AU2014262172B2

AU2014262172B2 - Methods and materials for assessing prostate cancer therapies and compounds

Info

Publication number: AU2014262172B2
Application number: AU2014262172A
Authority: AU
Inventors: Charlie D. Chen; Michael E. Jung; Samedy Ouk; Charles L. Sawyers; Derek Welsbie
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California San Diego UCSD
Priority date: 2004-02-24
Filing date: 2014-11-11
Publication date: 2017-04-06
Anticipated expiration: 2025-02-23
Also published as: AU2017201659A1; AU2014262172A1

Abstract

A modest (2-5 fold) increase in androgen receptor (AR) mRNA is the only expression change consistently associated with developing resistance to antiandrogen therapy. Increased levels of AR confer resistance to anti-androgens by amplifying signal output from low levels of residual ligand and altering the noral response to antagonists. This invention provides cell based assays for use in the examination of new therapeutic modalities and provides for the design of novel antiandrogen compounds.

Description

METHODS AND MATERIALS FOR ASSESSING PROSTATE CANCER THERAPIES AND COMPOUNDS rnammmmTmmmmm [0001] This invention was made with United States Government support under Department of Defense Grant DAMD17-98-1 -8569. The Government may have certain rights to the invention. 1. Field of the Invention.

[0002] The present invention relates to methods and materials for assessing prostate cancer therapies, as well as novel prostate cancer compositions and methods for making and using said compositions. 2. Description of Related Art.

[00©] Cancer is the second leading cause of human death next to coronary disease, Worldwide, millions of people die from cancer everyyo®. In fee United Statehalone^as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2millionnew cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In this ceriturf; dancer is predicted to become fee leading cause of death.

[0004] Worldwide, several cancels stand out as fee leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a cofemoft fefell feature. Wife very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, ;:evcn fef those caper patients who initially survive their primary canceis, common experience has shown feat their lives are dramatically altered, [000¾ Adenocarcinoma of fee prostate is themostfrequently diagnosed cancer in men in of male cancer deafes (Karp et al., Cancer :Ris. S6iS:M7-5556 (1996)). Therapy for prostate cancer is typicafryimtiated using hormone drugs that lower serum testosterone, often given, in combination with competitive androgen receptor (AR) antagonist. Although initially effective at blocking tumor growth, these therapies eventually fail,, leading to a drag resistant stage called afidtogen independent or hormone refractory (HR) disease that is uniformly lethal.

[0006] Postulated mechanisms to explam resistanee to hormonotherapy can beseparated into three general categories.1'3 The first includes DNA-based alterations in the AR gene such as amplification or point mutations, which collectively only occur in. a minority of patients.4'7 A subset of these AR mutations map to the ligand binding domain (LBD) and are proposed to cause resistance by altering the response of the receptor such that noncanonical ligands like estrogen or hydrocortisone, or even AR antagonists like flutamide, behave as agonists.8·9 Although their clinical association vrift anfianifegj^

Overall frequency of AR amplification or mutation cannot,account for most cases of hormone refractory disease.

[0007] The second category applies to the'maj ority of patients without AR gene mutation or amplification who retain active AR signaling. Increased mitogen-activated protein kinase signaling mediated by oncogenes such as ErbB2 or Ras can cause ligand-independent activation of AR.10,11 The kinases and substrates responsible for AR activation in this setting aretmtetovyn, but this is pressed to occurthrough downstreamphosphorylation ofAR-assodated proteins or AR itself, analogous to the estrogen receptor (BR).12'14 Similarly, alteration in the balance of coactivators or corepressors can affect AR activation,15·16 based M^i^®fiBdinpifrt¾.,7 Therelative frequency oftheseevents and toefr relationship to clinical drug resistance remain to be defined.

[Oi0$] The third calory ofhormcme resistance mechanisms is based on the concept that the pro-growth and survival functions of AR can be “bypassed” by alternative signaling p3iiways>siiSid^4R%M One exampleis u^gBlatibntffAg anti-ippi^c geimlE^ in late stage clinical samples,.IS'19 but functional proof ^ arofem hbimeiisiS^ The ARbypasshypQthesis is also consistent

With observations of AR gene niethylation leading to decreased or absent AR expression in ΤΑ some HR cancers,* as well as reports that androgen induces growth arrest or apoptosis in certain contexts.21'22 [0009] Collectively, these data implicate multiple mechanisms by which prostate cancers acquire resistance to hormone therapy and highlight the continuing debate about the role of AR in late stage disease progression. Consequently, there is a need in the art for models that reproduce clinically significant aspects of this disease progression, particularly the transition from the initial stage in the prostate cancer where the cancer cells are sensitive to hormone antagonists to the subsequent drug resistant stage. In particular, a well-defined and manipulatable cell based model is needed to dissect the molecular events associated with the progression from a drug sensitive to a drug resistant phase. There is also a need in the art for cell based prostate cancer models that reproduce the drug sensitive and/or drug resistant phases of cancers of the prostate that can be used, for example in the evaluation of new therapeutic modalities. In addition, there is need for the identification of novel compounds that can be used to inhibit the growth of prostate cancer cells, particularly hormone refractory prostate cancers. The present invention disclosed herein satisfies this need.

[0009a] Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment, or any form of suggestion, that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

[0009b] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude other additives, components, integers or steps.

SUMMARY OF THE INVENTION

[0009c] Embodiments of the invention are defined by each of the accompanying claims. For example, in one embodiment, the invention relates to a compound as defined by claim 1.

[00010] Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest (2-5 fold) increase in androgen receptor (AR) mRNA was the only expression change consistently associated with developing resistance to antiandrogen therapy. This increase in AR mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone sensitive to hormone refractory stage, and was dependent on a functional ligand-binding domain. Furthermore* AR antagonists displayed agonist activity in cells with increased AR levels, and this antagonist/agonist conversion was associated with alterations in the pattern of coactivators and corepressors recruited to the promoter of AR target genes. Increased levels of A R confer resistance to anti-androgens by amplifying signal output from low levels of residual ligand and altering the normal response to antagonists. The disclosure provided herein that is based upon these findin gs includes assays for examining the effects of therapeutic compounds on mammalian cells such as androgen independent prostate cancer eells and further provides insight towacl the design of novel antiandrogens.

IddftllJ One embodiment of feemventionisamefeod of testing compounds foran gfigci on a mammalian prostate plhpFceli wifi .a. mammalian prostate cancer cell wherein the mammalian cancer cell is selected for the test because it expresses an exogenous wild type androgen receptor (AR) polynucleotide that ensodesthe AR polypeptide such thatthe levels of mRNA in the cell that encode the AR polypeptide or AR polypeptide are at least about 2 fold higherthan normal/endogenous AR mRNA or AR polypeptide levels in a mammalian prostate cell; aid then comparing one or ffiOre characteristics of the mammalian prostate - cancer cell to which the compound #Sr administered with the same one or mote characteristics of a control mammalian prostate cancer cell to which the compound has not bean administered, wherein a difference in one or more of the one or more characteristics indicates that the compound has an effect Oil the mammalian prostate cancer cell.

[000X2] Another embodiment of the invention is a method of examining the physiological etihcd Of acoteipinhd * prostate cancer cell, the method comprising contacting the compound to be tested with a mammalian prostate cancer cell, wherein the mammalian prostate cancer ceil is selected for the method because it expresses either an exogenous wild type polynucleotide that encodes the AR polypeptide or a polynucleotide that encodes a variant of the AR polypeptide, wherein the variant has a deletion, insertion or substitution of at least one amino acid in the AR polypeptide amino acid sequence and wherein th&felajtRyeis of mRNA in the cell that encode the AR polypeptide variant or the total levels of AR polypeptide variant are at least 2 fold higher than normal/endogenous AR mRNA Of AR polypeptide in die cell; and then examining one or more physiological eharictefistics of the mammalian prostate cancer cell to which the compound is administered, examined.

[000:1¾ A related embodiment of the invention is&method further comprising examining thephysiolpgipl effect of a plurality of compounds On a mammalian prostate cancer cell selected as describedabove, whereinan observable difference in one or more physiological characteristics exerted by a first compound as compared to one or more physiological characteristics exerted by a second compound indicates that the first compound has a steph^isr or weaker physiological effect than the secondcompound oh.the fhammali an prostate cancer cell. Typically the method isperformed in a high throughput format. Alternatively, the method is performed in a low throughput format. Compounds tested m such assays are typically antagonists or agonists. Inpractiee we define a chug as ahAR antagonist when the drug inhibits or copipetesforthe binding of a ligand or a stimulus and inhibits die biological function of the androgen receptor. A drag is defined as an AR agonist when the drug stimulates or activates the biological function of the androgen receptor.

[00014] Yet another embodiment of the invention is a meft.od.of testing one or more

compounds for an effect on a mammalian cell, themethod comprising contacting at least one compound to be tested with the mammalian cell, wherein the mammalian cell is selected for the test because it expresses an exogenous wild type or mutated protein of interest, such as· the estrogen receptor, such that the total levels of mRNA in the cell that encode the protein of interest or the total protein levels of the protein ofmterest are at least 2 fold higher than normal/endogenous mRNA or polypeptide of the protein of interest, such as the estrogen receptor, in the cell, comparing one or more characteristics of the mammalian cell to which the compound is administered with the same one or more characteristics of a control mammalian cell to which the compound has not been administered, wherein a difference in one or mom dtem«$efi^ies;inftcafeft^ theeorapouiffhas» tiiamftaliaS cancer cell or iaaas»i®meelk in suehmeihads, the mammalian cel ls typically a cancer cell, for example a breast, ovarian or prostate cancer cell.

[00015] A related embodiment of the invention is a method further comprising examining the physiological effect of a plurality of compounds on a mammalian cell that is selected as described above, wherein an observable ftffereneemone or more physiological characteristics exerted by a first compound as compared to one or more physiological characteristics exerted by a second compound indicates that the first.compound has;a stronger or weaker physiological effect than the second compound on the mammalian cell. [00¾¾ Another embodiment ofthe invehtionis a method oftoeiting ,a hoimonetellafitoiy prostate cancer mapstieni, the method comprising administering to the patienfan ageint that decreases or affects the biological function of the androgen receptor by affecting the androgen receptor ligand-binding, nuclear translocation, cm- by affecting DNA-bmding, or through altering formation of coactmtor or compressor complexes associated with the androgen receptor.

[00017} Another embodiment of die invention is a method of treating ahormonerefractory prostate cancer in a patient, the method comprising administering to the patient and an agent that decreases or affects the biological function of the androgen receptor through affecting the. androgen receptor BNA levels,, androgen mRNA levels, or androgen protein levels. In such methods, the androgen receptor protein level can be decreased through modulation of signal transduction pathways such as targeting EGF receptors that crosstalk to the androgen receptor. Alternatively, the androgen receptor protein level is decreased by the induction of cellular degradation pathways such as proteosome degradation machinery. Alternatively, the androgen receptor protein level is decreased by dissociating the androgen receptor from heat shock proteins that maintain the androgen receptor integrity. Preferably the androgen receptor protein level is decreased using androgen receptor antisense or mRNA knockdown technology. We consider that any one of these above manipulations or combination of any pflhese manipulations would affect the biological function of the androgen reciptex; The preferred way of these methods would be to use an agent to disrupt or reduce the ligand binding of the androgen receptor. $0018] Anotherombodiment ofthe invention is a method of treating a hormone refractory prostate cancer hi a patient, the method comprising administering to the patient an agent that ded^ases or affects the biological function of the androgen receptor through, modifying the androgen receptor protein. Optionally, theandrogen receptorprotein ismodified by modifying the polynucleotide or polypeptide sequence of Ihe androgen receptor or by posttranslational modifications of the androgenreceptorincluding, but not restricted to, phosphorylation, acetylation, ubiquitination, andsuraolation.

[00019} Another embodiment ofthe invention is a method oftreating a disease or ©audition, which is resistant to a drug, or ateeatment,or combination of adrugand a tfeatoiem::tiy;to®easmg#e concentration of the protein that is the target ofthe drug or treetofehtotofemetitod cbtop^ that affects the biological function of the protein through means described in the paragraphs above. Typically the disease or condition is hormone refractory prostate cancer, in which the androgen receptor DNA, mRNA or protein levels is increased in prostate cancer cells after surgical or medical castration, or treatments with anti-androgen therapy or the combination of castration and anti-androgen therapy. In an illustrative embodiment, the disease or condition isftormone refractory breast cancer, in which ihe estrogen receptor DNA, mRNA, If protein levels is increased in keastcancer cells after hormone therapy such as treatments with tamoxifen or raloxifene, {00020) Yet another embodiment of the invention is a method of inhibiting prostate specific antigen production in a mamtnalian prostate cancer cell (e.g. a hormone refractory prostate cancel'), die method comprising contacting the cell with a composition comprising a compound selected from the group consisting of compounds 4 and 5 shown in Example 6, so that prostate specific antigen production in the prostate cancer cell is inhibited. A related embodiment of dm invenfitm is method of inhibiting die growth of ahurnan prostate cancer cell, die method comprising contacting the cell with a composition comprising a compound selected from the group consisting of compounds 4 and 5 shown in Example 6, such that growth of the prostate cancer cell is inhibited. Another related embodiment of the invention is a method of antagonizing the function of the ligand binding domain of the AR polypeptide in a prostate cancer cell, the method comprising contacting the cell with a compound selected from the group consisting of compounds 4 and 5 shown in Example 6, such that the function of the ligand binding domain of the AR is antagonized. {00021) Yet another embodiment of the invention is a method of antagonizing the effect of an androgen on a function of the ligand binding domain of the AR polypeptide in a prostate cancer cell, the method comprising contacting the cell with a androgen antagonist compound selected from lie group consisting of compounds 4 and 5 shown in Example 6, such feat the effect of an androgen on a function of tie ligand binding domain of the AR polypeptideis antagonized. 100022) Another embodiment of the inventionis a composition comprising a compound Sete^from-ttegro^ 4 and 5 shown mExamples, ^related embodanetifis a pharmaceutical composition comprising a compound selected from the group consisting of compounds 4 and 5 shown ip Example Certain embodiments of the of admMstermgmesecompounds, for example to an individual hay^a;pd^ee^oer. In view of their similarity with chemical structure offCasodex), certain methods used in administering this compound can be used in the administration of the novel compounds disclosed herein (see, e,g. U.S. Patent Nos. 5.985,868, 6,479 063 and 6,506,607 which are incorporated herein by reference).

[00023] In a further embodiment of the invention, there are provided articles of manufacture and kits containing materials usefid for examining compounds such as AR agonists or antagonists using the methods disclosed herein. The artiele of manufacture comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from, a variety of materials such as glass or plastic. The label on the container may indicate directions for either in vivo or in vitro use, such as those described above. The kit of the invention comprises the container described above and a second container comprising a buffer. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inst^Vi^

BRIEF DESCRIPTIQNOFTHE DRAWINGS

[(10024] FIG. 1 depicts the results of tests showing expression of AR mRNA in HS And HR Xenografts. Top, normalized microarray values for AR probe sets one and two (Asymetrix IDs 1577 and 1578, respectively) are shown for pools oftumors from each of thofrairieen xeflpgpaits. Bottom, AR Western blot from one tumor of each xenografts’ pool lysed m:2^,SEiS, AR protein longer exposures.

[0Q02SJ FIG. 2 diapammatically depicts a model ofprostate cancer progression. Hormone therapy. consisting of androgen-lowering drugs and competitive AR antagonists, decreases, fenamber of active, receptors a Mipieal re§ptmse (HS disease). Failure of therapy (HR disease) results from increased receptor level which inverts the response to antagonists and amplifies the^jp^^jsll' ligands -residual Mdrbgen& arrtagonists and other steroids;

DETAILED DESCRIPTION OF THE INVENTION

[00026] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology usedherein are intended to have the meanings commonly understood by those of skill ;la the art to which this invention pertains. Bisome cases, terms with commonly understood heaeiri for clarity and/or for ready reference, and the inclusion of such deffifofonsherem should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using.conventional methodology by thpse skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995) and Sambrook et at,, Molecular Cloning: A laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

[00027] As used herein, the term "polynucleotide" means a polymeric form of nucleotides of at least about 10 bases or base pairs in length, either ribonucleotides or deoxynueleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA- [00027a] A£tiredherein, except where the context requires otherwise,;the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to eMudtitiifaefadd^ves* «mpoaen^ Inters or steps.

[00027b] Reference to any prior art iu the specification is not, and should not be taken as, an asknowted^ent ©rapy form ^sugf^o»,^ forms part o f the common general tndwtedie hi Australiaor any other jurisdiction or that this prior art could reasonably be expeeted fofeasrertitined, understood and regarded as relevant by a person skilled in the art.

[00020] As used herein, foe term "polypeptide"means apolymer of at least about 6 aminoaeids. the fc^%ndmge® jecepfof of the polynucleotides that encode the atidregesrec^tor plypejaide, Sutih|3d|piufc Bor exampKsenCbang et al., Science 24i p850), 324-326 (1988). Also see NM 00QQ#<hta^wwwmebtmlm,n^^ db=nucleotide&list uids~213222Sl&dopt=GenBank&term=sapiens+AR+androgen+reoeptor+Qros tate+cancer&atv=- l >gi:213222511. Tire term "androgen receptor polypeptide" means any of the known androgen receptor polypeptides. For example, see Chang et al., Science 240 (4850). 324-326 (1988). Also see NM Q00044<htip://wwwj^i^1math.gog:80/ ' entrez/viewer.fcgi?cmd~Retrieve& db=nucteotide&list uids^l 322251 &dont" GerLBank&terni-sapiens+AR-t-aiidrogen+receptor-{-prostate+cancef&qtv^l> gi;21322251). The term “androgen receptor polypeptide variant” means a polypeptide that exhibits AR activity and which has a deletion, insertion or substitution of at least one amino acid in the AR polypeptide amino acid setpenee as: set forth in Ghang etal., Science 240 (4850), 324-' 326 (1988). (00029] The terms "agonist" and "agonistic’’ when use!herein refer to a molecule which is capable of, directly or indirectly, substantially inducing, promoting or enhancing biological activity or activation of a molecule such as AR The trams "antagonist" and "antagonistic" - amolecule which is capable of directly or indirectly, substantially

inhibiting biological activity or activation of a molecule such as AR (00030] "Treatment" or "therapy" refer to both therapeutic treatment and prophylactic or preventative measures. (00031] The term “therapeutically effective amount” refers to an amount, of a drug effective to treat a disease or disorder in a mammal. In tire-case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (ie., slow to some extent and preferably stop) cancer cell infiltration infeperipheral organs; inhibit {ie., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one. or more of the symptoms associated ysttte disorden To the extentthe drug may prevent growth and/Or Mllexisting cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for: example, be measuredby^sessmgtumorbmdenQr volume, thetime todisease progression (TTP|an®# defem^^ (RR).

[00032J “Growth inhibition" when used herein refers to the growth inhibition of a cell in vitro and/or f« vivo. The inhibition of celi growth can be measured by a wide variety of A "growth inhibitoiy agent" wheatsedhrar®niefetOh tiomf©und or compo^tkm^viMt a3hibifegtow4hfa<feti% vitro andhrm vivo. Thus, the growth inhibitory agent reduces the percentage of cells in S phase. Examples of growth inhibitory agents include: agents that block cell pycfepr^esmcm ^a|itoeeAer titan Sphase), such as agents that induce©! arrestandM-phase arrest ClassicalM-phaseblockers meliteethevincas (vincristine and vinblastine), TAXOL®, and topo H inhibitors such as doxorubicin» epirubidn, datmombiein, etoposide, andbleomydn. Sioseafett®i fiat arrest G1 also spill over into S-phase arrest, for example, BNA alkylating agents such as tamoxifen, prednisone, itedbtzin^mechloretomine, cisplatin, methotrexate, 5-fluofOuraeil, and ara-C. Further information can be found in The Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami etal. (WB Saunders: Philadelphia, 1995), especially p. 13.

[00033] "Mammal·' for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and fern animals, and zoo, sports, or pet animals, such as-dogs, horses, cats, cows, etc. Preferably, the mammal is human.

[00034] The terms "cancer", “cancerous", or “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include birt are hot limited to, carcinoma, lymphoma, blastema, sarcoma, and leukemia. More particular examples of such cancers include breast cancer, ovarian cancer, colon cancer, colorectal cancer, rectal cancer, squamous cell cancer, ceil lung cancer, non-small cell lung earner, Hodgkin’s andnon-Hodgkin’s lymphoma, testicular cancer.esophageal cancer, gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma, cervical cancer, glioma, liver cancer, bladder cancer, hepatoma, endometrial ca®gm<m9S>;salivaEy#Wd;i^^ prostate cancer, vulval cancer» thyroid cancer, hepatic caremoma and vatious types of head and neck cancer.

[00035] acceffeble^easier. As used herein the language "pharmaceutically acceptable carrierMs intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal a^B^is^onihani absorption deiayingagenis,andthe life, compatiblewith jipmaimUicaE The use of such media and agents for pharmaceutically ::aetiye,subStaul«^is Wei $a»qr^p.:.-twites? any conventional meditior agent isihcon^tibte v®i &e active compound, such media can be used in the compositions of the invention.. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the, fevtation is formulated to be compatible with its intended routeof administration.

[00036] Therapeutic compositions of the invention can be prepared by mixing the desired molecule having the appropriate degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (See, e.g. Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro (ed.) 1995, Mack Publishing Company, Easton, PA) in the form of lyophilized formulations, aqueous solutions or aqueous suspensions. Acceptable carriers, excipients, or stabilizers are preferably nontoxic to recipients at the dosages and concentrations employed, aodimclude buffers such as Tris, HEPES, PIPES, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine-preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; mono^odharides, disaccharides, and other carhohydm^ M mannose, or dextrins; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counter-ions suchas sodium; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00037] The invention is based on the discoveries disclosed herein that a modest (2-5 fold) increase in androgen receptor (AR) mRNA in prostate dancer cells was the only expression change consistently associated with developing resiffehce fe.antiSndrogee.to This increase in AR mRNA and protein was both necessary and sufficient to comtertprostafe: cancer growth from ahormone sensitive to hohrione refractory stage, and was dependent on a functional tigand-binding domain, Furthermore, AR antagonists displayed agonist activity in cells with antagonist/agonist conversion was associated with

efliafcttons-Mtelatiern.# to the promoter of AR targetgenes, Increasedleyeis of AR confer reristance to anti-androgens by amplifying signal output from low levels of residual ligand and altering the normal response to antagonists. The findings provide insight toward tip [000381 ©he embodiment of the invention disclosed herein is:'€iitetl|od testing compounds for an effect on a mammalian, prostate cancer cell comprising contacting the compound to be tested with a the mammalian prostate cancer cell, wherein the mammalian cancer cell is selected for the test because it expresses an exogenous wild type androgen receptor (AR) polynucleotide teat encodes the AR polypeptide such that the levels of mRNA in the cell that encode the AR polypeptide or the AR polypeptide are at least about 2 fold higher than normal/endogenous AR mRNA levels in a mammalian prostate cell; and then comparing one or more characteristics of the mammalian prostate cancer cell to which the compound was administered with the same one or mom characteristics of a. control mammalian prostate cancer cell to which the compound has not been administered, wherein a difference In one or more of the one or more characteristics indicates that the compound has an effect on the mammalian prostate cancer cell.

[00039] A test compound which binds AR may then be further screened for the inhibition of a specific physiological activity (e.g. tyrosine kinase activity). Such an embodiment includes, for example determining whether said test compound inhibits the signaling Of AR by utilizing molecular biological protocols to create recombinant contracts whose enzymological and biological properties can be examined directly. Enzymology is performed for example, by measuring tyrosine kinase activity in vitro or in ARE expressing cells using ..stepaard;^^..

[00040] Certain discoveries and physiological processes associated: with the invention are discussed below, foaddition, in the examples below, certain compounds useful for example mihbihli^ Methods for making and using these compounds are further disclosed herein.

[00041] To examine the range of HR meehanisms in a relatively unbiased manner, we peribrmed global gene expression profiling on seven “isogenie’’ hormone sensitive (HS) and HRhiiman prostate caneer xenograff pairs (14 total xenografts). AllHRsobiines were passage in castrated mice md compared with HS tumors with similar passage numbers in intact mice/'3'26 Th&:micfomrty daiaadt analyzed using a number of Moinfomiaiic strategies, including unsupervised and supervised learning. First, we asked if any subgroups Of HR prostate eapber could be identified using a» • unsupervised hierarchical clustering aigprite^the foilowing procedure was followed; (afSeven 0.5 cm3tumors fiom each xenograft, grown in either intact (HS) or castrated (HR) mice, were pooled and total KNA was extracted to generate a single sample. After, processing, the cRNA was hybridized to the Asymetrix U95A drip and the microarray data was analyzed by Microarray Suite. Background elements which did not significantly vary :(standard deviation < 1000 and coefficient of variation < 1) or werenot detectedin any of the samples (defined as perfect match hybridization not significantly different than mismatch control signal intensity) were filtered out. The remaining elements, representing 1,056 genes, were then used by an unsupervised leanring algorithm to generate a hierarchical clustering diagram, (b) The microatxay data, obtained as described in (a), was reanalyzed using Microarray Suite whereby each pair was condensed into a single dataset that represents the ratio in expression between each HS xenograft and its HR counterpart. In addition, using a number of parameters as defined by Microarray Suite software such as fold-change, absolute signal intensity and the confidence in each probe set given by perfect match/misnShtch ratios, elements from the condensed dataset were assigned an independent designation of increase (Γ), marginal increase (MI), no change (HC), marginal decrease (MD) or decrease (D). Elements assessed as NC in all of the xenograft pairs were filtered out to generate a list of 3,774 genes that was then used by an unsupervised learning algorithm to generate a hierarchical clustering diagram, (c) Each of the designations described in (b) was assigned a value ranging from +1 (I) to -1 (D) and a score for each element was produced by summing the values across each of the seven IIS/HR. xenograft pairs. This testing showed that each ®&,xia^aft#Q^pe| with its HS counterpart, consistent with the feet that isogenic. pPpf Since xenograft-specific expression signatures are likely to obscure gene expression changes responsible for the HS-to-HR transition,217 we condensed each HS/HR pair into a single dataset representing fold-change in expression. Again, no closeiy related sobgroups emerged, suggesting either thatdifferent meehariisras were responsible for the HS-ioT3R transition in each xenograft or that any common mechanism involved too few genes to 'influence the clusteritigpattem f^pexploj. cd telatter possibility using m algorithm to identify any elements that consistently changed during theHS-to-HR transition. Remarkably, out of 12,559 probe sets, only one r- directed against the AR cDNA - was differentially expressed in all seven HS/HR pairs (Mg. 1, top). Notably, the second highest ranked probe set, upregulated in five of seven pairs, was also directed against AR. Consistent with tire RNA data, immunoblots showed higher levels of AR protein in HR tumors than their parental HS counterparts (Fig, 1, bottom). The .fold-change in AR protein (based on analysis of individual xenograft tumors) did not perfectly correlate with the fold-change in AR mRNA (based on expression analysis of.pools of tumors lor each xenograft), possibly due to sample-to-sample variation. Alternatively, post-transcriptional mechanisms may affect steady state AR protein levels.28 Nonetheless, these data raise the possibility of a common final mechanism of resistance to hormone therapy, (00043] To determine if increased AR protein concentration plays a causal role in the HS-to-HR transition, wo introduced an epitope-tagged wildtype/4R cDNA by retrovirus infection into HS LNCaP human prostate cancer cells. A three-fold increase in AR levels in LNCaP-AR cells mimics the expression difference observed in the LNCaP HS/HR pair studied in the microarray experiment. To test whether increased AR egression causes the HS-tO-HR transition, we designed two in vitro assays to mimic tine clinical circumstances of HR disease. The first measures fete ability to grow in low androgen concentrations; the second measures growth in the presence of tire antiandrogen hicalutamide. As expected, LNCaP cells infected with the empty vector failed to grow in steroid-depleted, charcoal-stripped serum unless supplemented with 100 pM of the syn&etic androgen, Rl 881. In contrast, LNGaP-AReells grew in at least 80% lower concentrations of R1881. LNCaP-AR cells were also resistant to hicalutamide.

[00044] Next, we asked if increased AR expression was sufficient, in vivo, to confer resistance to hormone therapy achieved through surgical castration using two xenograft tnodelsi LNCaP and LAPC4. HS LAPC4 cells were infected with AR lentivirus (dBlttoiistrated^^ A&phtein^ and fiank? Of ititaetpf castled male SCID mice. AR overexpression shortened the latency of in ca^rateftflfinals LAPC4 and LNCaP models.

[00045] We used stable RNA interference to address the iiesiptocal question of whetor fe, increase in AR expression observed in HR xenografts was necessary for developing resistance to hormone therapy. Knockdown of AR levels in HR LAPC4 cells was achieved using a lentivirus vector expressing a short hairpin ENA (shRNA) against AR cA-linked with a GFP-expression cassette. After implantation into the flanks of castrated male mice, AR shKNA-infected tumors grew more slowly than vector-infected controls. Moreover, those tumors that did grow did not express GFP when compared to veetor-ihfected eontrols and still expressed AR protein, indicating selection for cells that escapedAEdmoelalown.

Parallel studies of AR knockdown in HR LNCaP sublines gave similar results.

[00046] To determine the mechanism by which increased AR levels cause HR disease, we considered two possibilities: (1) high levels of receptor lead to constitutive activation in the absence of ligand, consistent with a recent study of AR29 (a ligand-independent model), or (2) high levels of receptor sensitize the cell to the residual amounts of ligand remaining after testosterone lowering therapy (a ligand-dependent mass action model). To distinguish between these models, we introduced two AR mutations, N705$ andR752Q, independently into the LBD tor selectively impair ligand-binding without interfering withligand-independent functions. N7G5S is associated with complete androgen insensitivity syndrome, whereas R752Q is found in patients with partial androgen insensitivity syndrome.30

[00047] Details of the experimental procedure were as follows: Mutant car wildiype AR constructs ware transfected into AR-null cells (CGS7) and androgen-starved tor 48 h. Cells were then incubated with increasing amounts of 1 in the presence or absence of 100- fold excess .of cold R1881 and bound ligand was measured by scintillation counting. LNCaP cells expressing the various AR LBD or genotropic mutants were assayed for HR growth in : vito? using, either the low-androgen sensitization or: an^onist^msi$ta@ee assay US;pfsvteiSly described above. All experiments were done in duplicate, Lx J# LNCaP cells pyemxpressmgthe vapopg JJ? cDNAs («= Iff) or a vector control (n = 10) were implanted into lito&rfe^of castrated male SCID mice and tumor volume (± SEM) was measured over time. was performed with a FLAG-specific antibody on LNGaP cells stably expressing FLAG-tagged, triple point mutant,K.618,632, 633M (ANLS) or wildtype AR.

[00048] As exj^tei, both mutationgumpaired Hgand-binding in a radiolabeled R1881 -binding asssy. insistent with diseaMseverity, R752Q retained low levels of tigand-bin||®g; and transcriptional activity in a reporter assay, mid serves as an internal control to ensure aitoast«&LBD mutant. Overexpression of either LBD mutant AR construct in LNCaP cellsL, even at levels approximately 10-fold higher than endogenous AR, failed to promote HR growth at levels beyond the vector conttol in low androgen media, in the presence of bicalutamide or in surgically castrated mice. These data establish that AR must bind ligand to confer HR growth and imply that a modest increase in receptor concentration permits AR to utilize the lower levels of androgens present in castrated patients. This conclusion also suggests that the widely used tettp “androgen-independent” may be a misleading description ofHR prostate cancer.

[00049] The availability of these in vitro assays for AR function offered an opportunity to address the additional question of whether AR induces the HS-to-HR transition through genotropic or non-genotropic functions. Deletion of the nuclear localization signal (NLS) blocked nuclear translocation and abolished HR growth in low concentrations of androgen or to the presence of bicalutamide. Similar results were obtained using a second AR mutant V581F that lacks DNA banding activity.31 Finally, deletion of the poiyproline region of AR reported to bind the SH3 domain of Src (ΔΡ AR)32 had no inhibitory effect on AR function in these assays. Therefore, the previously reported non-genotropic effects of AR on bone growthand prostate cancer cell survival32'34 are unlikely to play a role ia resistarice to antiandrogen therapy.

[00050] The ligand-binding mutagenesis studies provide evidence for a mass action model to explain resistance to androgen-lowering hormone therapy. If this mechanism is the sole cause of resistanee,then suprapharmacologic concentrations of bicalutamide should overcome the excess levels of AR and block transcriptional activity. Surprisingly, we Observe fhenfp®#? the effectsof high-dose bicalutamide on prostate specific antigen (PSA) expression in cells expressing excess AR Bicalutamide

showed typical antagonist a#yl£y fcplrenta] LASC4'·· cells, as measured by inhibition of PSA mRNA and protein expression, but functioned as an agonist in LAPC4 cells expressing increased levels of AR. The antagonist-to-agonist conversion was not unique to bicaluMnide smem similar results were observed using other AR antagonists such as cyproterone acetate and flutamide, Similar results were observed with LNCaP cells. In addition, increased AR levels conferred responsiveness to noncanonical ligands such as estrogen, reminiscent of the effects of the T877A AR LBD mutation in LNCaP cells.8 [00051] Details of the above high-dose testing were as follows: LAPC4 cells over expressing AR or a GFP control were androgen-starved for 5 days with charcoal-stripped serum and then challenged with bicalutamide or cyproterone acetate or flutamide for 96 hour. PSA and β-actin message was assayed using semi quantitative RT-PCR. Secreted PSA after 48 hour challenge with bicalutamide or Πβ-estradiol was measured via ELISA. LNCaP cells stably infected with an AR-expressing or control virus (Neo) was androgen-starved for 5 days and then challenged for 48 hours with bicalutamide. PSA and β-actin were then measured. LNCaP ceils stably infected with the AR-expressing virusor the vector cpmrel were starved for 5 days and then challenged with either vehicle, 10 μΜ bicalutamide, 100 pM R1881 or 1 nM DHT. After 1 hour, cells mare harvested and processed for chromatin immunoprecipitation.11 [00052] To determine if the above-described conversion was peculiar to the PSA gene or was true for other androgen-regulated genes, we conducted a microarray experiment LNUaP cells infected with, an AR-expressing retrovirus or tfceempty-vector control were challenged with increasing doses of R1881 or 10 μΜ bicalutamide and then processed for hybridization

Bicalutamide inducedthe expression of 48 probe sets (defined as 2-fbld increase, P < 0.Q5,500 minimum expression) in LNCaP-AR cells, whereas no #gfeeflts Wire dgttii®mtly ip^ulated in control cells. The list was comprised mostly of an^geteregAted g^^iinclu^ng^feewefttaownm (KLK2)^. (Seethe TABLE at theendofthis description for a complete list of the genes tested. However, bicalutamide induced only a subset (<10%) of the total number of androgen-regulated genes (> 600 probe sets). Upon closer examination, induced genes overlapped, for the most part, wife the most highly induced, androgen-responsive genes. Consistent with this, unsupervised hierarchical ctostering of the samples grouped the profile of genes induced by 10 μΜ bicalutamide most closely with those in duced by low doses (10-30 pM) of R1881. Together, these data suggest that increased Alt expression globallyconverts antagonists to weak agonists, and therefore leads to the induction of only the most androgen-sensitive genes.

[00053} To address the mechanism by which bicalutamide gains agonist properties in the setting of increased AR Levels, we performed chromatin immunoprecipitation experiments to define the components of the AR transcription complex on the promoters of two genes, PSA and KLK2, activated by bicalutamide in the microarray experiment. After exposure to the classic agonists R1881 or dihydrotestosterone, AR and polymerase Π were recruited to both prOffidtiis regardless: ofAR^ ARwas alsoreeruited to both templates after exposure to bicalutamide in vector and AR-overexpressing cells, but polymerase Π was present only in the setting of exeess AR. However, in AR-overexpressing cells, the repertoire of cc^tivators recruited to these promoters by bicalutamide was more limited (e.g, SRC1 and not AJB1) when compared with R1881 or dihydrotestosterone. We also examined the effect of increased AR levels οη ροΰΜΛϊ OC»*pffi^ by itepre^<M^v As expected, NCoR was recruited to both promoters in parental cells following bicalutamide treatment without evidence of histone aeetylstion cmpo However, NCoR recruitment was reduced (PSA promoter) Or absent (KLK2 promoter) after bicalutamide treatment in cells witit increased AR protein levels. Therefore, a modest cfaafige® the lerelaifARpi^iu can shift the relative abundance ofcoactivators or corepressers psentt ARtiaget gi#^ with resultanteffects on J0pS4] The sinprising resultfrom our expression profiling was theuniversal upregulation the HR xenograft ffiodels, a finding likely to have clinical relevance based on surveys of AR levels in patient material.7,37 AR gene amplification could clearly result in increased AR levels, but this occurs in a minority of patients and cannot be invoked as the explanation for our xenograft findings, since there was no increase InARcopynumber

StudiesofHlgene regulation have implicated AR itself as a positive acting transcription factor that binds the AR gene and leads to increased AR mRNA .levels.39 Therefore, other mechanisms postulated to givemereaSedAMhM^ fncreas^Blta (ErbB2, Ras, MAPK.)oraWered cQafeiivator/corSpressor ratios, may also lead to mcreased^RmRNA levels, albeitindirectly. Thus, any one of a frumbehcffpriinaryM anincreaseinrii? 10¾¾ suggesting a final eomrnon pathway for escape from standardlionnone therapy. fM^SJ One caveat is that our conclusions about AR are based on studies of HSxenografr . models, nearly all Of w|tieh3¾¾¾ how HS growth is “restored” when such tumors are explanted into intact male mice is a long-debated paradox in die prostate cancer field, and the mechanism remains unknown. We previously provided evidence that such explants contain a mixture of HS and HR clones, and that HR subBnes develop through clonal expansion under the selective pressure of androgen deprivation,40 The HS clones that evolve from such explants may be, HS only in relative terms, reflecting a transition state in ft® continuum between truly hormone-naive prostate cancer (no prior exposure to antiandrogen therapy) and full blown HR disease. It remains to be determined whether AR upregulation is sufficient to confer HR growth to hormone-naive cells. Newer transgenic or knockoutmodels of murine prostate cancer may provide an opportunity to address this question in a “cleaner” experimental system.41,42 [00056] Hie simplest model to explain how increased expression of AR can confer resistance to anti-androgen therapy is mass action. According to this model, the 3-5 fold increase in receptor levels observed in our xenograft models can compensatefor low ligand levels and restore AR signaling. However, ,the fact that increased receptor levels cause antagonists to function as agonists suggests another level of complexity. Our comparison of genes induced by bicalutamide to those induced by a range of androgen doses indicates that antagonjsteiuneteasv^afc:^ Comparative hnalyris ^phhlsd 'C®the promoters of AR tatrpt:geh^; revealed a potential mechanism. Specifically, a more limitea repertoire of eqaqt|v^fe®:::i$: recruited to AR target genes after Stimulation with bicalutamide, suggesting that suboptimai h^dsb^tdt^bssenableihe cpihaal array ofcofacfors formaximaltranseriptional activity Because steroid receptor-antagonist complexes adopt a variety of eenfoiSja^^ aipigbni^bbH&<f ARmay be unable to bind the fell complement of coactivator machinery.

[00057] The molecular basis for loss of antagonism is less apparent. Previous work has demonstrated that the agonist versus antagonist response of nuclear receptors like EUs altered by increased expression of coactivators (increased SRC1) or by decreased expression of compressors (deafe^sedNCoR).45,46 Our 4tt^#st|||teh that increased expression of the nuclear receptor itself causes a similar outcome, perhaps by upsetting, the balance of compression and coactivation in theeell. Additional experiments examining each ofthese components are required to sort through these or alternative explanations.

[00058] The clinical relevance of antagonist/agonist conversion should also be considered. About 30% of men whose disease progresses during treatment with AR antagonists experience a paradoxical fall in serum PSA levels when the antagonist is discontinued, called antihandrogen withdrawal syndrome.47 One proposed mechanism is mutation in the AR gene, based on the fact that flutamide functions as an agonist in cells expressing the T877A AR mutation.® Although compelling, this mechanism cannot account for all cases because recent estimates of the frequency of AR mutations in HR patients are too low.5 Our findings suggest that patients with antiandrogen withdrawal syndrome may be those with the highest level of AR upregulation.

[00059] Perhaps the most important implicatioja of the present invention is toward the development of novel antiandrogens. The faet that an intact LBD is required for AR to cause resistance to hormone therapy provides compelling rationale for the design ofnovel antagonists that exploit existing knowledge of this well defined hinding pocket45 Because AR action appears tp be mediated exclusively through genotropic mechanisms, one can also envision drugs that prevent AR nuclear translocation or impair assembly of AR ttShsMpion complexes on target genes. Finally, it will be important to determine if the mechanisms of antiandrogen resistance: - ft* otherh<^<meid^eRdent diseases such as breast cancer.

[00060] Host cells, such.as.jrosMteasesrbells can be transfected or hamformed witli expression or cloniiigwedtom d^iteled h^dih for the expression of the human AR proteins aadmfetredm coiwei^qalmteteiffiedteM selecting transformants, or amplifying the genes encoding the desired sequences. The culture without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found m Mammalian Cell Bfoietfedfofe a Practical Approach. M. Butler. ed. (ERE Press, 1991) and SambrooketaL, ' supra, 10006¾ A wMevarietyofmefoQds of tiansducmg maxrunalian Gells areifoown in the art, for example, using reagents and methods such as viral vectors (e.g. the retroviral vectors disclosed in the Examples below), lipids (e.g, lipofection), CaP04 and electroporation etc. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al,,;:supra. or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology. 52:456-457 (19?8)cah beemployed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4,399,216. However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymofogv. 185:527-537 (1990) and Mansour et al., Nature. 336:348-352 (1988).

[00062] Suitable host cells for cloning iff expressing the AR DNA in the vectors herein include various prostate cancer cell lines such as LNCaP lines, DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrBC), as well as a number of mammalian cells routinely used for foeexpressionofrecombinantproteins (e.g., OS, CEO, 293,293T cells).

[00063] The nucleic acid {e.g., cDNA or genomic DNA) encoding AR may be inserted info a:replica6te:veefor,.fe .qfoplng^ or for expression. Various vectors;, are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, orphage. the appropriate nucleic acid sequence may be inserted into foe vectorby avariety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one

Of moi^Mariter genes, an enheiteef d^lnt, a promoter, and a transcription teimmaiion sequence. Construction of suitablevectors containing one or more of these components employs standard ligation techniques which areknownto the skilled artisan.

[00064] The AR proteins may be ptbdbcesd recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide (e.g., the FLAG tag disclosed herein), which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component ofthe vector, or it may be a part of the AR DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin Π leaders.

For yeast secretion the signal sequence inay be, e.g., the yeast iavertase leader, alpha factor leader (including Sacchmomyces and Khtyveromyces α-fector leaders, the latter described in U.S. Patent No. 5,010,182), or add phosphatase leader, the C. albicans glucoamyiase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion ofthe protein, such as signal sequences from secreted polypeptides of the same» retold speeies, as well as viral secretory leaders.

[00065¾ Both expression and cloning vectors typically contain a nucleic add sequence that enables the vector to replicate in one or more selected host cells. Such sequence ms well known for a variety of bacteria, yeast* and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or RPV) are usefiil for cloning vectors in mammalian cells. A wide range of host-vector systems suitable for the expression of AR are available, see for example, Sambrook et al,, 1989, supra;

Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian exja^bh include butare not limited to pcDNA 3.1 myc-ifis-tag (Inyitrogen) and the retroviral vector pSRatkneo (Muller et al., 1991, MGB11:1785). Using these expression Vectors, AR can be exprKsed in prostate eaneer and hon-prostatecell lines, including for exp^le:ifNC#, 293,293T, rat-1, ΜΗ 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of an ARprotein or fragment thereof. Such host- vector functional properties of AR. and AR mutations eranalpfsi [00066] Expression and cloning vectors will typically contain a selection gene, also termed a selectable market. Typical selection gen#,encode proteins that.($§confer resistance ::fo antibiotics or other toxins, e.g., ampiciliin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies or (c)' supply critical nutrients complex media, e.g., die gene encoding D-alanine racemase for Bacilli.

[00067] Samples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take uptheAR nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl, Acad. Sci. USA. 77:4316 (198(¾. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature. 382:39 (1979); Kingsman etaL Gene. 7:141 (1979); Tschemper et al, Gene, 10:157 (1980)). Tbefrpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATGC No. 44076 or PEP4-1 (Jones, Generics. 85.:12 (1977)).

[00068] Additional details regarding the practice of the invention are as follows; Oligonucleotide U95A and U133 A gene arrays were purchased from Aftymetrix. Charcoal-stripped dextran-treated fetal bovine serum was obtained from Omega Scientific. Bicalutamide was obtained from the UCLA Investigational Drag Pharmacy and dissolved in acetone. Cold and 3H-R1881 were obtained from NBN Life Sciences. AR antibody N-30 (Santa Cruz) aid Flag antibody M2 (Sigma) were used in the immunoblot assays. Secreted PSA was measured by ELISA (American Quaiex). Protein extracts were prepared in high detergent bui#:i§%:fDS)to ensuretotateell lysis.

[00069] Typical ®1A constracds were prepared as follows;· pCSUACG (U6-shRNAcAfoCMV-GFP) was constructed by HgatingtheB^HI/AcoRIdigests ofpCSCG Slid the U6-shRNAoARPCR product. The U6-shRNAaAR PCR was performed «fog a hURcontainingplasmid ai a 60°C annealing temperaiure with suitable primers: pCSCA (CMV-AR) Whs created by subclohing the Xbal fragment afpSRa-AR into the Nhel site of pCSCG. M. mutants were made by standard PCR-based site-directed mutagenesis using the

QuikChange Kit (Stratagene). QNLS contains three point mutations (K618M, K632M. K633M) previously shown, to' disrupt nuclear.UJfrb contains a. deletion of amino acids 372-381, based on prior work.32 ARRjPb-Luciferase was kindly provided by Robert Matusik (Vanderbilt). PSA RT-PCR was also perfbrmed nsing suitable primers* [00070] Details of epical In vitro and 1« vivo Growth experiments are as follows: LNC&P (ATCC) and LAPC4 cells were maintained inlscove’s medium supplemented with 10% fetal bovine serum. LNCaP-AR and LNCaP-vector were derived try infection with the pSRO -AR or pSRP retrovirus,, respectively, and selection in 500 ng/ral of G418. LNCaP-AR, LNCaP-vector, LAPC4-AR, and LAPC4-vector in other experiments were derived by infection with the pCSCA or pCSC lentivirus, respectively, without selection (>90% infection). For in vitro experiments, LNCaP or LAPC4 cells stably infected with different constructs were androgen-starved by growth in charcoal-stripped serum for 3-5 d. 5x104 cells were plated per well in media containing 10% charcoal-stripped sentm supplemented with various concentration ofR1881 or in media containing 10% full serum with various concentration of bicalutamide. Colonies were visualized with crystal vjolet staining 2 weeks later. Ji vrvo tnmorigenieity was measured by injection of 5 x 103 LAPC4 or 1 x 106 LNCaP cells in 100 μΐ ofMatrigel (Collaborative Biomedical) subcutaneously into the flanks of intact or castrated male SCID mice. Tumor size was measured weekly in three dimensions, using caliber as described.26 AR knockdown was performed by infection of HR LAPC4 with shRNA AR lentivirus. Tumors which grew in castrated mice were explained, and analyzed by flaw cytometry for tire percentage of GFP-positive cells. All mouse experiments were performed in compliance with the guideKneg of the Animal Research Committee (ARC) of the UCLA. p007l) HS andHR xenograftpairs for the microarray study were collected from three institutions. LUCaP23,35, and 41 were developed at University of Washington; CWR22 was::de5sfeped% Case Western ReserveUniversity and kmdly provided by University of NoA CttolinaatUhap^^ and 9 Were developed at the University of California at Los AngelesrLNCaP was purchased from ATCC and implanted into mice. The HS Xenografts were either grown in intact nude mice or SCID male mice, and their HR Gounferplfts Were developed by dedal passagpjn eapfedmalemiee. Microarray «^pefifeeats weaB performed and data analyzed according to manufacture’s instructions (Affymetrix). We extracted total KNA from a pool of two to eight tumors with comparable sizes and serum PSA leveisfor each xenograft using TriReagent (Molecular Research Center) and RNeasy (Qiagen). For each sample, 15 pgoftotalKNA was used to generate double stranded cDNA and the cRNA Was transcribed with biotin-labeled nucleotides (KiSiO Dia^iostics). The eRMAwas fragmented and hybridized to U95A microarray (Affymetrix). Scanned images were used for absolute and comparison analysis (Aflymetrix manual). The microarray data were generated through the Genespringprogram (Silicon {000721 Details of typical chromatin immunoprecipitation are as follows: LNCaP-AR or LNCaP-vector were androgen-starved and challenged with either vehicle, 100 pM of R1881, 1 nM ofDHT, or 10 μΜ ofbicalutamide for ί hour. Soluble chromatin was prepared after formaldehyde crosslinkmg and sonication. Specific IgGs against AR, N-CoR, FolU, Ae-• H3/4, SRC1, TIF2, ABB 1, and PCAF were used to immunoprecipitate protein-bound DN A fragments. After reversing the crosslinldng, PGR reactions were performed to amplify the

Duplicates for bicalutamide or vehicle treatment were averaged and queried for the number of probe sets induced 2-fold, P < 0.05 wife a minimum expression of 500 in at least one sample. The dataset was analpeti he titeaumber of probe sets that induced a minimum 2-fold increase. The datashowedthat increased AR expression globally converts antagonists to weak agonists.

[00073} Throughout this application, various publications are referenced (within parentheses for example). The disclosures of these publications are hereby mcorppratedby reference herein in their entireties. In order to facilitate an understanding of various typical aspects of fee invention, certain aspects of these incorporated materials are reproduced herein. {00074] The following examples provide an illustration of how feesa-Beningmethod of fee present invention was used in the testing, development and idmtification of new chemical compounds fer the freatmliRqfcancer, espeqia%:l^^ (androgen- independent) prostate cancer).

[00071] ;Tbfere aremksy compounds which have been binding domain (LBD) androgen receptor (AR). Several have been UsedastirUp to Wat prostate cancer, e.g., bicalutamide (Casodex) 1, which is the current drug of choice for

* prostate cancer. S everal more potent binders of the AR LBD have been identified. e,g., the thiohydantoins, RU59063 2 and BUD, 3 (see, e.g. Teutsch et al., J Steroid Bfackem. Molec. Biol 1994,48,111-119 and VanDortetal., J. Med. Chem. 2000,45,3344-3347). We decided to carry out a study using the screening method of the present invention to identify novel analogues of compounds such as 2 and 3 which would have other alkyl groups with final polar functionality in place of the 4-hydroxybuty! group (Rin2 and 3) which might form much stronger interactions, and even in some cases covalent interactions, with the various polar amino acid side chains of the LBD of the AR Thus the state of the ait was that some strong binders had been discovered but they had not been yet developed into prostatecancer drugs.

[00076] We have prepared a series of compounds 4 based on tfegeneral structure 2 aid have carried out initial screening using the method of the present invention and additional biological testing which Shows that these compounds inhibit the growth of a prostate cancer cell line, including hoimone-sensitive and honnone-refractory variants. The biologicaldata allows us to measure both agonist and antagonist effects and we find that the best compounds are pure antagonists, namely they bind strongly to the LBD of the AR. and shut down the natural process of active cancer growth. In particularthe 4-azido- phenyl derivative 5 has shown the best antagonist effects without any strong agonist effects.

[0007¾ The preferred sequence for the chemical synthesis of these compounds is shown below. The commercially available aniline 6 is converted in one step into the isothiocyanate 7. Commercially available acetone cyanohydrin 8 is converted into the two cyanoamines lOab by reaction with the amines 9a (n=3-8) and die aniline 9b. Addition of lOab to 7 followed by treatment with mild acid produces the desired thiobydantoins 4 (n=3-8) and 5 in good yield.

[00078] These compounds 4 and 5 show remarkable anti-prostate cancer activity. The .i biological testing was carried out by measurement of the amoimt of prostate-specific anfigsh (PSA) produced by LNCaP cells. The 1¾ v^uals bf 4 and 5 for PSA expression areten-fold lower that that for bicalutamide (2-3 times stronger) and the compounds seem to be foil antagonists. Therefore they offer foe potential for a better drug for the treatment of prostate cancer, namely they antagonize the effect of androgens on the AR and thus should greatly slow theppWtt of prostate cancer.

[00079} shown above, wehaveprepared the compounds 4and5and cmriedofofoe initial set of biological assays (including initial screening using the method of foe present invention) that show their potent binder and antagonist ability. This exciting set of compounds is relatively simple to synthesize and already have more potential than the well known heavily used antiprostate cancer drug Casodex.

[00080] Thepreseat invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within foe scope of the invention. Various modifications to foe models and methods of the invention, in addition to those described herein, will become those skilled in the art fiomfoe foregbing description and teachings, and are similarly intaadedtoiffl Such modifications or other embodiments can be practiced without departing from the true scope and spirit of foe invention. However, the invention is only limited by the scope of foe appended claims. TABLE ' ,

Affv ID Gen Bank tD »

' ' Afty ID Gen Bank ID 212005 AL5828Q8 — - ...... 206205 NM. 022782.1 213017 NML138340 . “ „ „„„ nonn„, 221965 NM 022782 201662 D89053.1 “ , 208309 NM 0087851 216323 XM 054284 ~ uu,OJ·' 21,689 AR70487.1 ^ NM-002465 205102 NM 006656 211548 2,50« 887779.1 205040 NM-°“»7·1 , 20,358 NM 0142,8 205041 **.000607.1 222201 «037736,1 ^ “*-013440·1 il'oia NtooQeqe., Φ* 222121 NVL015505 *\«* , i . 218782 NM 01410S1 2126Θ5 NC*?1^Sq8 · -1 20,369 j«W ^ ^005035^ 20,560 «^04,17., 11975 ΝΜ-°°?35?·1 21,802 801004472.1 "*-<»»"» mm NM.023920.1 2!6“ M27331·1 ; 215806 Ml3231i 219478 NMJ324115.1 * 209813 M16768.1 2103ij9 BC005196.1 ; 1 ί. ‘: 1 ' : ' 211144 M308941 g09854 NMJ30ij551 . 20,583 8,7040.1 201103 NM-003248 ,04582 LmW 222,18 ""“T1 ' ^ 219655 NM-018455 '205^62 NM 014668.1 ί"Ρ Ν“-01?456·1 20155, 8032«., ?°?°53 ^39 209309 D90427.1

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Claims

CLAIMS WHAT IS CLAIMED IS:

1. A compound having the formula 1/”r Me wherein R is (CH2)nN3 and where n is 4.
2. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier for said compound.
3. A method of inhibiting prostate specific antigen production in a mammalian prostate cancer cell, the method comprising contacting said mammalian prostate cancer cell with a sufficient amount of a compound according to claim 1, such that prostate specific antigen production in said mammalian prostate cancer cell is inhibited.
4. A method of inhibiting the growth of a human prostate cancer cell, the method comprising contacting said human prostate cancer cell with a therapeutically effective amount of a compound according to claim 1, such that growth of said human prostate cancer cell is inhibited.
5. A method of antagonizing the function of the ligand binding domain of the androgen receptor polypeptide in a prostate cancer cell, said method comprising the step of contacting said prostate cancer cell with a sufficient amount of a compound according to claim 1, such that the function of the ligand binding domain of the androgen receptor is antagonized.
6. A method of antagonizing the effect of an androgen on a function of the ligand binding domain of the androgen receptor polypeptide in a prostate cancer cell, the method comprising the step of contacting said prostate cancer cell with a sufficient amount of a compound according to claim 1, such that the effect of an androgen on a function of the ligand binding domain of the androgen receptor polypeptide is antagonized.
7. A method for examining the physiological effect of the compound of claim 1 on a mammalian prostate cancer cell, wherein said prostate cancer cell expresses an exogenous wild type androgen leceptor polynucleotide that encodes an androgen receptor polypeptide or an androgen receptor polypeptide variant, said cell further comprising an abnormal level of mRNA that encodes said androgen receptor polypeptide or said androgen receptor polypeptide variant when compared to the level of mRNA that encodes said androgen receptor polypeptide or said androgen receptor polypeptide variant in a normal prostate cell, the method comprising: (a) determining that the abnormal level of mRNA in the prostate cancer cell is at least two fold higher than the level of mRNA in the normal prostate cell; (b) contacting the compound with the prostate cancer cell to provide a treated prostate cancer cell; and (c) examining one or more physiological characteristics of the treated prostate cancer cell.
8. A method of examining the physiological effect of the compound of claim 1 on a mammalian prostate cancer celh wherein the prostate cancer cell expresses an exogenous wild type androgen leceptor polynucleotide that encodes an abnormal level of androgen receptor polypeptide or an abnormal level of androgen receptor polypeptide variant when compared to the level of androgen receptor polypeptide or androgen receptor polypeptide variant encoded by a normal prostate cell, the method comprising: (a) determining that the abnormal level of androgen receptor polypeptide or the abnormal level of androgen receptor polypeptide variant is at least two fold higher than the level of androgen receptor polypeptide or androgen receptor polypeptide variant in the normal prostate cell; (b) contacting the compound with the prostate cancer cell to provide a treated prostate cancer cell; and (c) examining one or more physiological characteristics of the treated prostate cancer cell.