trans 3FNMA-Ar1 clusters in the S0, S1 and D0 states with good accuracy, and the values estimated by dispersion-corrected DFT calculations compare well with the experimental results. From our studies, the ωB97X-D and TD-ωB97X-D methods using high quality basis set are recommended for studying the intermolecular interaction of such vdW clusters in the ground and excited states.

Synthesis and basic evaluation of 7α-(3-[18F]fluoropropyl)-testosterone and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone.

7α-Substituted androgen derivatives may have the potential to visualize androgen receptors with positron emission tomography. In the present study, we synthesized fluoropropyl derivatives of 7α-(3-[18F]fluoropropyl)-testosterone ([18F]7) and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone ([18F]15), and characterized their in vitro binding, in vivo biodistribution, and performed blocking studies in mature androgen deprived male rats. We synthesized [18F]7 and [18F]15. In vitro binding to recombinant rat AR ligand binding domain protein was determined using a competitive radiometric ligand-binding assay with the high-affinity synthetic androgen [17α-methyl-3H]-methyltrienolone ([3H]R1881). In vivo biodistribution was performed in mature male rats treated with diethylstilbestrol (chemical castration). A blocking study was performed by co-administration of dihydrotestosterone (36µg/animal). 7α-(3-Fluoropropyl)-testosterone (7) and 7α-(3-fluoropropyl)-dihydrotestosterone (15) showed competitive binding to recombinant rat AR ligand binding domain protein. The IC50 value of 15 (13.0±3.3nM) was higher than 7 (47.8±10.0nM). In contrast to the AR binding affinity, the ventral prostate uptake of [18F]7 and [18F]15 at 2h post-injection was similar (0.07% injected dose/g of tissue). A blocking study indicated that specific binding of [18F]15 is observed in the ventral prostate. [18F]7 and [18F]15 showed moderate levels of bone uptake, which indicates moderate metabolic de-fluorination in rodents. [18F]15 is better than [18F]7 in terms of radiochemical yield, in vitro binding affinity, prostate specific binding and stability against in vivo metabolic de-fluorination. However, the net uptake level of [18F]15 in prostate might be insufficient for in vivo visualization. Although [18F]7 and [18F]15 improved in vivo stability against de-fluorination, other basic characterization data in rodents were not superior to the current standard tracer 16β-[18F]fluoro-5α-dihydrotestosterone. It is also revealed that the shorter side chain length of 7α-[18F]fluoromethyl-dihydrotestosterone is superior to the longer three carbon chain in [18F]15, in terms of net prostate uptake and in vivo metabolic stability.

Mixed triorganobismuthines RAr2Bi [Ar = C6F5, 2,4,6-(C6F5)3C6H2] and hypervalent racemic Bi-chiral diorganobismuth(iii) bromides RArBiBr (Ar = C6F5, Mes, Ph) with the ligand R = 2-(Me2NCH2)C6H4. Influences of the organic substituent.

Triorganobismuthines R(C6F5)2Bi (1) and R[2,4,6-(C6F5)3C6H2]2Bi (2) [R = 2-(Me2NCH2)C6H4] were synthesized by reaction of RBiBr2 with C6F5MgBr and 2,4,6-(C6F5)3C6H2Li, respectively, in a 1 : 2 molar ratio. The Bi-chiral bromides R(C6F5)BiBr (3), R(Mes)BiBr (4), and R(Ph)BiBr (5) were obtained from RBiBr2 and C6F5MgBr, MesMgBr or PhMgBr, or from PhBiBr2 and RLi, in a 1 : 1 molar ratio. The molecular structures of 1-5 were determined by single-crystal X-ray diffraction and are discussed taking into account the chirality of the species. Supramolecular aspects in the solid state are presented. The solution behaviour of the title compounds, including dynamic aspects, are discussed on the basis of multinuclear ((1)H, (13)C, (19)F) NMR spectroscopy.

Quasi-three-coordinate iron and cobalt terphenoxide complexes {Ar(iPr8)OM(μ-O)}2 (Ar(iPr8) = C6H-2,6-(C6H2-2,4,6-(i)Pr3)2-3,5-(i)Pr2; M = Fe or Co) with M(III)2(μ-O)2 core structures and the peroxide dimer of 2-oxepinoxy relevant to benzene oxidation.

The bis(μ-oxo) dimeric complexes {Ar(iPr8)OM(μ-O)}2 (Ar(iPr8) = C6H-2,6-(C6H2-2,4,6-(i)Pr3)2-3,5-(i)Pr2; M = Fe (1), Co (2)) were prepared by oxidation of the M(I) half-sandwich complexes {Ar(iPr8)M(η(6)-arene)} (arene = benzene or toluene). Iron species 1 was prepared by reacting {Ar(iPr8)Fe(η(6)-benzene)} with N2O or O2, and cobalt species 2 was prepared by reacting {Ar(iPr8)Co(η(6)-toluene)} with O2. Both 1 and 2 were characterized by X-ray crystallography, UV-vis spectroscopy, magnetic measurements, and, in the case of 1, Mössbauer spectroscopy. The solid-state structures of both compounds reveal unique M2(μ-O)2 (M = Fe (1), Co(2)) cores with formally three-coordinate metal ions. The Fe···Fe separation in 1 bears a resemblance to that in the Fe2(μ-O)2 diamond core proposed for the methane monooxygenase intermediate Q. The structural differences between 1 and 2 are reflected in rather differing magnetic behavior. Compound 2 is thermally unstable, and its decomposition at room temperature resulted in the oxidation of the Ar(iPr8) ligand via oxygen insertion and addition to the central aryl ring of the terphenyl ligand to produce the 5,5'-peroxy-bis[4,6-(i)Pr2-3,7-bis(2,4,6-(i)Pr3-phenyl)oxepin-2(5H)-one] (3). The structure of the oxidized terphenyl species is closely related to that of a key intermediate proposed for the oxidation of benzene.