WO2015150921A2

WO2015150921A2 - Methods for treating prostate cancer

Info

Publication number: WO2015150921A2
Application number: PCT/IB2015/001187
Authority: WO
Inventors: Xavier SALVATELLA; Eva DE MOL; Carlos W. BERTONCINI; Christopher T.W. PHANG; Iain J. MCEWAN; Antoni RIERA
Original assignee: Fundacio Institut De Recerca Biomedica (Irb Barcelona); Institucio Catalana De Recerca I Estudis Advancats; University Court Of The University Of Aberdeen; Unversitat De Barcelona
Priority date: 2014-04-03
Filing date: 2015-04-03
Publication date: 2015-10-08
Also published as: WO2015150921A3

Abstract

A method for inhibiting activity of the human androgen receptor in a cell, and a method for treating prostate cancer in a subject in need thereof, using a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF. A method of inhibiting androgen receptor activity in a cell, and a method for treating prostate cancer in a subject in need thereof, using an inhibitor of the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser426, Ser430, Ser431, Ser432, Thr435 and Thr438. A method of diagnosing a subject having, or at risk of having, castration-resistant prostate cancer, comprising determining the phosphorylation status of one or more of residues Ser422, Ser424, Ser426, Ser430, Ser431, Ser432, Thr435 and Thr438.

Description

METHODS FOR TREATING PROSTATE CANCER

BACKGROUND OF THE INVENTION

Prostate cancer

[0001] Prostate cancer (PC) is the second most frequent cancer in men and the fifth most frequent cause of cancer related deaths. When the cancer is localized it can be treated by surgery or radiotherapy but in about 30% of the cases PC cancer cells will have spread to other tissues before the disease is detected and treated. This scenario is usually detected by increases of the blood levels of prostate specific antigen (PSA) after surgery or radiotherapy in a scenario known as biochemical failure.

[0002] The first line of treatment in these cases is hormone blockade, which causes important decreases in the blood levels of androgens and the apoptosis of prostate cells as these rely on the activation of androgen receptor (AR) by androgens for their proliferation. AR is a transcription factor that in its androgen bound active form enhances the transcription of genes related to the development of the male phenotype.¹

[0003] Hormone blockade is often combined with the administration of anti-androgens, which are AR antagonists with high affinity for the androgen binding site in the ligand binding domain (LBD) of AR. Although this combined therapeutic approach is initially successful patients inevitably become refractory to it in a condition called castration resistant prostate cancer (CRPC) that inevitably leads to metastatic PC and is responsible for 30.000 yearly deaths in the USA and 70.000 in Europe.

Castration resistant prostate cancer

[0004] The molecular basis of CRPC is not fully understood but a substantial body of evidence points towards AR having an important role by becoming active by mechanisms other than hormone binding. Several such mechanisms have been put forward, including the expression of AR mutants active at very low levels of androgen, the expression of constitutionally active AR isoforms lacking the LBD^2"4 and the activation of signaling cascades boosting the activity of the receptor by post translational modifications (PTMs) such as phosphorylations.⁵'⁶ [0005] Understanding how AR is activated in CRPC is key for the development of molecular approaches for this indication but research in this area is hampered by the presence of intrinsically disorder (ID) regions⁷ in AR.⁸ ID proteins as well as ID regions in multi-domain proteins are functional regions of sequence that fail to fold into conformational states with well-defined secondary and tertiary structures.

Intrinsic disorder

[0006] Proteins displaying ID are particularly frequent in highly evolved organisms and often play the role of hubs in protein-protein interactions (PPIs) networks. These observations have led to the suggestion that ID is a molecular feature that has evolved to facilitate signaling by allowing proteins to bind to a wide range of ligands in different conformations.⁹ Given that ID proteins and ID regions are also over-represented among proteins involved in cancer there is substantial interest in targeting them with small molecules.¹⁰

[0007] Unlike globular proteins, the structural properties of which are well accounted for by a single conformation, ID proteins and regions are instead best described by structurally heterogeneous ensembles of rapidly interconverting conformations. In some cases proteins displaying ID become sufficiently structured for structure determination by conventional means upon interaction with binding partners¹¹ but in many cases the resulting complexes continue to display substantial structural heterogeneity, which makes the characterization of their structure challenging.¹²'¹³

[0008] Nuclear magnetic resonance (NMR) is a very powerful experimental technique for the structural characterization of ID proteins and regions.¹⁴ Its most remarkable feature is that it yields residue specific parameters that can be related to specific structural properties such as inter-atomic distances and torsion angles. That this type of protein cannot be described by a single conformation has lead to the development of different approaches for the generation of conformational ensembles by using NMR parameters to bias molecular simulations. ^{15 18}

Androgen receptor

[0009] The amino acid sequence of human AR1 (obtained from Uniprot: accession number P10275) corresponds to that represented by SEQ ID NO.l, as follows:

MEVQLGLGRVYPRPPSKTYRGAFQNLFQSVREVIQNPGPRHPEAASAAPPGASLLLLQQQ QQQQQQQQQQQQQQQQQQETSPRQQQQQQGEDGSPQAHRRGPTGYLVLDEEQQPSQPQSA LECHPERGCVPEPGAAVAASKGLPQQLPAPPDEDDSAAPSTLSLLGPTFPGLSSCSADLK DILSEASTMQLLQQQQQEAVSEGSSSGRAREASGAPTSSKDNYLGGTS I SDNAKELCKA VSVSMGLGVEALEHLSPGEQLRGDCMYAPLLGVPPAVRPTPCAPLAECKGSLLDDSAGKS TEDTAEYSPFKGGYTKGLEGESLGCSGSAAAGSSGTLELPSTLSLYKSGALDEAAAYQSR DYYNFPLALAGPPPPPPPPHPHARIKLENPLDYGSAWAAAAAQCRYGDLASLHGAGAAGP GSGSPSAAASSSWHTLFTAEEGQLYGPCGGGGGGGGGGGGGGGGGGGGGGGGEAGAVAPY GYTRPPQGLAGQESDFTAPDVWYPGGMVSRVPYPSPTCVKSEMGPWMDSYSGPYGDMRLE TARDHVLPIDYYFPPQKTCLICGDEASGCHYGALTCGSCKVFFKRAAEGKQKYLCASRND C IDKFRRKNCPSCRLRKCYEAGMTLGARKLKKLGNLKLQEEGEASSTTSPTEETTQKLT VSHIEGYECQPIFLNVLEAIEPGVVCAGHDNNQPDSFAALLSSLNELGERQLVHVVKWAK ALPGFRNLHVDDQMAVIQYSWMGLMVFAMGWRSFTNVNSRMLYFAPDLVFNEYRMHKSRM YSQCVRMRHLSQEFGWLQI PQEFLCMKALLLFS I I PVDGLKNQKFFDELRMNYIKELDR I IACKRKNP SCSRRFYQLTKLLDSVQPIARELHQF FDLLIKSHMVSVDFPEMMAEI I S VQVPKILSGKVKPIYFHTQ

The aforementioned SEQ ID NO. l is written from the N-terminal end to the C-terminal end, whereby residue 1 corresponds to M (methionine) and residue 919 corresponds to Q (glutamine). The amino acid sequences and residues mentioned throughout the patent specification respectively refer to sequences and residues of SEQ ID NO. l (e.g. Ser422 refers to the 422nd amino acid residue of SEQ ID NO. l , namely serine, while residues 421 to 446 refer to the amino acid sequence of SEQ ID NO. l from the 421st to 446th amino acid residues, namely GSGSPS AAAS S S WHTLFT AEEGOL YG [also identified as ⁴²¹GSGSPSAAASSSWHTLFTAEEGQLYG⁴⁴⁶]). The ID regions in AR correspond to the N-terminal transactivation domain (NTD, residues 1 to 559) and to the hinge region connecting the DNA binding domain (DBD, 559 - 623) with the LBD (670 - 919). The NTD plays a fundamental role in AR function by recruiting, after DNA binding by the DBD, the transcription machinery to promoters and enhancers of genes regulated by AR. The hinge region also plays an important role by interacting with proteins, such as importin, responsible for the nuclear translocation of androgen-bound AR.¹⁹'²⁰

The structure of AR undergoes substantial changes upon activation by androgens.

Inactive AR is a monomeric cytosolic protein stabilized by molecular chaperones that bind to the ID NTD. Androgen binding to the LBD causes an intra-domain conformational change that leads to the formation of activation function 2 (AF-2) in the surface of this domain. As AF-2 has high affinity for a well-defined hydrophobic motif (FQNLF, 23 - 27) near the N-terminus of AR androgen binding leads to the formation of head-to-tail dimers stabilized by this interaction, that translocate to the nucleus.²¹ [0011] Once in the nucleus AR interacts with the DNA via interactions of the DBD with specific regions of sequence called androgen response elements (AREs) found near the promoters and enhancers of genes regulated by AR.²² AREs are organized as pairs of DNA sequences with affinity for DBD dimers, stabilized by interactions between DBDs. These AR dimers recruit the transcription machinery at the promoter by transient PPIs of the NTD with general transcription factors (GTFs) and nuclear receptor co-activators (NCOAs).

Currently available treatments for CRPC

[0012] Current therapeutic approaches for CRPC focus, like those for PC, on preventing the activation of AR by androgens. Abiraterone, marketed as abiraterone acetate under the trade name Zytiga by Janssen, inhibits the 17 a-hydroxylase and CI 7,20 lyase activity of cytochrome P450 17 (CYP17), an enzyme expressed in prostate tumors which catalyzes the intracrine synthesis of androgen precursors, that contributes to tumor growth during hormone blockade.²³ Abiraterone was approved for the treatment of CRPC in April 2011 by the FDA.

[0013] Enzalutamide, marketed under the trade name Xtandi by Medivation, is an anti- androgen that binds to the LBD of AR with higher affinity than flutamide and bicalutamide and is effective in prostate tumors where increased AR levels, typically due to gene amplification, contribute to the onset of CRPC.²⁴ In addition enzalutamide is an antagonist of the relatively common W741C mutant of the AR that can be activated by other anti-androgens. This drug was approved in August 2012 by the FDA and received EMA approval in April 2013.²⁵

[0014] Although abiraterone and enzalutamide represent important developments in the treatment of CRPC they offer modest outcomes, increasing survival for only 4 and 5 months, respectively. In addition they address a fraction of the mechanisms suspected to fuel the proliferation of prostate cells in CRPC, namely the intracrine synthesis of androgens, AR gene amplification and mutations in the LBD.

[0015] Furthermore, tumors treated with abiraterone and enzalutamide have already been reported to develop resistance against these therapeutics. This has been linked to the overexpression of constitutively active splice variants following treatment of both abiraterone and enzalutamide.²⁶'²⁷ In addition, promiscuous activation of AR by exogenous glucocorticoids that are co -administered to reduce the side effects of abiraterone, and by steroid precursors upstream of CYP17 have been observed in patients treated with abiraterone.²⁸ Additionally, a new mutation in the AR LBD (F876L) has been reported that confers resistance to enzalutamide by converting it into an AR agonist.²⁹'³⁰

[0016] In order to increase the range of therapeutic options for CRPC it is key to develop therapeutic strategies that address other important mechanisms such as the expression of NTD mutants and constitutively active splicing variants of AR lacking the LBD as well post translational modifications (PTMs) boosting the activity of the receptor.

The transactivation domain of AR as therapeutic target

[0017] Contrary to what is the case for the LBD the NTD is indispensable for AR function and as a consequence represents in principle an attractive target for the treatment of both PC and CRPC by inhibiting with small molecules or biologicals the PPIs that this domain must establish to recruit the transcription machinery at promoters and enhancers.³¹

[0018] Specific observations that suggest that the NTD is a target of particularly high potential for CRPC include the inhibition of cell growth and proliferation by NTD (1- 558) decoys,³² the high frequency of NTD mutations in patients of hormone blockade,³³ the ability of small molecules targeting the NTD to cause the regression of CRPC in cell lines and animal models (see below) and the large number of actual and potential phosphosites in the NTD.³⁴'³⁵

[0019] Due to this potential efforts have been directed at identifying the regions of sequence of the NTD that play particularly important roles for AR function in a CRPC context. These led to the finding that a small and well-defined motif in the NTD (residues 426 to 446, S AAAS S S WHTLFT AEEGQL YG) mediates androgen depletion independent transactivation in CRPC cell lines.³⁶ This identifies this region of sequence as one likely to be involved in PPIs, with yet to be identified binding partners, that could be targeted for drug discovery for CRPC.

The transactivation domain of AR interacts with TFIIF

[0020] TFIIF is a GTF tightly associated with RNA polymerase II (RNAPII) and therefore considered part of the transcription machinery. Its exact role is not known but it is thought to directly or indirectly contact with the transactivation domains of gene regulatory proteins bound to promoters and enhancers of transcription such as AR.³⁷ TFIIF is also known to play an important role in the termination of transcription by recruiting FCP1, a phosphatase that dephosphorylates the ID C-terminal tail of RNAPII and in doing so decreases it affinity for DNA and enables a subsequent round of transcription. 38

[0021] TFIIF is a heterodimeric protein stabilized by interactions between the N-terminal domains of subunit 1, also known as RAP74, and subunit 2, also known as RAP30.³⁹ The C-terminal domain of RAP74, also known as RAP74CTD, is a small 68-residue globular protein of known structure that recruits FCP1 by interacting with either of the two ID motifs that bind to it in a a-helical conformation which has been characterized both by X- ray crystallography³⁸ and NMR.⁴⁰

[0022] It is well established that AR binds to TFIIF through interactions of its transactivation domain with RAP74CTD both in vitro and in vivo.⁴¹ Although the region of sequence of the NTD involved in this interaction has not yet been characterized in detail an investigation of the effect of point mutations in NTD on the affinity between AR and TFIIF identified two PSTLSL motifs (residues 159 to 164 and 340 to 345) as key for the interaction⁴² Residues Met 245, Leu 247 and V 249 were also suggested to be part of the binding site of RAP74CTD.^{43 44}

EPI-001 is an experimental drug for the treatment of CRPC that targets the NTD

[0023] EPI-001 is a derivative of bisphenol A identified in a phenotypic high throughput screening campaign as a potent inhibitor of AR in CRPC cell lines as well as in a xenograft mouse model of this disease. Unlike all AR inhibitors used in the clinic or EPI- 001 does not target the androgen binding site in the LBD of AR and instead targets the ID NTD.³⁴

[0024] The identity of the specific residues of the NTD that interact with EPI-001 is not known mainly due to the ID nature of this domain, that renders its structural characterization challenging. A recent publication, however, indicates that this small molecule inhibits AR by the irreversible reaction of one or several nucleophilic side chains of the NTD EPI-001 to yield an adduct that is transcriptionally silent.³⁵

[0025] The discovery of EPI-001 is an important development for the development of therapeutic approaches for CRPC because it indicates that targeting the NTD with small molecules is possible and leads to very desirable outcomes in preclinical studies. However, many questions regarding the mechanism of action of EPI-001 remain to be answered, including the identity of the side chain(s) of the NTD that react with EPI-001 and the source of specificity for the NTD of AR.

SUMMARY OF THE INVENTION

[0026] In order to understand the mechanisms by which the NTD contributes to AR function and to identify new therapies for castration-resistant prostate cancer, the present inventors have carried out a comprehensive characterization of the structural properties of this domain. In addition, the inventors have studied how these change upon interaction with RAP74 and EPI-001. Given the intrinsically disordered nature of the NTD, these studies have been mainly performed by solution nuclear magnetic resonance (NMR) in combination with other biophysical tools and cell biology experiments.

[0027] All therapies currently available for PC and CRPC focus on preventing the activation of AR by androgens either by inhibiting the paracrine and intracrine secretion of androgens or by inhibiting androgen binding to AR. We here describe novel methods for treating CRPC that instead aim at inhibiting AR function after the receptor has been activated by androgen binding or by the various aberrant mechanisms of activation that occur in CRPC. In addition we describe a novel method to identify CRPC patients particularly likely to benefit from the novel treatment methods disclosed herein.

[0028] The new therapeutic approaches rely on weakening or altogether inhibiting the interaction between the NTD of AR and RAP74 after activation and translocation to the nucleus and on modifying the chemical structure of the NTD with reactive small molecules in ways that prevent it from being functional. The diagnostic approach relies instead on identifying specific phosphorylations of the NTD that strengthen its interaction with RAP74CTD, thus allowing prostate cancer cells to proliferate in patients undergoing hormone blockade and contributing to the onset of CRPC.

[0029] These inventions involve the straightforward identification of small molecules, for therapy, and of biologicals, for therapy and diagnosis, by using standard techniques such as screening methods for identifying kinase inhibitors and for raising antibodies against phosphopeptides. More specifically the inventions enable the design of efficient screening methods because they allow the design of a suitable screening assay and the identification of the kinase(s) phosphorylating the AR motif. They also enable raising specific antibodies for diagnosis and therapy because they provide the identity of the phosphopeptides that must be recognized by the antibody.

[0030] The present inventors have discovered that phosphorylating Ser residues N- terminal to the motif of AR that interacts with RAP74CTD increases the affinity between these two proteins. Thus, the invention relates in one embodiment to inhibiting the kinases that are responsible for these phosphorylations and/or activating the phosphatases that can dephosphorylate these residues as potential therapeutic avenues for CRPC.

[0031] Another aspect of the invention relates to a treatment of CRPC by administration of small molecules or biologicals that interact with the motif 4²¹GSGSPSAAASSSWHTLFTAEEGOLYG⁴⁴⁶ of the NTD of WT or mutant AR in either of the 2⁸ -1 = 255 phosphorylated forms which are possible by combining all possible Ser or Thr potential underlined phosphosites and excluding the non phosphorylated form.

[0032] Still another aspect of the invention relates to a method to diagnose CRPC by extracting circulating tumor cells from the prostate cancer patient and determining the phosphorylation status of the androgen receptor that these express by using an antibody that specifically recognize either of the 255 phosphorylated motifs in the NTD of AR.

[0033] Another aspect of the invention relates to the administration of small molecules or biologicals that interact with (a dimeric form of) Tau-5 and/or react with the SH group of the side chain of Cys 404 for the treatment of CRPC with the specific exclusion of EPI- 001.

[0034] Thus, in one embodiment, the invention is directed to a method for inhibiting activity of the human androgen receptor in a cell, said method comprising contacting said androgen receptor with a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF. In one embodiment, the molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein. In a particular embodiment, the molecule is not EPI-001. In certain embodiments of the invention, the androgen receptor is wild-type while in other embodiments it is a mutant form. In a particular embodiment, the molecule selectively inhibits the interaction of amino acids 433-448 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74. [0035] In one embodiment, the invention is directed to a method for treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF. Equally, the present invention is directed to a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF for use in treating prostate cancer. In an embodiment, the prostate cancer is castration-resistant prostate cancer. Preferably, the molecule is selected from the group consisting of a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein in the foregoing aspects of said method of treating prostate cancer and molecule for use in treating prostate cancer. In another embodiment of the foregoing aspects of said method of treating prostate cancer and molecule for use in treating prostate cancer, the molecule used in this method is not EPI-001. The androgen receptor can be mutant or wild-type. In certain embodiments of the foregoing aspects of said methods of treatment of prostate cancer and said molecule for use in treating prostate cancer, the molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

[0036] In another embodiment, the invention is directed to a method for inhibiting androgen receptor activity in a cell, the method comprising contacting the androgen receptor with an inhibitor of the phosphorylation of one or more residues of the N- terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438. In one embodiment, phosphorylation of residues Ser430, Ser431, and Ser432 is inhibited. In general, the molecule can be selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, or an antibody fusion protein. The androgen receptor can be mutant or wild-type in this method.

[0037] In a further embodiment, the invention is directed to a method of treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438. Equally, the present invention is directed to a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438 for use in treating prostate cancer. In one embodiment, the molecule inhibits phosphorylation of Ser430, Ser, 431 , and/or Ser432. In general, the molecule can be a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, or an antibody fusion protein. The androgen receptor can be mutant or wild-type in the methods of treating prostate cancer and molecule for use in treating prostate cancer of the invention. In another embodiment, the cancer is castration- resistant prostate cancer. In one embodiment of the foregoing aspects of said method of treating prostate cancer and molecule for use in treating prostate cancer, the molecule inhibits a kinase that phosphorylates one or more of said residues. Preferably, the kinase is the CK1 kinase or the GSK3beta kinase. In certain embodiments, the kinase is the CK1 kinase or the GSK3beta kinase and the molecule is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l ,4-benzodioxin-6-yl)-5-(2-pyridinyl)-l -imidazol- 2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2-pyridinyl)phenyl]amino]-9H-purin-2- yljamino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl- 1H- imidazol-2-yl)-2 pyrimidinyl] amino] ethyl] amino] -3 -pyridinecarbonitrile; Myr-N- GKEAPPAPPQSpP-NH₂, Myr-N-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Gln- Ser(P0₃H)-Pro-NH₂ trifluoroacetate salt; or 4-Benzyl-2-(naphthalen-l-yl)- 1 ,2,4- thiadiazolidine-3,5-dione.

In a further embodiment, the invention is directed to a method of diagnosing a subject having, or at risk of having, castration-resistant prostate cancer, the method comprising obtaining a tumor cell sample from a patient with prostate cancer, and determining the phosphorylation status of the androgen receptor in the tumor cell sample at one or more of residues Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438, wherein phosphorylation of one or more of these residues is indicative of a subject having, or at an increased risk of having, castration-resistant prostate cancer. [0039] In another embodiment, the invention is directed to a method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor. Equally, the present invention is directed to at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor for use in treating prostate cancer. In one embodiment, the cancer is castration-resistant prostate cancer. The receptor can be the wild-type or a mutant form of the human AR in the foregoing method of treating prostate cancer and molecule for use in treating prostate cancer. In a certain embodiment, the invention is directed to a method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 433-446 of the N-terminal domain of the androgen receptor with RAP74. Equally, the present invention is directed to a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446, preferably the motif of amino acids 433-446, of the N-terminal domain of the androgen receptor with RAP74 for use in treating prostate cancer. In one embodiment of the foregoing method of treating prostate cancer and molecule for use in treating prostate cancer, said androgen receptor is wild- type, while in another embodiment said androgen receptor is a mutant form.

[0040] In another embodiment, the invention is directed to a method for treating prostate cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor. Equally, the present invention is directed to a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N- terminal domain of the androgen receptor for use in treating prostate cancer.

[0041] The molecule can be a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein. In one preferred embodiment of the foregoing aspects of the method of treating prostate cancer and molecule for use in treating prostate cancer, the molecule is not EPI-001. BRIEF DESCRIPTION OF THE FIGURES

[0042] FIG. 1. Chemical shift perturbation results revealing the identity of the AF-1 motif that interacts with RAP74CTD as ⁴³³ WHTLFTAEEGQL YG⁴⁴⁶. The average chemical shift perturbations were calculated as [(5Hb₀und-6Hf_ree)²+((5Nbound-6Nf_ree)/5)²]^1/2

[0043] FIG. 2. Titration of AF-1 with RAP74-CTD used to determine that the affinity between these two molecules is ca lmM. The x axis represents the concentration of RAP74CTD whereas the y axis represents the ¹⁵N chemical shift. The experimental data is shown as dots and the fitted equation in red. The chemical shift changes of twelve residues were used for the fitting but only eight are shown here for clarity.

[0044] FIG. 3. Alignment of the sequences of the C-terminal and central intrinsically disordered motifs of FCP1 that interact with RAP74CTD with the sequence of the AR motif.

[0045] FIG. 4. NMR structure (pdb code lonv) of the complex between RAP74CTD and the C-terminal motif of FCP1 with an indication of the salt bridges involving negatively charged side chains of FCP1 with positively charged side chains in RAP74CTD. The termini of the motif are shown as N and C to illustrate its orientation.

[0046] FIG. 5. Illustration that removing the two negatively charged residues of the proposed charge clamp decreases the affinity between AF-1 and RAP74CTD and therefore leads to essentially no chemical shift changes in this domain of TFIIF. The spectra of 50 μΜ free RAP74CTD, that of the same domain in the presence of 500 μΜ WT peptide and that of the same domain in the presence of 500 μΜ mutated (E440K,E441K) peptide are shown. The x axis represents the 1H chemical shift whereas the y axis represents the ¹⁵N chemical shift.

[0047] FIG. 6. Transcriptional activity of the wild type and E440K, E441K charge reversal mutant in PC3 cells (see Materials and Methods).

[0048] FIG. 7. Alignment of the sequences of the C-terminal and central intrinsically disordered motifs of FCP1 that interact with RAP74CTD with the sequence of the AR motif with an illustration of the presence of Ser residues (underlined) in a region rich in negatively charged residues in the FCP1 motifs.

[0049] FIG. 8. Titration of RAP74CTD with a peptide simultaneously phosphorylated at

Ser 430 and 432, which yields an affinity of K_D = 80 μΜ. The x axis represents the 1H chemical shift whereas the y axis represents the ¹⁵N chemical shift. The spectra corresponding to 50 μΜ RAP74CTD and to 50 μΜ RAP74CTD and 1 niM peptide are shown.

[0050] FIG. 9. Transcriptional activity in PC3 cells of the wild type and AR mutants used to investigate the effect of replacing the ⁴³⁰SSS⁴³² motif present in WT AR with phosphorylation mimics (see Materials and Methods).

[0051] FIG. 10. Chemical structure of compound 1, also known as EPI-001.

[0052] FIG. 11. Chemical shift perturbations of the ¹⁵N chemical shift caused by 10 molar equivalents of EPI-001 on the resonances of AF-1.

[0053] FIG. 12. 1H Chemical shift perturbations and line broadening effects caused by

AF-1 on the resonances of 1. The resonance of DSS-d6, used to reference the chemical shift, is shown as a control.

[0054] FIG. 13. Secondary structure in AF-1 according the analysis of the ¹³Ca chemical shifts, where regions with substantial helical secondary structure are shaded.

[0055] FIG. 14. Comparison of the ¹⁵N chemical shift changes caused by interaction with

1 (top) with those caused by an increase in concentration (bottom). The discontinuous horizontal line in the upper plot represents the minimal chemical shift change considered significant.

[0056] FIG. 15. Results of the MS experiments obtained after incubation of AF-1 with 1 for 1, 2.5 and 4 hours at 315 K, which indicate that the only Cys residue that appears to react with 1, among the 8 present in AF-1, is Cys 404.

DETAILED DESCRIPTION

Definitions

[0057] Inhibitor. Any chemical compound, nucleic acid molecule, or peptide/polypeptide, such as a small organic molecule, a nucleic acid (such as an RNAi nucleic acid), or an antibody, specific for a gene product that can reduce activity of the gene product. An inhibitor of the disclosure, for example, can inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a protein and thereby preventing the protein from binding a target (such as a receptor or binding partner) or preventing protein activity (such as enzymatic activity). Indirect inhibition can be accomplished, for example, by binding to a protein's intended target, such as a receptor or binding partner, thereby blocking or reducing activity of target protein.

[0058] Prostate cancer. A malignant tumor, generally of glandular origin, of the prostate.

Prostate cancers include adenocarcinomas and small cell carcinomas. Many prostate cancers express prostate specific antigen (PSA). Prostate cancer initially grows in an androgen-dependent manner, and androgen deprivation therapy (ADT) is an effective treatment in many cases of prostate cancer. However, prostate cancer often will eventually become refractory to ADT. "Castration-resistant prostate cancer" (CRPC, also known as hormone- refractory prostate cancer) is prostate cancer that has become androgen-independent and progresses despite low levels of androgens (for example, in a subject undergoing ADT).

[0059] Subject: Living multi-cellular vertebrate organisms, a category that includes both human and non-human mammals. Subjects include veterinary subjects, including livestock such as cows and sheep, rodents (such as mice and rats), and non-human primates.

[0060] Therapeutically effective amount: An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response. A therapeutic agent, such as a chemotherapeutic agent, is administered in therapeutically effective amounts. Effective amounts of a given therapeutic agent can be determined in many different ways, such as assaying for a reduction in tumor size or improvement of physiological condition of a subject having cancer, such as prostate cancer. Effective amounts also can be determined through various in vitro, in vivo or in situ assays.

[0061] Treating a disease: "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such as a sign or symptom of prostate cancer. Treatment can also induce remission or cure of a condition, such as prostate cancer. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient. Administration of Inhibitors

[0062] In some embodiments, the disclosed methods include administering a therapeutically effective amount of an inhibitor (e.g., an NTD inhibitor or a kinase inhibitor) to a subject with cancer (such as prostate cancer).

[0063] In some examples, the method includes selecting a subject with CRPC and administering a therapeutically effective amount of an inhibitor (e.g., NTD inhibitor or kinase inhibitor) to the subject.

[0064] One of skill in the art can identify a subject with CRPC. CRPC is generally defined as prostate cancer with disease progression despite androgen deprivation therapy and castrate serum levels of testosterone. CRPC can present as a rise in serum levels of prostate-specific antigen (with or without symptoms), progression of pre-existing disease, appearance of new metastases, or a combination thereof (see, e.g., Hotte and Saad, Curr. Oncol. 17:S72-S79, 2010). Prognosis of CRPC is generally poor.

[0065] Therapeutic agents (such as an NTD inhibitor of the invention or a kinase inhibitor of the invention) can be administered to a subject in need of treatment using any suitable means known in the art. Methods of administration include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, intratumoral, vaginal, rectal, intranasal, inhalation, oral, or by gene gun. In some examples, the therapeutic agent is administered intravenously. In other examples, the therapeutic agent is administered orally. If two or more agents are administered to a subject, the agents can be administered by the same route or by different routes.

[0066] Parenteral administration is generally achieved by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Administration can be systemic or local.

[0067] Therapeutic agents can be administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present disclosure. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21^st Edition (2005) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic agents Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like.

[0068] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.

[0069] Appropriate dosages for treatment with one or more of the inhibitors of the invention (e.g., NTD inhibitor that binds the desired motif, or a kinase inhibitor with specificity to the desired residues) can be determined by one of skill in the art. In general, an effective amount of a therapeutic agent that includes one or more of the inhibitors of the invention administered to a subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject, the condition to be treated, or the severity of the condition. An effective amount of an inhibitor can be determined by varying the dosage of the compound and measuring the resulting therapeutic response, such as an increase in survival (such as overall survival, progression-free survival, or metastasis-free survival) or a decrease in the size, volume or number of tumors.

[0070] The inhibitors of the invention can be administered in a single dose, or in several doses, as needed to obtain the desired response. However, the effective amount can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration. In particular examples, the inhibitor is administered intravenously, intraperitoneally, or orally. In some non-limiting examples, the dose of an inhibitor administered to a subject can be about 0.1 mg/kg to about 1000 mg/kg. In particular examples, the dose can be about 0.5 mg/kg to about 100 mg/kg, such as about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg. In other examples, the dose can be about 10 to 800 mg, for example, about 50 mg to 800 mg, or about 100 mg to 600 mg of an inhibitor.

[0071] In other examples, the inhibitor is administered intravenously, orally, or intraperitoneally. In some non-limiting examples, the dose of an inhibitor administered to a subject can be about 0.1 mg/kg to about 1000 mg/kg. In particular examples, the dose can be about 1 mg/kg to about 100 mg/kg, such as about 1 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg. In other examples, the dose can be about 10 to 800 mg, for example, about 50 mg to 800 mg, or about 100 mg to 600 mg of an inhibitor.

[0072] The combined administration of two or more inhibitors of the invention includes administering them either sequentially or administering both agents at substantially the same time (e.g., an overlap in performing the administration). With sequential administration a subject is exposed to the agents at different times so long as some amount of the first agent remains in the subject (or has a therapeutic effect) when the other agent is administered. The treatment with both agents at the same time can be in the same dose, for example, physically mixed, or in separate doses administered at the same time.

[0073] In some examples, a therapeutically effective dose of an inhibitor includes daily, weekly, bi-weekly, or monthly use for at least about 2 weeks, such as at least about one month, two months, three months, six months, one year, two years, three years, four years, five years, or more. The disclosed methods include an inhibitor of the invention, which can be administered alone, in the presence of a pharmaceutically acceptable carrier, in the presence of other therapeutic agents (for example other anti-cancer therapeutic agents), or both. Such anti-cancer therapeutics include, but are not limited to, chemotherapeutic drug treatment, radiation, gene therapy, hormonal manipulation, immunotherapy and antisense oligonucleotide therapy. Examples of useful chemotherapeutic drugs include, but are not limited to, microtubule binding agents (such as paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine, epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin, rhizoxin, and derivatives or analogs thereof), DNA intercalators or cross-linkers (such as cisplatin, carboplatin, oxaliplatin, mitomycins such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, and derivatives or analogs thereof), DNA synthesis inhibitors (such as methotrexate, 5-fluoro-5'- deoxyuridine, 5'fluorouracil, gemcitabine, and analogs thereof), DNA and/or RNA transcription inhibitors (such as actinomycin D, daunorubicin, doxorubicin, and derivatives or analogs thereof), enzyme inhibitors, gene regulators, enzymes, antibodies (such as trastuzumab, bevacizumab, cetuximab, and panitumumab), angiogenesis inhibitors, enzyme inhibitors (such as camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof), kinase inhibitors (such as imatinib, gefitinib, sunitinib, and erolitinib), and gene regulators (such as raloxifene, 5-azacytidine, 5-aza-2'- deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof) or combinations of two or more thereof. In some examples, the inhibitor of the invention is administered prior to, concurrent with, or subsequent to the one or more additional chemotherapeutic agents. In some examples, an inhibitor is administered in combination with one or more of cisplatin, docetaxel, gemcitabine, 5-fluorouracil, bevacizumab, erlotinib, or sunitinib..

[0074] In certain embodiments, provided herein are methods for improving the Prostate-

Specific Antigen Working Group 2 (PSAWG2) Criteria for prostate cancer (see Scher, H., Halab, S., Tannock, S., Morris, M., Sternberg, C. N., et al. Design and end points of clinical trials for patients with progressive prostate cancer and castrate levels of testosterone: Recommendations of the Prostate Cancer Clinical Trials Working Group. J Clin Oncol. 2008; (26) 148-1159) of a patient, comprising administering an effective amount of a inhibitor of the invention (e.g., an NTD motif inhibitor or an inhibitor of a kinase of the invention) to a patient having castration- resistant prostate cancer.

[0075] In one embodiment, provided herein are methods for inhibiting phosphorylation of

Ser430, Ser431, and/or Ser432 in the NTD of the AR in a patient having castration- resistant prostate cancer, comprising administering an effective amount of a kinase inhibitor to said patient. In one embodiment, provided herein are methods for inhibiting phosphorylation of Ser422, Ser424, and/or Ser426 in the NTD of the AR in a patient having castration-resistant prostate cancer, comprising administering an effective amount of a kinase inhibitor to said patient. In some such embodiments, the inhibition of phosphorylation is assessed in a biological sample of the patient, such as in circulating blood and/or tumor cells, skin biopsies and/or tumor biopsies or aspirate. In such embodiments, the amount of inhibition of phosphorylation is assessed by comparison of the amount of phosphorylated residues before and after administration of the kinase inhibitor. In certain embodiments, provided herein are methods for measuring inhibition of phosphorylation of NTD Ser residues in a patient having castration-resistant prostate cancer, comprising administering an effective amount of a kinase inhibitor to said patient, measuring the amount of phosphorylated NTD Ser residues in said patient, and comparing said amount of phosphorylated Ser residues to that of said patient prior to administration of an effective amount of a kinase inhibitor.

[0076] In some embodiments, the kinase inhibitor is a compound as described herein. In one embodiment, the kinase inhibitor is selected from the group consisting of : is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2- pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l ,4-benzodioxin- 6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2- pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4- dichlorophenyl)-5 -(5 -methyl- 1 H-imidazol-2-yl)-2 pyrimidinyl] amino] ethyljamino] -3 - pyridinecarbonitrile; Myr-N-GKEAPPAPPQSpP-NH₂, Myr-N-Gly-Lys-Glu-Ala-Pro-Pro- Ala-Pro-Pro-Gln-Ser(P03H)-Pro-NH₂ trifluoroacetate salt; and 4-Benzyl-2-(naphthalen-l- yl)-l,2,4-thiadiazolidine-3,5-dione.

[0077] A kinase inhibitor can be combined with radiation therapy or surgery.

[0078] In certain embodiments, a kinase inhibitor is administered to patient who is undergoing radiation therapy, has previously undergone radiation therapy or will be undergoing radiation therapy. In certain embodiments, a kinase inhibitor is administered to a patient who has undergone tumor removal surgery.

[0079] Further provided herein are methods for treating patients who have been previously treated for castration-resistant prostate cancer, but are non- responsive to standard therapies, as well as those who have not previously been treated. Further provided herein are methods for treating patients who have undergone surgery in an attempt to treat the condition at issue, as well as those who have not. Because patients with castration-resistant prostate cancer can have heterogeneous clinical manifestations and varying clinical outcomes, the treatment given to a patient can vary, depending on his/her prognosis. The skilled clinician will be able to readily determine without undue experimentation specific secondary agents, types of surgery, and types of non-drug based standard therapy that can be effectively used to treat an individual patient with castration- resistant prostate cancer.

[0080] In one embodiment, the castration-resistant prostate cancer is that in which the kinase pathway is activated.

[0081] The present invention now provides the means for generation of molecules

(including small molecule compounds, peptides and peptide mimetics and antibodies) for inhibiting androgen-independent activation of the human AR. Such inhibitory compounds, used in combination with androgen deprivation would more effectively limit androgen mediated disease progression..

[0082] This invention also provides nucleic acid constructs encoding peptides of this invention as well as said constructs in an expression vector. This invention also provides cells and pharmaceutical compositions comprising the peptides, nucleic acid constructs and expression vectors of this invention. This invention also provides a method of inhibiting androgen-independent activation of AR by introducing into said cell, a peptide, nucleic acid construct or expression vector of this invention.

[0083] This invention also provides the use of a peptide, nucleic acid construct, expression vector or cell of this invention for the preparation of a medicament for the treatment of androgen mediated diseases including prostate tumors. This invention also provides the use of a peptide, nucleic acid construct, expression vector or pharmaceutical composition of this invention for treatment of androgen mediated diseases including prostate tumors, particularly in patients undergoing androgen deprivation therapy. This invention also provides a method of determining whether a compound or a mixture of compounds affects androgen-independent activation of androgen receptor (AR) comprising the steps of:

(1) contacting a compound or a mixture of compounds with a peptide comprising one or more tracts of amino acids derived from at least 10 contiguous amino acids of amino acids 433-446 of the N-terminal domain of the human AR; and,

(2) detecting whether a compound of said compound or mixture of compounds binds to said one or more tracts. In the preceding method, said one or more tracts of amino acids can be derived from differing lengths of contiguous amino acids of amino acids 433-446, as described above for the peptides of this invention. [0084] Peptides of this invention can be synthesized when convenient by any number of known peptide synthesis techniques. Alternatively, the peptides can be expressed in any suitable host cell into which an expression vector for the peptide has been introduced. The host cell and expression vector components will typically be selected to allow for expression of the peptide in the host. Peptides derived from residues 433-446 of the NTD of the AR are useful as a tool for screening compounds which affect androgen- independent activation of AR.

[0085] Methods of this invention for determining whether a compound affects androgen- independent activation of the AR can be used to detect compounds that potentially inhibit activation of the AR. In one embodiment, this method involves determining whether a compound binds to amino acid tracts derived from 433-446 of the NTD of human AR. This can be accomplished by a variety of known methods which can also facilitate the separation and recovery of the binding compound. Detection of an apparent change in conformation or molecular weight of a peptide when bound to a compound can be carried out, for example by gradient ultra-centrifugation or by SDS PAGE. The peptide could be labeled (for example by a fluorescent compound) to facilitate separation. Alternatively, the peptide could be immobilized to facilitate separation of binding compounds. An example of such immobilization is attachment of the peptide to a suitable activated substrate (e.g., beads of a chromatography gel), or by immunological techniques. Antibodies to AR and methods of producing anti-AR antibodies have been described in the art.

[0086] Alternate methods of screening which are part of this invention involve monitoring a change in the function of a peptide of this invention. In such embodiments, one can take advantage of the transactivation/repression characteristics of the N-terminal region of the AR and employ methods whereby enhancement or repression of such characteristics are observed as being indicative of the presence of a compound that interact with the peptide in such a way as to affect such characteristics.

EMBODIMENTS

[0087] El . A method for inhibiting activity of the human androgen receptor in a cell, said method comprising contacting said androgen receptor with a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.

[0088] E2. The method of embodiment El wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

[0089] E3. The method of embodiment El or E2, wherein said molecule is not EPI-

001.

[0090] E4. The method of embodiment El or E2, wherein said androgen receptor is the wild-type receptor.

[0091] E5. The method of embodiment El or E2, wherein said androgen receptor is a mutant form of the receptor.

[0092] E6. The method of embodiment El or E2, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

[0093] E7. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.

[0094] E8. The method of embodiment E7, wherein said prostate cancer is castration- resistant prostate cancer.

[0095] E9. The method of embodiment E7, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

[0096] E 10. The method of embodiment E9, wherein said molecule is not EPI-001.

[0097] El l . The method of embodiment E7, wherein said androgen receptor is the wild-type receptor.

[0098] E12. The method of embodiment E7, wherein said androgen receptor is a mutant form of the receptor.

[0099] E13. The method of embodiment E7, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

[0100] El 4. A method of inhibiting androgen receptor activity in a cell, said method comprising contacting said androgen receptor with an inhibitor of the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.

[0101] E15. The method of embodiment E14, wherein phosphorylation is inhibited for

Ser430, Ser, 431, and Ser432.

[0102] El 6. The method of embodiment E14 wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, or an antibody fusion protein.

[0103] E17. The method of embodiment E14, wherein said androgen receptor is the wild-type receptor.

[0104] E18. The method of embodiment E14, wherein said androgen receptor is a mutant form of the receptor.

[0105] El 9. A method of treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.

[0106] E20. The method of embodiment E19, wherein said molecule inhibits phosphorylation of Ser430, Ser, 431, and/or Ser432.

[0107] E21. The method of embodiment El 9, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

[0108] E22. The method of embodiment E19, wherein said androgen receptor is the wild-type receptor.

[0109] E23. The method of embodiment E19, wherein said androgen receptor is a mutant form of the receptor.

[0110] E24. The method of embodiment El 9, wherein said cancer is castration- resistant prostate cancer. [0111] E25. The method of embodiment E19, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.

[0112] E26. The method of embodiment E25, wherein said kinase is the CK1 kinase or the GSK3beta kinase.

[0113] E27. The method of embodiment E26, wherein the molecule is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9- isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro- 1 ,4-benzodioxin-6-yl)-5-(2- pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2- pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]- 1 -butanol dihydrochloride; 6-[[2-[[4-(2,4- dichlorophenyl)-5 -(5 -methyl- 1 H-imidazol-2-yl)-2 pyrimidinyl] amino] ethyljamino] -3 - pyridinecarbonitrile; Myr-N-GKEAPPAPPQSpP-NH₂, Myr-N-Gly-Lys-Glu-Ala-Pro-Pro- Ala-Pro-Pro-Gln-Ser(P0₃H)-Pro-NH₂ trifluoroacetate salt; or 4-Benzyl-2-(naphthalen-l- yl)-l,2,4-thiadiazolidine-3,5-dione.

[0114] E28. A method of diagnosing a subject having, or at risk of having, castration- resistant prostate cancer, said method comprising obtaining a tumor cell sample from a patient with prostate cancer, and determining the phosphorylation status of the androgen receptor in said tumor cell sample at one or more of residues Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438, wherein phosphorylation of one or more of said residues is indicative of a subject having, or at an increased risk of having, castration-resistant prostate cancer.

[0115] E29. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor.

[0116] E30. The method of embodiment E29, wherein said cancer is castration- resistant prostate cancer.

[0117] E31. The method of embodiment E29, wherein said receptor is the wild-type receptor.

[0118] E32. The method of embodiment E29, wherein said receptor is a mutant form of the receptor. [0119] E33. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N- terminal domain of the androgen receptor with RAP74.

[0120] E34. The method of embodiment E33, wherein said androgen receptor is wild- type.

[0121] E35. The method of embodiment E33, wherein said androgen receptor is a mutant form.

[0122] E36. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor.

[0123] E37. The method of embodiment E36, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

[0124] E38. The method of embodiment E36, wherein said molecule is not EPI-001.

[0125] E39. A molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF for use in treating prostate cancer.

[0126] E40. The molecule for use in treating prostate cancer of embodiment 39, wherein said prostate cancer is castration-resistant prostate cancer.

[0127] E41. The molecule for use in treating prostate cancer of embodiments 39 or 40, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

[0128] E42. The molecule for use in treating prostate cancer of embodiment 41, wherein said molecule is not EPI-001.

[0129] E43. The molecule for use in treating prostate cancer of embodiments 39 to 42, wherein said androgen receptor is the wild-type receptor.

[0130] E44. The molecule for use in treating prostate cancer of embodiments 39 to 42, wherein said androgen receptor is a mutant form of the receptor. [0131] E45. The molecule for use in treating prostate cancer of claims embodiments to

44, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

[0132] E46. A molecule that inhibits the phosphorylation of one or more residues of the

N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438 for use in treating prostate cancer.

[0133] E47. The molecule for use in treating prostate cancer of embodiment 46, wherein said molecule inhibits phosphorylation of Ser430, Ser, 431, and/or Ser432.

[0134] E48. The molecule for use in treating prostate cancer of embodiments 46 or 47, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

[0135] E49. The molecule for use in treating prostate cancer of embodiments 46 to 48, wherein said androgen receptor is the wild-type receptor.

[0136] E50. The molecule for use in treating prostate cancer of embodiments 46 to 48, wherein said androgen receptor is a mutant form of the receptor.

[0137] E51. The molecule for use in treating prostate cancer of embodiments 46 to 50, wherein said cancer is castration-resistant prostate cancer.

[0138] E52. The molecule for use in treating prostate cancer of embodiments 46 to 51, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.

[0139] E53. The molecule for use in treating prostate cancer of embodiment 52, wherein said kinase is the CK1 kinase or the GSK3beta kinase.

[0140] E54. The molecule for use in treating prostate cancer of embodiment 53, wherein the molecule is selected from the group consisting of: 2-( ?)-(l-Ethyl-2- hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4- [4- (2,3-Dihydro-l,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9- (l-Methylethyl)-6-[[3-(2-pyridinyl)phenyl]amino]-9H-purin-2-yl]amino]-l-butanol dihydrochloride; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2 pyrimidinyl] amino] ethyl] amino]-3 -pyridinecarbonitrile; Myr-N-GKE APPAPPQSpP- NH₂, Myr-N-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Gln-Ser(P0₃H)-Pro-NH₂ trifluoroacetate salt; or 4-Benzyl-2-(naphthalen-l-yl)-l,2,4-thiadiazolidine-3,5-dione.

[0141] E55. At least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor for use in treating prostate cancer.

[0142] E56. The at least one molecule for use in treating prostate cancer of embodiment 55, wherein said cancer is castration-resistant prostate cancer.

[0143] E57. The at least one molecule for use in treating prostate cancer of embodiments 55 or 56, wherein said receptor is the wild-type receptor.

[0144] E58. The at least one molecule for use in treating prostate cancer of embodiments 55 or 56, wherein said receptor is a mutant form of the receptor.

[0145] E59. A molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N-terminal domain of the androgen receptor with RAP74 for use in treating prostate cancer.

[0146] E60. The molecule for use in treating prostate cancer of embodiment 59, wherein said androgen receptor is wild-type.

[0147] E61. The molecule for use in treating prostate cancer of embodiment 59, wherein said androgen receptor is a mutant form.

[0148] E62. A molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor for use in treating prostate cancer.

[0149] E63. The molecule for use in treating prostate cancer of embodiment 62, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

[0150] E64. The molecule for use in treating prostate cancer of embodiments 62 or 63, wherein said molecule is not EPI-001.

[0151] All documents cited in the present disclosure are herein incorporated by reference in their entireties. The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described. EXAMPLES

Example 1

Materials and methods

Materials

[0152] 1 was purchased from Sigma- Aldrich and all peptides were synthesized by the combinatorial chemistry unit (UCQ) of the Barcelona Science Park (PCB).

Protein expression

AF-1 construct

[0153] A plasmid codifying for full length AR was obtained from Addgene and the gene codifying for AF-1 was cloned in a Gateway pDONR entry vector. The sequence codifying for a TEV protease cleavage site was introduced by PCR and the resulting gene was transcloned to a pDEST17 vector by recombination. The His-tagged AF-1 protein was expressed in Rosetta cells in conventional or minimal medium as inclusion bodies, re-dissolved in 8M urea buffer (pH 7.8 20 mM Tris pH 7.8 500 mM NaCl lmM imidazole 0.05% NaN₃ 1/1000 beta-mercaptoethanol and 8M urea) and purified by affinity chromatography in an Akta purifier system by using a Ni²⁺ column. The urea was removed by dialysis against 50 mM Tris buffer at pH 8.0 with 1 mM DTT and the His- tag was removed by overnight incubation in the cold room (277 K) with His-tagged TEV protease followed by a reverse Ni²⁺ column. The resulting solution was concentrated and further purified by size exclusion before NMR analysis. In this last step, the buffer was exchanged to 20 mM sodium phosphate at pH 7.4 with 1 mM TCEP and 0.05% NaN₃.

RAP74CTD construct

[0154] A plasmid codifying for full length RAP74 was obtained from Addgene and the gene codifying for RAP74CTD was cloned in a Gateway pDONR entry vector. The sequence codifying for a TEV protease cleavage site was introduced by PCR and the resulting gene was transcloned to a pDEST-HisMBP vector obtained from Addgene by recombination. The HisMBP-tagged RAP74CTD protein was expressed in Rosetta cells in conventional or minimal medium and purified by affinity chromatography in an Akta purifier system by using a Ni²⁺ column. The HisMBP-tag was removed by overnight incubation in the cold room (277 K) with His-tagged TEV protease followed by a reverse Ni²⁺ column. The resulting solution was concentrated and further purified by size exclusion. In this last step, the buffer was exchanged to 20 mM sodium phosphate at pH 6.5 with 0.05% NaN₃.

NMR experiments

r1H¹⁵N1-HSOC experiments

[0155] For AF-1, the [1H,¹⁵N]-HSQC NMR experiments were carried out at 278K, pH

7.4, ImM TCEP, 20 mM phosphate buffer, 0.5% dioxane or 5% DMSO in a Bruker Avance 600 MHz or 800 MHz spectrometers equipped with a 5mm CryoProbe. For RAP74CTD, the [1H,¹⁵N]-HSQC NMR experiments were carried out at 298K, pH 6.5, 20 mM phosphate buffer, in a Bruker Avance 600 MHz spectrometer equipped with a 5mm CryoProbe. For the interaction study of AF-1 and RAP74CTD, the concentration of NTD construct was 50 μΜ when spectra were recorded of AF-1, and when spectra were recorded of RAP74CTD, the concentration of RAP74CTD was 50 μΜ and that of the peptides was 500 μΜ. For the interaction study of AF-1 with 1, the concentration of AF-1 was 25 μΜ and that of 1 was 250 μΜ. The pulse sequences used for these experiments were the standard ones provided by Bruker and the number of increments in tl used in the experiments was either 256 (RAP74CTD) or 512 (AF-1). The chemical shifts were referenced to DSS-d₆.

1H ID experiments

[0156] The 1H ID NMR experiments were carried out at 278K, pH 7.4, ImM TCEP, 20 mM phosphate buffer, 0.5%> dioxane or 5%> DMSO in a Bruker Avance 800 MHz or 600 MHz spectrometer equipped with a 5mm CryoProbe. Concentrations of 5 μΜ, 10 μΜ and 25 μΜ AF-1 were added to 100 μΜ 1 to probe the effect of AF-1 on the signals of this small molecule. The concentration of NTD construct or peptide was 25 μΜ and that of 1 was 250 μΜ for the STD experiments. The chemical shifts were referenced to DSS-d₆. MS experiments

[0157] Samples of 25 μΜ AF-1 construct were incubated at 315 K with 250 μΜ 1 in 20 mM phosphate buffer at pH 7.4 containing lmM TCEP and 5% DMSO. Prior to incubation the buffer was degassed and placed under inert atmosphere. After 4 h of incubation the reaction was stopped by flash freezing and the resulting frozen solution stored at 258 K.

[0158] The analysis by MS was carried out by diluting the solution 1 :2 in ammonium bicarbonate, treating it with 2 mM DTT during 2hr and with 5 mM iodoacetamide for 30 min in the dark. The reaction was quenched with reaction with 2mM DTT and the mixture digested with trypsin overnight at 315 K. The digestion was quenched by 1% formic acid.

[0159] Samples were loaded to a C18 trap column using a nanoAcquity Ultra

Performance LCTM chromatographic system and the peptides were separated using a CI 8 analytical column. The column outlet was directly connected to an Advion TriVersa NanoMate fitted on an LTQ-FT Ultra mass spectrometer.

[0160] The mass spectrometer was operated in a data-dependent acquisition mode.

Preliminary MS scans were acquired in the FT with the resolution set to 100.000. Up to six of the most intense ions per scan were then fragmented and detected in the linear ion trap. The spectrometer was set-up in positive polarity mode.

[0161] A database search was performed with Bioworks using Sequest search engine and the SwissProt database. Peptide mass tolerance was 10 ppm and the MS/MS tolerance was 0.8 Da. Peptides with a q- value lower than 0.1 and a FDR < 1% were considered as positive identifications with a high confidence level.

Functional assays

[0162] PC-3 cells were trans fected in triplicate with plasmids expressing WT or mutant

AR together with a PSA reporter gene and treated with 0 or 1 nM R1881 for 24 h. The mean fold activation to the WT receptor +/- SD for at least three replicate experiments was measured. In Figs. 6 and 9 * is equivalent to p < 0.05, ** to p < 0.01 and *** to p < 0.001 (student t-test). Example 2

[0163] As discussed in Background the NTD of AR binds RAP74CTD in TFIIF and this interaction can contribute to positioning RNAPII at the transcription start site of genes regulated by AR. Direct or indirect PPIs between gene regulatory proteins and members of the transcription machinery such as GTFs are key for activating transcription in both healthy and cancer cells. Weakening or inhibiting the interaction between the NTD of AR and RAP74CTD is therefore a likely therapeutic approach for PC and CRPC.

[0164] To identify the residues of the NTD that bind to RAP74CTD we carried out NMR experiments in which we measured how the 1H and ¹⁵N chemical shifts of the backbone amides of an ¹⁵N enriched construct of the NTD spanning activation function 1 (AF-1, residues 142 to 448 of AR) changed upon incubation with increasing concentrations of RAP74CTD (residues 450 to 517 of RAP74). The chemical shift changes were monitored by measuring [¹H,¹⁵N]-HSQC spectra of the AF-1 construct at 800 MHz.

[0165] These experiments revealed the motif consisting of residues 433 to 446, with sequence WHTLFTAEEGQLYG, as that binding to RAP74CTD (Fig. 1). Contrary to what we expected we did not observe significant chemical shift changes in regions of sequence of the NTD previously reported, by using a different experimental approach, to contribute to this interaction (residues 159 to 164, 340 to 345, M245, L247 and V249). Importantly for this invention the motif instead coincides almost exactly with that found in gene reporter experiments in androgen depletion independent cell lines (C4-2 and 22Rvl) to be important for the proliferation of prostate cells in CRPC by a, to date, unknown mechanism.³⁶

[0166] Plots of the size of the chemical shift perturbations in AF-1 as a function of the concentration of RAP74CTD indicated that the affinity between these two proteins was modest (1 μΜ, Fig. 2). The high value of the dissociation constant of the complex can be explained by the absence in our in vitro experimental setup of factors occurring in vivo that increase the stability of this complex. These include transcriptional co-activators, the DNA-associated nature of both AR and RAP74CTD and the presence of PTMs in AR.¹²

[0167] The activity of the transactivation domains of gene regulatory proteins is regulated by transcriptional co-activators. In vivo these multi-domain proteins act as mediators of the interaction between transactivation domains and the transcription machinery in part by causing disorder to order transitions in the latter that poise them for binding.¹² Transcriptional co-activators are no indispensable for transcription to occur but they assist the process and offer opportunities for regulation.

[0168] AR and TFIIF are both DNA-associated proteins. The former directly binds to

DNA through the DBD and the latter is part of the pre initiation complex that is also associated with DNA. In vivo, therefore, these two proteins can only diffuse relative to one in one dimension. This constraint can significantly reduce the entropic cost of AR binding to RAP74CTD and thus strengthen the interaction between these to proteins in vivo. Similar effects are known to operate in the binding of membrane-bound proteins.⁴⁵

[0169] PTMs are also known to regulate the interaction between gene regulatory proteins and GTFs. Phosphorylations play a particular important role in this mechanism of regulation by modulating the affinity between binding partners and the specificity of PPIs via the interplay of kinases and phosphatases.¹² In the context of aberrant AR activation in CRPC there has been much discussion of the potential role of phosphorylations in cell proliferation by strengthening PPIs that are of modest importance in healthy cells but become crucial for proliferation during hormone blockade.⁶

[0170] None of these factors is replicated in the in vitro experiments that we have carried out. This, coupled to the need of the PPIs initiating transcription to be transient, contribute to explaining the weak interaction between AF-1 and RAP74CTD measured in vitro and prompted a more detailed analysis of the structural basis of the interaction by investigating the binding epitope of AR on the surface of RAP74CTD.

Example 3

[0171] For this we measured the changes in the 1H and ¹⁵N chemical shifts of the backbone amides of a ¹⁵N enriched sample of RAP74CTD upon incubation with increasing concentrations of the AF-1 construct. To ensure that no region of AF-1 other that the motif identified in the chemical shift perturbation experiments played an important role in the interaction we also carried out equivalent experiments by using a synthetic peptide with a sequence spanning the binding motif, Ac- SAAAS SS WHTLFT AEEGQL YG-NH₂.

[0172] The chemical shift changes were monitored by measuring [¹H,¹⁵N]-HSQC spectra of RAP74CTD at 600 MHz and the results in both cases revealed that the WHTLFT AEEGQL YG motif binds a hydrophobic cleft in the structure of RAP74CTD which, according to previous reports, has affinity for two different but related motifs in an ID domain of the RNAPII phosphatase FCPl (Fig. 3). A comparison of the sequences of the two FCPl motifs interacting with RAP74CTD with that of the AR NTD motif revealed by our experiments to bind to a very similar epitope shows clear similarities.³⁸'⁴⁰'⁴⁶

[0173] The three motifs possess a i,i+3/4 pattern of hydrophobic residues followed by two glutamic acid (Glu) residues. An analysis of the structure of the complex formed by RAP74CTD with the C-terminal ID region of FCPl shows that the motif appears to adopt, on average, an a-helical secondary structure where the hydrophobic residues define the surface of the helix in contact with the hydrophobic cleft of RAP74CTD. In addition the Glu and Asp side chains form salt bridges with Lys or arginine Arg chains in RAP74CTD (Fig. 4).

[0174] Experiments aimed at determining the structure of the complex between the

WHTLFTAEEGQLYG motif and RAP74CTD were not successful presumably due to its low stability and transient nature. By analogy with the structure of RAP74CTD bound to the C-terminal ID motif of FCPl we postulated that the structure of the complex between AR and RAP74CTD involved the formation of a short 9-residue helix with sequence WHTLFTAEE stabilized by a C-terminal charge clamp formed by the two Glu side chains with relatively small contributions to the affinity of residues C-terminal to the charge clamp, with sequence GQLYG.

[0175] Evidence for the fundamental contribution of residues N-terminal to the charge clamp to the affinity of the C-terminal FCPl motif for RAP74CTD stems from Ala scanning results,³⁸ from the observation that the residues C-terminal to the charge clamp have completely different conformations in the crystal and solution structures of the complex,³⁸'⁴⁰ which indicates that these residues have structural heterogeneity, and from molecular simulations, which again indicate that this complex is particularly flexible and likely to be fuzzy ⁴¹

[0176] In order to validate this structural model we introduced mutations in the sequence of the NTD that should lead to decreases in the stability of the complex. A repetition of the chemical shift perturbation experiments showed, in agreement with our predictions, that charge reversal mutations (Glu to Lys) in the two charge clamp residues prevented the motif from binding to RAP74CTD (Fig. 5). These experiments indicated that the complex between RAP74CTD and the C-terminal FCP1 motif is a reliable representation of the average structure of the complex between the NTD of AR and this GTF.

Example 4

[0177] To investigate the physiological relevance of this new PPI we measured the transcriptional activity of AR in PC-3 cells transfected with a plasmid encoding either wild type (WT) AR or the mutants of AR that do not bind to RAP74CTD in vitro. We chose to use PC-3 cells because they do not express AR, ensuring that the transcriptional activity measured is the luciferase assay stems from the AR encoded by the plasmid. A comparison of the activity of the charge reversal mutant with that of the WT clearly indicated that abrogating complex formation leads to a substantial decrease in transcription, supporting the notion that the interaction between AR and RAP74 via the motif is important for the function of AR (Fig. 6).

[0178] A detailed comparison of the sequences of the FCP1 motifs and of the

WHTLFTAEEGQLYG motif in the NTD of AR revealed an important difference in amino acid composition in the region immediately N-terminal to the binding motif. Whereas both the central and C-terminal ID FCP1 motifs, that bind to RAP74CTD with strong affinity (ca. 5 μΜ),⁴⁸ possess a large number of negatively charged side chains that are not present in the AR sequence, which is instead rich in serine (Ser) residues (Fig. 7). This led us to propose that phosphorylations in these Ser residues can strengthen the interaction between the NTD of AR and RAP74CTD, which is rich in Arg and Lys residues in its surface, and act as a regulatory mechanism for the interaction between AR and RAP74CTD.

[0179] Several phosphorylations in the NTD of AR have been reported in the literature.

The phosphorylation closest to the motif binding to RAP74CTD is that of Ser 424⁴⁹ by a not yet identified kinase upon activation of AR by androgens. To determine whether this phosphorylation significantly increases the affinity between the AR motif and RAP74CTD we measured the [¹H,¹⁵N]-HSQC spectrum of a sample of ¹⁵N-enriched RAP74CTD in the presence of a synthetic peptide Ac- GSG(pS)PSAAASSSWHTLFTAEEGQLYG-NH₂ that spanned the binding motif and included position 424 in the phosphorylated form. A comparison of the chemical shift changes caused by this peptide with those caused by the peptide Ac- SAAASSSWHTLFTAEEGQLYG-NH₂ indicated that the phosphorylation of Ser 424 caused a detectable but very modest increase in the stability of the complex.

[0180] An analysis of the potential phosphosites in AR using bioinformatics tools highlighted Ser 430 a very likely candidate for phosphorylation adjacent to the binding motif, that starts at Trp 433 according to the chemical shift perturbation experiments carried out with ¹⁵N-enriched AF-1. To determine whether phosphorylating this or the adjacent positions (Ser 431, Ser 432) leads to an increase in affinity we carried out chemical shift perturbation experiments with ¹⁵N-enriched RAP74CTD in the presence of three different variants of the WT synthetic peptide (Ac- SAAASSSWHTLFTAEEGQLYG-NH₂) phosphorylated at position Ser 430, Ser 431 or Ser 432.

[0181] These experiments indicated that phosphorylating either of these positions leads to substantial increases in affinity, giving support to the notion that introducing negative charges in this region of sequence is a possible mechanism of regulation of the interaction between AR and RAP74CTD. Given that the most common distance between phosphosites in the human proteome is two residues⁵⁰ we also studied the interaction between RAP74CTD and the Ac-S AAAS S S WHTLFT AEEGQL YG-NH₂ peptide phosphorylated at positions 430 and 432. The result of this experiment led to an affinity of 80 μΜ, which is ca one order of magnitude larger than that of the non phosphorylated peptide (Fig. 8).

Example 5

[0182] To investigate whether such phosphorylations can increase the transcriptional activity of AR we carried out additional measurements of the activity of the receptor in PC-3 cells. In this case we compared the activity of the WT AR to that of a mutant where Ser residues 430, 431 and 432 were simultaneously mutated to Ala, preventing their phosphorylation, or to Glu, mimicking it. The results that we obtained clearly indicated that introducing negative charges in this region of sequence causes a substantial increase in transactivation, again supporting the hypothesis that phosphorylation of these Ser residues is a possible regulatory mechanism of the interaction (Fig. 9).

[0183] As discussed in Background the motif of the NTD of AR interacting with

RAP74CTD is key for the ability of prostate cells to proliferate during hormone blockade in CRPC. A key observation of the researchers that discovered this was that the relative importance of this motif relative to that of the rest of the domain depended on whether the prostate cells used in the experiment could proliferate in a hormone-independent fashion or whether they instead relied on androgens.³⁶ That the motif was only key for the proliferation of androgen depletion independent cell lines indicates that the PPI formed by the motif is regulated by a yet to be characterized mechanism that only occurs in CRPC.

[0184] Our structural analysis of the interaction between AR and TFIIF indicates that a possible mechanism of up regulation in CRPC cells of the interaction between these proteins through the motif WHTLFTAEEGQLYG is the phosphorylation of Ser or Thr residues present either in the motif or in its vicinity.⁶

Example 6

[0185] As discussed in Background a small molecule irreversible inhibitor of the AR

NTD active against androgen depletion independent cell lines and a CRPC animal model was recently identified in a high throughput phenotypic screening (EPI-001, 1 in Fig. 10).³⁴'³⁵ Irreversible drugs act by recognizing a structural feature of the surface of the target and by reacting irreversibly with a functional group of the protein molecule in the vicinity of the site of recognition. Given the lack of detail about how 1 recognizes the sequence of the NTD and about the identity of the functional group(s) that react with it we have investigated these two processes in vitro by using NMR and mass spectrometry (MS) among other biophysical techniques.

[0186] We initially carried out experiments to investigate whether the recognition of the

NTD by 1 and its irreversible reaction with the domain could be studied independently. The results indicated that incubation of the AF-1 construct with 1 in a 1 : 10 ratio at 278K leads to the formation of a complex stabilized by non covalent interactions (see below) without formation of the adduct according to MS analysis. In order to investigate the nature of the complex formed by 1 and AF-1 we carried out NMR experiments, under these conditions, in which we measured to what extent the 1H and ¹⁵N chemical shifts of the backbone amides of AF-1 were perturbed by incubation with 1.

[0187] A comparison of the [¹H,¹⁵N]-HSQC spectra of AF-1 obtained in the absence and in the presence of 1 indicated that this small molecule causes small but reproducible changes in 1H and, especially, ¹⁵N chemical shifts (Fig. 11) in three well-defined regions of sequence, centered around residues 360, 400 and 435, that overlap well with a sub- domain of AF-1 known as transactivation unit 5 (Tau-5, residues 360 to 530).⁵¹ Tau-5 is one of two regions of the NTD reported in the literature to mediate the transcriptional activity of AR, the other one being Tau-1 (residues 170 to 240),⁵¹ with the former playing a particularly important role in the function of the constitutively active splicing AR variants, lacking a LBD, that occur in CRPC.

[0188] To obtain independent evidence of the formation of an activated complex we measured the effect on the 1H ID spectrum of 1 of its incubation with AF-1 under the same conditions used for the HSQC experiments described above. These revealed the presence of small but reproducible chemical shift perturbations, that depended on the concentration of AF-1, as well as to the line broadening effects commonly observed in the 1H spectra of small molecules interacting with targets of apparent high molecular weight when these are present in sub -stoichiometric concentrations (Fig. 12).⁵²

[0189] Further and conclusive evidence for the interaction of 1 with the AF-1 construct was obtained by using an NMR technique known as saturation transfer difference spectroscopy (STD) in which the transfer of saturation from the target to the ligand is detected by 1H ID NMR. That the AF-1 construct causes line broadening and allows saturation transfer is highly indicative of the NTD adopting, despite its ID character, a conformation of low rotational diffusion correlation time upon interaction with 1 ,⁵² This observation is compatible with a folding induced by binding scenario - common in ID proteins interacting with other proteins, nucleic acids or small molecules - in which the ligand causes a population shift in the conformational ensemble of the ID protein to favor, by the law of mass action, the conformations to which it has affinity.⁵³

[0190] The observation of chemical shift perturbations in residues of AF-1 that are quite apart in sequence upon incubation with 1 is compatible with such a scenario and identifies the region of sequence corresponding to Tau-5 as that undergoing a population shift upon interaction with 1. To confirm that 1 interacts with a compact conformation of Tau-5 stabilized by inter-residue interactions equivalent to those that stabilize the structures of globular proteins we carried out STD experiments under denaturing conditions (8 M urea) and observed, as expected, no interaction between 1 and the AF-1 construct, in agreement with observations reported in the literature.³⁴ [0191] ID proteins undergoing folding by binding processes can populate the bound state in the absence of the ligand although with a lower population than in its presence. To investigate this possibility we analyzed the ¹³Ca of the AF-1 construct and identified substantial nascent a-helical structure in the core of Tau-1 (residues 175 to 205)⁵¹ as well as in the three regions of Tau-5 undergoing chemical shifts in the presence of 1, which in addition presented substantial line broadening. These results confirmed that 1 interacts with a region of sequence that is partially folded in the free state and, what is more important, provides a rationale for the binding specificity of 1 (Fig. 13).

[0192] Given the dimeric nature of the DNA-bound state of AR²² we carried out experiments aimed at investigating the oligomeric state of the AF-1 construct under the set of conditions used to study its interaction with 1. A comparison of the [¹H,¹⁵N]-HSQC spectra obtained at concentrations ranging from 10 to 700 μΜ indicated that the chemical shifts of a substantial number of residues of AF-1 are concentration dependent, which is evidence for a monomer-dimer equilibrium, that was confirmed by a native gel.

[0193] A detailed analysis of the residues that experience the largest changes in chemical environment upon dimerization indicated that they correspond to those that experience chemical shift changes upon interaction with 1, indicating that the compact state of AF-1 that is recognized by 1 is the dimeric form of the domain. This was confirmed by a comparison of the sign of the chemical shifts caused by 1 with that of the shifts caused by increases in the concentration of AF-1, in the absence of 1, which were equivalent (Fig. 14).

[0194] Following this characterization of the reversible interaction between 1 and the dimeric form of Tau-5 in the NTD of AR we proceeded to characterize its irreversible reaction with a yet to be identified functional group of the receptor. We found that increasing the temperature from 278K to 298 or 315K leads, after 4 h of incubation, to the detection by MS of an adduct with a mass (M_add_uct) corresponding to the product of a nucleophilic attack, by a AF-1 functional group, of the CH₂-C1 bond present in 1 i.e. M_adduc, = MAF-I + MI - MHCI.

[0195] We then used a top down MS approach described in Materials and Methods to identify the AF-1 functional group reacting with the carbon as the SH group of the side chain of Cys 404 (Fig. 15). That the Cys residue reacting with 1 is the only one, out of 8 present in AF-1, that is part of the region of sequence that experiences chemical shift changes upon incubation with 1 at low temperature provides evidence that the reversible complex identified by NMR is an on pathway intermediate in the mechanism of formation of the adduct. In this scenario the specificity of the interaction between 1 and the dimeric form of Tau-5 in the formation of the intermediate is the source of the specificity of the reaction between 1 and the side chain Cys 404.

Our analysis of the interaction between the AF-1 construct and 1 indicates that this experimental drug for CRPC reacts specifically with the SH group of the side chain of residue Cys 404. In addition it indicates that the origin of this specificity is the affinity that 1 has for a relatively structured dimeric form of Tau-5. Given this newly acquired knowledge we wish to put forward the following invention for the treatment of CRPC, because it enables the design of an optimized construct of AR suitable for the identification of electrophilic ligands of dimeric AF-1 by high throughput screening or fragment screening, which are two standard tools in drug discovery.

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doi: 10.1016/j.sbi.2011.09.010

Claims

WHAT IS CLAIMED IS:

1. A method for inhibiting activity of the human androgen receptor in a cell, said method comprising contacting said androgen receptor with a molecule which specifically inhibits interaction of amino acids 433-438 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.

2. The method of claim 1 wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

3. The method of claim 1 or 2, wherein said molecule is not EPI-001.

4. The method of claim 1 or 2, wherein said androgen receptor is the wild-type receptor.

5. The method of claim 1 or 2, wherein said androgen receptor is a mutant form of the receptor.

6. The method of claim 1 or 2, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

7. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF.

8. The method of claim 7, wherein said prostate cancer is castration-resistant prostate cancer.

9. The method of claim 7, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen- binding fragment of an antibody, and an antibody fusion protein.

10. The method of claim 9, wherein said molecule is not EPI-001.

11. The method of claim 7, wherein said androgen receptor is the wild-type receptor.

12. The method of claim 7, wherein said androgen receptor is a mutant form of the receptor.

13. The method of claim 7, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

14. A method of inhibiting androgen receptor activity in a cell, said method comprising contacting said androgen receptor with an inhibitor of the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.

15. The method of claim 14, wherein phosphorylation is inhibited for Ser430, Ser, 431, and Ser432.

16. The method of claim 14 wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen- binding fragment of an antibody, or an antibody fusion protein.

17. The method of claim 14, wherein said androgen receptor is the wild-type receptor.

18. The method of claim 14, wherein said androgen receptor is a mutant form of the receptor.

19. A method of treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule that inhibits the phosphorylation of one or more residues of the N-terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438.

20. The method of claim 19, wherein said molecule inhibits phosphorylation of Ser430, Ser, 431, and/or Ser432.

21. The method of claim 19, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen- binding fragment of an antibody, and an antibody fusion protein.

22. The method of claim 19, wherein said androgen receptor is the wild-type receptor.

23. The method of claim 19, wherein said androgen receptor is a mutant form of the receptor.

24. The method of claim 19, wherein said cancer is castration-resistant prostate cancer.

25. The method of claim 19, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.

26. The method of claim 25, wherein said kinase is the CK1 kinase or the GSK3beta kinase.

27. The method of claim 26, wherein the molecule is selected from the group consisting of: 2-(R)-( 1 -Ethyl-2-hydroxyethylamino)-6-(4-(2-pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l ,4-benzodioxin-6-yl)-5-(2-pyridinyl)-lH-imidazol-2- yljbenzamide; 2- [ [9-( 1 -Methylethyl)-6-[ [3 -(2-pyridinyl)phenyl]amino] -9H-purin-2-yl]amino] - 1 - butanol dihydrochloride; 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-lH-imidazol-2-yl)-2 pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile; Myr-N-GKEAPPAPPQSpP-NH₂, Myr- N-Gly-Lys-Glu-Ala-Pro-Pro-Ala-Pro-Pro-Gln-Ser(P0₃H)-Pro-NH₂ trifluoroacetate salt; or 4- Benzyl-2-(naphthalen- 1 -yl)- 1 ,2,4-thiadiazolidine-3 ,5-dione.

28. A method of diagnosing a subject having, or at risk of having, castration-resistant prostate cancer, said method comprising obtaining a tumor cell sample from a patient with prostate cancer, and determining the phosphorylation status of the androgen receptor in said tumor cell sample at one or more of residues Ser422, Ser424, Ser 426, Ser430, Ser431 , Ser432, Thr435 and Thr438, wherein phosphorylation of one or more of said residues is indicative of a subject having, or at an increased risk of having, castration-resistant prostate cancer.

29. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically-effective amount of at least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor.

30. The method of claim 29, wherein said cancer is castration-resistant prostate cancer.

31. The method of claim 29, wherein said receptor is the wild-type receptor.

32. The method of claim 29, wherein said receptor is a mutant form of the receptor.

33. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N-terminal domain of the androgen receptor with RAP74.

34. The method of claim 33, wherein said androgen receptor is wild-type.

35. The method of claim 33, wherein said androgen receptor is a mutant form.

36. A method for treating prostate cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N- terminal domain of the androgen receptor.

37. The method of claim 36, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

38. The method of claim 36, wherein said molecule is not EPI-001.

39. A molecule which specifically inhibits interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF for use in treating prostate cancer.

40. The molecule for use in treating prostate cancer of claim 39, wherein said prostate cancer is castration-resistant prostate cancer.

41. The molecule for use in treating prostate cancer of claims 39 or 40, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

42. The molecule for use in treating prostate cancer of claim 41, wherein said molecule is not EPI-001.

43. The molecule for use in treating prostate cancer of claims 39 to 42, wherein said androgen receptor is the wild-type receptor.

44. The molecule for use in treating prostate cancer of claims 39 to 42, wherein said androgen receptor is a mutant form of the receptor.

45. The molecule for use in treating prostate cancer of claims 39 to 44, wherein said molecule selectively inhibits the interaction of amino acids 433-446 of the N-terminal domain of the receptor with the RAP74 subunit of TFIIF, but does not inhibit interaction of amino acids 433-437 of the N-terminal domain of the receptor with RAP74.

46. A molecule that inhibits the phosphorylation of one or more residues of the N- terminal domain of the androgen receptor selected from the group consisting of: Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 and Thr438 for use in treating prostate cancer.

47. The molecule for use in treating prostate cancer of claim 46, wherein said molecule inhibits phosphorylation of Ser430, Ser, 431, and/or Ser432.

48. The molecule for use in treating prostate cancer of claims 46 or 47, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, an antigen-binding fragment of an antibody, and an antibody fusion protein.

49. The molecule for use in treating prostate cancer of claims 46 to 48, wherein said androgen receptor is the wild-type receptor.

50. The molecule for use in treating prostate cancer of claims 46 to 48, wherein said androgen receptor is a mutant form of the receptor.

51. The molecule for use in treating prostate cancer of claims 46 to 50, wherein said cancer is castration-resistant prostate cancer.

52. The molecule for use in treating prostate cancer of claims 46 to 51, wherein said molecule inhibits a kinase that phosphorylates one or more of said residues.

53. The molecule for use in treating prostate cancer of claim 52, wherein said kinase is the CK1 kinase or the GSK3beta kinase.

54. The molecule for use in treating prostate cancer of claim 53, wherein the molecule is selected from the group consisting of: 2-(i?)-(l-Ethyl-2-hydroxyethylamino)-6-(4-(2- pyridyl)benzyl)-9-isopropylpurine trihydrochloride; 4-[4-(2,3-Dihydro-l,4-benzodioxin-6-yl)-5- (2-pyridinyl)-lH-imidazol-2-yl]benzamide; 2-[[9-(l-Methylethyl)-6-[[3-(2- pyridinyl)phenyl] amino] -9H-purin-2-yl]amino] - 1 -butanol dihydrochloride; 6- [ [2- [ [4-(2,4- dichlorophenyl)-5 -(5 -methyl- 1 H-imidazol-2-yl)-2 pyrimidinyl] amino] ethyl] amino]-3 - pyridinecarbonitrile; Myr-N-GKEAPPAPPQSpP-NH₂, Myr-N-Gly-Lys-Glu-Ala-Pro-Pro-Ala- Pro-Pro-Gln-Ser(P0₃H)-Pro-NH₂ trifluoroacetate salt; or 4-Benzyl-2-(naphthalen-l-yl)- 1,2,4- thiadiazolidine-3,5-dione.

55. At least one molecule that activates a phosphatase that dephosphorylates one or more of Ser422, Ser424, Ser 426, Ser430, Ser431, Ser432, Thr435 or Thr438 in the N-terminal domain of the androgen receptor for use in treating prostate cancer.

56. The at least one molecule for use in treating prostate cancer of claim 55, wherein said cancer is castration-resistant prostate cancer.

57. The at least one molecule for use in treating prostate cancer of claims 55 or 56, wherein said receptor is the wild-type receptor.

58. The at least one molecule for use in treating prostate cancer of claims 55 or 56, wherein said receptor is a mutant form of the receptor.

59. A molecule that specifically inhibits the interaction of a phosphorylated form of the motif of amino acids 421-446 of the N-terminal domain of the androgen receptor with RAP74 for use in treating prostate cancer.

60. The molecule for use in treating prostate cancer of claim 59, wherein said androgen receptor is wild-type.

61. The molecule for use in treating prostate cancer of claim 59, wherein said androgen receptor is a mutant form.

62. A molecule that binds to the dimeric form of Tau-5 and/or the SH group of the side chain of Cys 404 of the N-terminal domain of the androgen receptor for use in treating prostate cancer.

63. The molecule for use in treating prostate cancer of claim 62, wherein said molecule is selected from the group consisting of: a small molecule compound, a peptide, a peptidomimetic, an antibody, and antigen-binding fragment of an antibody, or an antibody fusion protein.

64. The molecule for use in treating prostate cancer of claims 62 or 63, wherein said molecule is not EPI-001.