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PDF file, 162K, AR mRNA levels are unaffected upon treatment with VPC-3022 and VPC-3033. Total RNA was extracted after treatment of LNCaP and MDV3100-resistant cells with VPC-3022 and VPC-3033 for 24 hours at the indicated concentrations in the presence of 0.1 nM R1881. Quantitative RT-PCR analysis of AR expression was performed and normalized to 18S rRNA levels. Error bars indicate standard deviation.

PDF file, 122K.

PDF file, 2136K, (A) Effect of VPC-3022 on AR activity in HeLa-AR cells in the presence of cyclohexamide. HeLa-AR cells were treated with serially-diluted concentrations of VPC-3022 (0 - 100 ?M) with or without 10 ?M of cyclohexamide for 8 hours, and then lysed with passive lysis buffer. (B) AR degradation caused by VPC-3031 and VPC-3041 in HeLa-AR cells. HeLa-AR cells were treated by VPC-3031 and VPC-3041 at different concentrations in the presence of 0.1 nM R1881.

PDF file, 1976K, (A) Inhibition of in vitro polymerization of tubulin at 37 {degree sign}C by various concentrations of VPC-3033; turbidity was recorded at 360 nM.The steady-state tubulin assembly level in the absence of inhibitor was set at 100%. (B) IC50 values were determined by sigmoidal fitting of the plot of the steady state levels of tubulin assembly (taken after 45 min) aginst drug concentration and represent the concentration for 50% inhibition of the maximum tubulin polymerization level.

PDF file, 3480K, The binding pose of DHT (A) and VPC-3022 with AR by molecular docking (B) and binding pose of VPC-3022 with AR after 10 ns molecular dynamics (C); RMSD values of the protein backbone, protein-ligand, ligand in the 10 ns molecular dynamics for AR-DHT (D) and AR-VPC-3022 (E).

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer. Mol Cancer Ther; 12(11); 2425–35. ©2013 AACR.

PDF file, 109K, Inhibition of VPC-3022 against wild-type AR in HeLa-AR cells. HeLa-AR cells transfected with ARR3tk-luciferase reporter were treated with VPC-3022 for 24 hours in various concentrations in the presence of 0.1 nM R1881. Error bars indicate standard deviation.

Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition. Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4’,6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell

Acute liver injury secondary to sepsis is a major challenge in intensive care unit. This study was designed to investigate potential protective effects of pomegranate against sepsis-induced acute liver injury in rats and possible underlying mechanisms. Pomegranate was orally given (800mg/kg/day) for two weeks before sepsis induction by cecal ligation and puncture (CLP). Pomegranate improved survival and attenuated liver inflammatory response, likely related to downregulation of mRNA expression of toll like recptor-4, reduced nuclear translocation and DNA binding activity of proinflammatory transcription factor NF-κB subunit p65, decreased mRNA and protein expression of tumor necrosis factor-alpha and reduction in myeloperoxidase activity and mRNA expression. Pomegranate also decreased CLP-induced oxidative stress as reflected by decreased malondialdehyde content, and increased reduced glutathione level and superoxide dismutase activity. These results confirm the antiinflammatory and antioxidant effects of pomegranate in CLP-induced acute liver injury mediated through inhibiting TLR4/NF-κB pathway, lipid peroxidation and neutrophil infiltration.

// Valentina R. Minciacchi 1 , Sungyong You 1 , Cristiana Spinelli 1 , Samantha Morley 2 , Mandana Zandian 1 , Paul-Joseph Aspuria 3 , Lorenzo Cavallini 1,4 , Chiara Ciardiello 1,5 , Mariana Reis Sobreiro 1 , Matteo Morello 1 , Geetanjali Kharmate 6 , Su Chul Jang 7 , Dae-Kyum Kim 7 , Elham Hosseini-Beheshti 6 , Emma Tomlinson Guns 6 , Martin Gleave 6 , Yong Song Gho 7 , Suresh Mathivanan 8 , Wei Yang 1 , Michael R. Freeman 1,2 and Dolores Di Vizio 1,2 1 Division of Cancer Biology and Therapeutics, Departments of Surgery, Biomedical Sciences and Pathology and Laboratory Medicine, Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA 2 The Urological Diseases Research Center, Boston Children’s Hospital, Boston, MA, Department of Surgery, Harvard Medical School, Boston, MA, USA 3 Women’s Cancer Program, Cedars-Sinai Medical Center, Los Angeles, CA, USA 4 Department of Experimental and Clinical Biomedical Science, University of Florence, Florence, Italy 5 Experimental Pharmacology Unit, Department of Research, IRCCS-Istituto Nazionale Tumori G. Pascale, Naples, Italy 6 Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, BC, Canada 7 Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea 8 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Australia Correspondence to: Dolores Di Vizio, email: // Keywords : Extracellular Vesicles, SILAC Proteomics, Cancer metabolism, Tumor progression, Amoeboid blebbing Received : January 05, 2015 Accepted : February 22, 2015 Published : March 14, 2015 Abstract Large oncosomes (LO) are atypically large (1-10µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep TM ) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5 th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

Summary There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1 H -indol-2-yl)-1 H -indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

The human androgen receptor plays a major role in the development and progression of prostate cancer and represents a well-established drug target. All clinically approved androgen receptor antagonists possess similar chemical structures and exhibit the same mode of action on the androgen receptor. Although initially effective, resistance to these androgen receptor antagonists usually develops and the cancer quickly progresses to castration-resistant and metastatic states. Yet even in these late-stage patients, the androgen receptor is critical for the progression of the disease. Thus, there is a continuing need for novel chemical classes of androgen receptor antagonists that could help overcome the problem of resistance. In this study, we implemented and used the synergetic combination of virtual and experimental screening to discover a number of new 10-benzylidene-10H-anthracen-9-ones that not only effectively inhibit androgen receptor transcriptional activity, but also induce almost complete degradation of the androgen receptor. Of these 10-benzylidene-10H-anthracen-9-one analogues, a lead compound (VPC-3033) was identified that showed strong androgen displacement potency, effectively inhibited androgen receptor transcriptional activity, and possesses a profound ability to cause degradation of androgen receptor. Notably, VPC-3033 exhibited significant activity against prostate cancer cells that have already developed resistance to the second-generation antiandrogen enzalutamide (formerly known as MDV3100). VPC-3033 also showed strong antiandrogen receptor activity in the LNCaP in vivo xenograft model. These results provide a foundation for the development of a new class of androgen receptor antagonists that can help address the problem of antiandrogen resistance in prostate cancer. Mol Cancer Ther; 12(11); 2425–35. ©2013 AACR.

Dimethyl sulfoxide (DMSO) is currently being used by many people, both topically and orally, to treat inflammatory conditions as well as cancer. However, little is known about the efficacy, safety and mechanism of action of DMSO in humans. We therefore examined the effects of this polar solvent on whole human blood, stimulated or not, ex vivo, with E. coli. We found that, between 1 and 2%, DMSO markedly inhibited cytokine production without affecting cell viability. However, at greater than 5%, significant death of leukocytes was observed, suggesting that DMSO has only a narrow window of efficacy. The two in vivo generated metabolites of DMSO, dimethyl sulfide (DMS) and dimethylsulfone (DMSO2), required higher concentrations than DMSO to be anti-inflammatory. Mechanism of action studies using purified human monocytes revealed that DMSO's anti-inflammatory properties were due, at least in part, to inhibition of the ERK1/2, p38, PI3K and JNK pathways. In vivo mouse model studies revealed that topical admini...

Indoleamine 2,3 dioxygenase (Ido1), the first and rate-limiting enzyme of the kynurenine pathway (KP), is a striatally enriched gene with increased expression levels in the YAC128 mouse model of Huntington disease (HD). Our objective in this study was to delineate age-related KP alterations in this model. Three enzymes potentially catalyze the first step of the KP; Ido1 and Indoleamine 2,3 dioxygenase-2 were highly expressed in the striatum and Tryptophan 2,3 dioxygenase (Tdo2) in the cerebellum. During development, Ido1 mRNA expression is dynamically regulated and chronically up-regulated in YAC128 mice. Kynurenine (Kyn) to tryptophan (Trp) ratio, a measure of activity in the first step of the KP, was elevated in YAC128 striatum, but no change in Tdo2 mRNA levels or Kyn to Trp ratio was detected in the cerebellum. Ido1 induction was coincident with Trp depletion at 3 months and Kyn accumulation at 12 months of age in striatum. Changes in downstream KP metabolites of YAC128 mice generally followed a biphasic pattern with neurotoxic metabolites reduced at 3 months and increased at 12 months of age. Striatally specific induction of Ido1 and downstream KP alterations suggest involvement in HD pathogenesis, and should be taken into account in future therapeutic developments for HD.

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called Binding Function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in-silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and x-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its anti-androgen resistant forms.

Interest in developing androgen receptor (AR) inhibitors with novel mechanism of action is on the rise since the commercial anti-androgens (including recently approved drug, Enzalutamide) face clinical limitations. The current therapies fail over a period of time because they all target mutation-prone hormone binding pocket on AR to which the receptor has already developed effective resistance mechanisms. Hence, there is a pressing need for novel therapeutics that inhibit the AR through alternative modes of action. Recent studies have identified a novel binding pocket on the surface of AR called binding function-3 (BF3) that is critical for the AR transcriptional activity. In order to develop promising drug-like candidates, we performed structural optimization of our lead AR BF3 inhibitor, followed by a series of experimental validation of the synthetic analogues. As a result, we have discovered a chemical series of quinoline as new lead BF3 inhibitors. One of the most potent inhibitors identified, VPC-13566, demonstrated an IC50 of 0.071μM in AR eGFP transcriptional assay. Confirming it as a true BF3 binder, VPC-13566 displaced Bag-1L peptide, a co-regulator protein that binds to AR BF3 pocket. Additionally, the Biolayer Interferometry assay detected direct reversible interactions between the AR ligand binding domain and the inhibitor. To confirm VPC-13566 binding to the BF3 pocket, a mutagenesis study was performed. Results with mutants F673E, E837A and N833W confirmed that compound did not show any binding to the protein compared with the wild type in a BLI assay. VPC-13566 demonstrated strong anti-proliferative activity against LNCaP and Enzalutamide-resistant prostate cancer cell lines (MR49F) whereas it did not affect the growth of AR independent PC3 cell line. It also reduced prostate specific antigen (PSA) in both LNCaP and MR49F and reduced expression of AR target genes, PSA and TMPRSS2. These findings suggest that VPC-13566 exhibits AR BF3 specific mechanism of action. Furthermore, VPC-13566 significantly reduced AR-dependent growth of tumors and PSA levels in LNCaP and MR49F xenograft models. Based on these outcomes, it can be anticipated that such drug prototypes will lay a foundation for the development of alternative or supplementary small-molecule therapies capable of combating PCa even in its drug resistant forms. Because the emergence of castration resistance is the lethal end stage of the disease, we anticipate that the proposed research will eventually have a substantial impact on patient survival. Citation Format: Ravi Shashi Nayana Munuganti, Mohamed DH Hassona, Eric Leblanc, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. Targeting binding function-3 site on the androgen receptor to treat Enzalutamide-resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3654. doi:10.1158/1538-7445.AM2015-3654

The androgen receptor (AR) is a hormone-activated transcription factor implicated in the development and progression of prostate cancer. The AR contains an N-terminal domain (NTD), followed by DNA binding (DBD) and ligand-binding (LBD) domains. Upon binding of androgens to the LBD, the AR moves into the nucleus where it interacts with target genes via the conserved AR-DBD. Prostate cancer treatment involves the use of small-molecules to block androgen production or to compete with androgens for binding to the AR-LBD. Drug-resistance occurs when LBD mutations render small-molecule treatment ineffective or when constitutively active AR splice variants, lacking the LBD, become overexpressed. We recently identified a surface pocket on the AR-DBD that may serve as an alternative drug-target site. The DBD-specific, small molecule compounds presented here block transcriptional activity of full-length and splice variant AR forms across multiple cell lines and show no cross-reactivity with related steroid nuclear receptors. The inhibition is lost when residues in the binding pocket are mutated or when the AR-DBD is replaced with a different DNA binding domain. Biophysical techniques were used to explore the interaction of these compounds with the purified, recombinant DBD protein. Gene expression analysis demonstrated the down-regulation of AR target genes following compound treatment, without any obvious expression signature associated with toxicity. The compounds affected DNA binding in vitro and also blocked interactions of the AR with chromatin inside the nucleus. Finally, the lead compound suppressed the growth of LNCaP tumor xenografts and was accompanied by down-regulation of AR target genes, such as PSA. These results suggest a mechanism whereby our developed inhibitory compounds interfere with DNA binding by the AR, potentially leading to effective treatment of advanced, castration-resistant prostate cancers. Citation Format: Kush Dalal, Mani Roshan-Moniri, Aishwariya Sharma, Huifang Li, Fuqiang Ban, Mohamed D. Hassona, Michael Hsing, Kriti Singh, Eric LeBlanc, Scott Dehm, Emma Tomlinson Guns, Artem Cherkasov, Paul S. Rennie. Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1778. doi:10.1158/1538-7445.AM2015-1778

The androgen receptor (AR) is one of the most validated therapeutic targets in prostate cancer (PCa). Conventional anti-androgens lose effectiveness as cancer therapeutics because anti-androgen resistance usually develops after long term treatment. The challenge is that the current therapeutics bind to the same site of the AR (hormone binding pocket) and act via the same mode, to which the receptor has already developed effective resistance mechanisms. Hence, there is a pressing need for novel therapeutics that inhibit the AR through novel, alternative modes of action. Recent studies have identified a novel binding pocket on the surface of AR called binding function 3 (BF3) that is important for the AR transcriptional activity. In order to identify compounds that specifically bind to BF3 site and inhibit the AR, we conducted a systematic in silico screen (that included large-scale docking, in-site rescoring, and consensus voting procedures) followed by experimental validation of the identified hit molecules. As a result, we have discovered a novel chemical series of indoles as lead BF3 inhibitors. One of the most potent inhibitors identified, VPC-13163, demonstrated an IC50 of 0.31µM in AR eGFP transcriptional assay. Confirming it as a true BF3 binder, VPC-13163 neither displaced the co-activator from an alternative coactivator binding site, activation function 2 site, nor androgen from the hormone binding pocket. Additionally, the Biolayer Interferometry assay detected direct reversible interactions between the AR ligand binding domain and the inhibitor. VPC-13163 demonstrated strong anti-proliferative activity against LNCaP and Enzalutamide-resistant prostate cancer cell lines (MR49F) whereas it did not affect the growth of AR independent PC3 cell line. It also inhibits prostate specific antigen (PSA) in both LNCaP and MR49F and reduces expression of AR target genes, PSA and TMPRSS2. These findings suggest that VPC-13163 exhibits AR BF3 specific mechanism of action. Furthermore, VPC-13163 reduces AR-dependent growth of xenograft tumors in vivo. Based on these outcomes, it can be anticipated that such drug prototypes will lay a foundation for the development of alternative or supplementary small-molecule therapies capable of combating PCa even in its drug resistant forms. Because the emergence of castration resistance is the lethal end stage of the disease, we anticipate that the proposed research will eventually have a substantial impact on patient survival. Citation Format: Ravi Shashi Nayana Munuganti, Mohamed DH Hassona, Eric Leblanc, Fuqiang Ban, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. The development of anti-androgens with a novel mechanism of action for treatment of castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2014-2508

The human androgen receptor (AR) represents a well-established drug target for prostate cancer treatment. All clinically used antiandrogens, such as Bicalutamide and Enzalutamide, possess similar chemical structures and bind to the AR in the androgen binding site (ABS). These AR antagonists are initially effective, but resistance invariably developed in the castration-resistant prostate cancer (CRPC). Even in late-stage patients, the AR activity is still persistent in the progression of the disease. Thus, there is a continuing need for novel chemical classes of AR antagonists that could overcome the drug resistance. In this study, we performed a virtual screening against the AR ABS, and identified novel AR antagonists with chemical structures completely different from existing antiandrogens. A 10-(4-Hydroxybenzylidene)anthracen-9(10H)-one compound was discovered that not only effectively inhibits AR transcription and strongly displaces androgen in the ABS, but induces AR degradation in prostate cancer cells. Starting from the initial hit compound, a series of 10-benzylidene-10H-anthracen-9-ones were synthesized and evaluated by in vitro assays. A close analogue with enhanced potency was identified as a lead compound, which demonstrated strong androgen displacement potency, effective AR transcriptional inhibition, and a profound ability to cause degradation of AR. Notably, it exhibited significant activity against MDV3100-resistant prostate cancer cells. This lead compound was evaluated in both non-castrated and castration-resistant LNCaP xenograft models, and demonstrated significant effect on inhibiting tumor growth and reducing prostate specific antigen (PSA). This series of compounds were predicted to adopt a different binding mode from current antiandrogens, which may circumvent the resistance rendered by identified gain-of-function mutations. Further development of this series of AR antagonists may have great therapeutic potential in CRPC. Citation Format: Huifang Li, Mohamed DH Hassona, Nathan A. Lack, Peter Axerio-Cilies, Eric Leblanc, Emma T. Guns, Paul S. Rennie, Artem Cherkasov. Discovery of a novel series of androgen receptor antagonists with potential therapeutic applications in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2014-1632

In the development and progression of prostate cancer, the androgen receptor (AR) plays a critical role and its hormone/ligand-binding site is the primary therapeutic target for all antiandrogens (eg Bicalutamide, MDV3100) currently used to treat advanced, metastatic forms of this disease. While treatment with these AR inhibitors initially suppresses prostate tumor growth, resistance to these drugs invariably emerges. Accordingly, there exists a clinical need for the development of new AR antagonists with different chemical structures and different mechanisms of action for targeting and inhibiting AR transactivation. To this end, we have focused on a surface pocket on the AR called Binding Function 3 (BF3). Applying iterative in silico screening to millions of chemicals in the ZINC database, coupled with biological screening for AR binding and activity, we identified several compounds that can bind directly to the BF3 pocket and inhibit AR transactivation. Cell-based screening assays for inhibition of a fluorescent ARE-reporter and PSA expression, together with Biolayer Interferometry analysis for measuring binding to the AR, revealed several chemically distinct BF-3 binders that showed significant IC50 inhibitions of the AR in the nano-molar to low micro-molar range. Validation for binding to the BF3 site was performed by x-ray crystallography. Furthermore, none of these compounds were able to displace DHT from the hormone binding site even at concentrations more than 10-fold higher than their IC50 values. In addition to wild type LNCaP prostate cancer cells, these BF3 binders effectively inhibited cell proliferation (MTS assay) and PSA expression in derived MDV3100-resistant prostate cancer cell lines, but had no effect on cell viability of AR-negative prostate cancer cell lines. In summary, we have identified an entirely new class of anti-androgens that bind to the BF3 surface site of the AR and inhibit its transactivation by a mechanism which does not involve binding to its hormone/ligand binding site and thereby bypasses the treatment resistance seen with conventional antiandrogens. (Supported by funds from the PCF, Canada Safeway, and CIHR). Citation Format: Ravi Munuganti, Fuqiang Ban, Huifang Li, Eric LeBlanc, Peter Axerio, Kate Frewin, Emma Tomlinson Guns, Artem Cherkasov, Paul S. Rennie. New potent inhibitors of the androgen receptor that target its BF3 surface binding site. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2013-1070