International Journal of Molecular Sciences

Review An Overview of Next-Generation Androgen Receptor-Targeted Therapeutics in Development for the Treatment of Prostate Cancer

Michael L. Mohler 1 , Arunima Sikdar 2 , Suriyan Ponnusamy 2, Dong-Jin Hwang 1, Yali He 1, Duane D. Miller 1 and Ramesh Narayanan 2,*

1 Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA; [email protected] (M.L.M.); [email protected] (D.-J.H.); [email protected] (Y.H.); [email protected] (D.D.M.) 2 Department of Medicine, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38103, USA; [email protected] (A.S.); [email protected] (S.P.) * Correspondence: [email protected]; Tel.: +1-901-448-2403; Fax: +1-901-448-3910

Abstract: Traditional endocrine therapy for prostate cancer (PCa) has been directed at suppression of the androgen receptor (AR) signaling axis since Huggins et al. discovered that diethylstilbestrol (DES; an estrogen) produced chemical castration and PCa tumor regression. Androgen deprivation therapy (ADT) still remains the first-line PCa therapy. Insufficiency of ADT over time leads to castration-



resistant PCa (CRPC) in which the AR axis is still active, despite castrate levels of circulating
 androgens. Despite the approval and use of multiple generations of competitive AR antagonists

Citation: Mohler, M.L.; Sikdar, A.; (antiandrogens), antiandrogen resistance emerges rapidly in CRPC due to several mechanisms, mostly Ponnusamy, S.; Hwang, D.-J.; He, Y.; converging in the AR axis. Recent evidence from multiple groups have defined noncompetitive or Miller, D.D.; Narayanan, R. An noncanonical direct binding sites on AR that can be targeted to inhibit the AR axis. This review Overview of Next-Generation discusses new developments in the PCa treatment paradigm that includes the next-generation Androgen Receptor-Targeted molecules to noncanonical sites, proteolysis targeting chimera (PROTAC), or noncanonical N-terminal Therapeutics in Development for the domain (NTD)-binding of selective AR degraders (SARDs). A few lead compounds targeting each of Treatment of Prostate Cancer. Int. J. these novel noncanonical sites or with SARD activity are discussed. Many of these ligands are still in Mol. Sci. 2021, 22, 2124. preclinical development, and a few early clinical leads have emerged, but successful late-stage clinical https://doi.org/10.3390/ijms22042124 data are still lacking. The breadth and diversity of targets provide hope that optimized noncanonical inhibitors and/or SARDs will be able to overcome antiandrogen-resistant CRPC. Academic Editor: Chendil Damodaran Keywords: prostate cancer; castration-resistant prostate cancer (CRPC); androgen receptor (AR);

Received: 2 February 2021 selective AR degraders (SARD); non-canonical Accepted: 17 February 2021 Published: 20 February 2021

Publisher’s Note: MDPI stays neutral 1. Background with regard to jurisdictional claims in Prostate cancer (PCa) is the most common non-cutaneous cancer among men. Al- published maps and institutional affil- though it is slow growing, often with no symptoms and being highly treatable in early iations. stages, the American Cancer Society predicts in 2020 that there will be around 33,000 deaths due to PCa [1]. Most of these deaths occur from advanced forms of PCa called castration-resistant PCa (CRPC). CRPC can be metastatic (mCRPC) or non-metastatic CRPC (nmCRPC), which grows aggressively and results in shorter overall survival (OS). Copyright: © 2021 by the authors. Treatment for early PCa includes attempts at curative therapies such surgery, cryotherapy, Licensee MDPI, Basel, Switzerland. proton therapy, and/or radiation therapy, sometimes followed by adjuvant pharmacother- This article is an open access article apy in high-risk patients such as chemotherapy, androgen-deprivation therapy (ADT), distributed under the terms and and/or hormone therapies. However, when curative and adjuvant therapies fail, as judged conditions of the Creative Commons by rising prostate-specific antigen (PSA) levels or metastasis, currently available hormone Attribution (CC BY) license (https:// therapies (various forms of indirect or direct androgen receptor (AR) antagonism) are creativecommons.org/licenses/by/ employed to delay progression of (but cannot cure) PCa. 4.0/).

Int. J. Mol. Sci. 2021, 22, 2124. https://doi.org/10.3390/ijms22042124 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2124 2 of 20

More than 90% of the early stage PCas are primarily dependent on the androgens, namely, testosterone (1) and dihydrotestosterone (DHT) (2) (Figure1), for growth [ 2]. Androgenic hormones bind to the AR, a member of the hormone receptor family of ligand- activated transcription factors, in the cytoplasm and promote translocation of the AR into the nucleus. In the nucleus, the AR binds to DNA regions called androgen response elements (AREs), which are palindromic sequences, recruits cofactors, and general tran- scription machinery, and that promote transcription and translation of the target genes [3]. AR is the primary therapeutic target in PCa and CRPC [4,5]. Although the early stage PCa is AR-driven in more than 90% of the cases, this percentage decreases in later stages of CRPC and mCRPC, where still around 70–80% of the cases require AR for growth [2,6,7]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 21 Still, this is considered as a high percentage reliant on a single therapeutic target, and hence

the preponderance of therapeutic modalities target antagonism of the AR axis.

FigureFigure 1. Overview 1. Overview of canonical of canonical androgen androgen receptor receptor (AR) ligands. (AR) ligands.

Resistance to ADT such as gonadotropin‐releasing hormone agonist or antagonist (or alternatively surgical castration) results in progression to CRPC. Subsequent or concomi‐ tant resistance to AR blocking agents such as the androgen synthesis inhibitor; abi‐ raterone; and/or antiandrogens such enzalutamide (3), apalutamide (4), or darolutamide is inevitable. Several mechanisms have been attributed to these resistances including over‐ expression of the AR, mutations in the AR ligand‐binding domain (LBD), loss of AR (neu‐ roendocrine PCa), constitutively active AR splice variants (AR‐SVs), increase in intra‐ tumoral hormonal synthesis, and activation of growth factor pathways [8]. Over time, as

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as judged by rising prostate‐specific antigen (PSA) levels or metastasis, currently available hormone therapies (various forms of indirect or direct androgen receptor (AR) antago‐ nism) are employed to delay progression of (but cannot cure) PCa. More than 90% of the early stage PCas are primarily dependent on the androgens, namely, testosterone (1) and dihydrotestosterone (DHT) (2) (Figure 1), for growth [2]. An‐ drogenic hormones bind to the AR, a member of the hormone receptor family of ligand‐ activated transcription factors, in the cytoplasm and promote translocation of the AR into the nucleus. In the nucleus, the AR binds to DNA regions called androgen response ele‐ ments (AREs), which are palindromic sequences, recruits cofactors, and general transcrip‐ tion machinery, and that promote transcription and translation of the target genes [3]. AR is the primary therapeutic target in PCa and CRPC [4,5]. Although the early stage PCa is AR‐driven in more than 90% of the cases, this percentage decreases in later stages of CRPC Int. J. Mol. Sci. 2021, 22, 2124 and mCRPC, where still around 70–80% of the cases require AR for growth [2,6,7].3 of 20 Still, this is considered as a high percentage reliant on a single therapeutic target, and hence the preponderance of therapeutic modalities target antagonism of the AR axis. Resistance to ADT such as gonadotropin‐releasing hormone agonist or antagonist (or Resistance to ADT such as gonadotropin-releasing hormone agonist or antagonist alternatively surgical castration) results in progression to CRPC. Subsequent or concomi‐ (or alternatively surgical castration) results in progression to CRPC. Subsequent or con- tant resistance to AR blocking agents such as the androgen synthesis inhibitor; abi‐ comitant resistance to AR blocking agents such as the androgen synthesis inhibitor; abi- raterone; and/or antiandrogens such enzalutamide (3), apalutamide (4), or darolutamide raterone; and/or antiandrogens such enzalutamide (3), apalutamide (4), or darolutamide is inevitable. Several mechanisms have been attributed to these resistances including over‐ is inevitable. Several mechanisms have been attributed to these resistances including overexpressionexpression of of the the AR, AR, mutations mutations in in the the AR AR ligand ligand-binding‐binding domain domain (LBD), (LBD), loss lossof AR of (neu‐ AR (neuroendocrineroendocrine PCa), PCa), constitutively constitutively active active AR AR splice splice variants variants (AR (AR-SVs),‐SVs), increase increase in in intra‐ intratumoraltumoral hormonalhormonal synthesis,synthesis, and and activation activation of of growth growth factor factor pathways pathways [8 [8].]. Over Over time, time, as as thethe AR AR milieu milieu present present in thein the PCa PCa becomes becomes complex complex and and heterogeneous, heterogeneous, patients patients become become refractoryrefractory to AR to blocking AR blocking agents agents [9]. The [9]. AR-SVsThe AR‐ haveSVs have emerged emerged as critical as critical players players in the in the developmentdevelopment and progression and progression of mCRPCs. of mCRPCs. Among Among AR-SVs AR‐ identifiedSVs identified to date, to date, AR-V7, AR also‐V7, also knownknown as AR3, as AR3, is one is ofone the of most the most abundant abundant and frequentlyand frequently found found forms forms in both in both PCa cellPCa cell lineslines and humanand human prostate prostate tumors tumors [10]. Notably, [10]. Notably, the lack the of LBDlack indicatesof LBD indicates that all Food that andall Food Drugand Administration Drug Administration (FDA)-approved (FDA)‐approved AR blocking AR agents blocking will agents have no will efficacy have inno inhibit- efficacy in ing AR-SVs.inhibiting Genomic AR‐SVs. events Genomic leading events to AR-SV leading expression to AR‐SV could expression act as novelcould biomarkersact as novel bi‐ of diseaseomarkers progression of disease that progression may guide that the may optimal guide usethe optimal of current use and of current next-generation and next‐gen‐ AR-targetederation AR therapy‐targeted [9]. therapy [9]. EndocrineEndocrine therapy-resistant therapy‐resistant PCa PCa cells cells generated generated by chronic by chronic treatment treatment with with3 or abi-3 or abi‐ rateroneraterone showed showed enhanced enhanced AR-V7 AR protein‐V7 protein expression expression [11,12 [11,12].]. Knockdown Knockdown of AR-V7 of AR by‐V7 by smallsmall interfering interfering RNA RNA (siRNA) (siRNA) in abiraterone-resistantin abiraterone‐resistant CWR22Rv1 CWR22Rv1 and and C4-2B C4‐2B enzalu- enzalutam‐ tamideide (MDV3100)-resistant (MDV3100)‐resistant (MDVR)(MDVR) cells restored their sensitivity sensitivity to to abiraterone, abiraterone, indicating indi- catingthat that AR AR-V7‐V7 is involved is involved in abiraterone in abiraterone resistance. resistance. Moreover, Moreover, an FDA an‐approved FDA-approved anthelmin‐ anthelminthicthic drug, drug,niclosamide niclosamide (5) [13], (5 has)[13 been], has previously been previously identified identified as a potent as a inhibitor potent in- of AR‐ hibitorV7, of re AR-V7,‐sensitizes re-sensitizes resistant cells resistant to 3 or cells abiraterone to 3 or abirateronetreatment in treatment vitro and inin vitrovivo [13,14].and in vivo [13,14].

2. Structural2. Structural Basis Basis for Antagonist for Antagonist Development Development ElucidationElucidation of the of crystal the crystal structures structures of the of AR the DNA AR DNA binding binding domain domain (DBD) (DBD) and theand the LBDLBD provides provides a new a new framework framework for understanding for understanding the functions the functions of this of receptor this receptor and hasand has led toled the to development the development of rational of rational drug design drug design for the treatmentfor the treatment of prostate of prostate cancer. Despite cancer. De‐ the lackspite of the any lack specific of any structural specific structural characterization, characterization, N-terminal N‐ domainterminal (NTD) domain inhibitors (NTD) inhib‐ are alsoitors in are preclinical also in preclinical development. development. Early ligand-based drug design was based on modification of structure of testosterone, the most prevalent endogenous androgen. Starting in the 1930s, many steroidal agonists such as methyltestosterone (6) and oxandrolone (7) and steroidal antagonists such as cyproterone acetate (8) were approved. Unfortunately, cross-reactivity with other nuclear hormone receptors, liver toxicity, and inability to separate anabolic (muscle/bone building) from androgenic (growth of primary and accessory sexual organs) activity has limited the utility of steroidal ligands. In the 1960s, it was discovered that 5α-reductase converted testosterone (1) to DHT (2) as a local amplification of the AR axis in certain tissues such as the skin and prostate [15]. Not until 2001 was the first ligand co-crystal structure of AR elucidated. This wild-type androgen receptor (wtAR)–DHT (Figure2A) structure was able to elucidate the binding mode for 2 and other the steroidal AR ligands [16]. The endogenous steroid 2 bound to the AR via an internalized, i.e., not solvent-exposed, hormone binding pocket (HBP) in the C-terminal LBD. The first nonsteroidal AR ligand, flutamide (9), was discovered as an AR antagonist in the 1980s [17]. Flutamide and its active metabolite, hydroxyflutamide (10), were the first in Int. J. Mol. Sci. 2021, 22, 2124 4 of 20

a series of agents termed as antiandrogens, including bicalutamide (11), the first diphenyl nonsteroidal AR ligand [17,18]. In the 1990s, the first nonsteoridal AR agonists were Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWdiscovered as derivatives of 11 in which the sulfonamide of 11 was replaced with thioethers5 of 21 (not shown); however, the thioethers lacked in vivo agonist activity [19]. Conversion of the thioethers to ethers resulted in the tissue-selective AR modulators (SARMs) such as enobosarm (12), which acted as agonists in anabolic tissues such as the musculoskeletal (orsystem antagonists) (as reflected of the by anabolic the levator tissues ani muscle)such as ventral and weak prostate partial and agonists seminal (or vesicles antagonists) [20– 22].of the anabolic tissues such as ventral prostate and seminal vesicles [20–22].

(A)

(B)

(C)

(D)

Figure 2. Conserved binding model for canonical ligands of the AR. Panel A: Cartoon model of a Figure 2. Conserved binding model for canonical ligands of the AR. Panel A: Cartoon model of crystal structure of dihydrotestosterone (DHT) (2) bound to the ligand‐binding domain (LBD) a crystal structure of dihydrotestosterone (DHT) (2) bound to the ligand-binding domain (LBD) (Protein Database (PDB) ID: 4OEA [23]). Panel B: Cartoon model of a crystal structure of a selec‐ tive(Protein androgen Database receptor (PDB) modulator ID: 4OEA (SARM) [23]). Panel 13 (PDBB: Cartoon ID: 5V8Q model [24]. of Panel a crystal C: Cartoon structure model of a selective of a crystalandrogen structure receptor of hydroxyflutamide modulator (SARM) (1013) (PDBbound ID: to 5V8Qthe T877A [24]. mutant Panel C AR: Cartoon LBD (PDB model ID: of 2AX6 a crystal [25]).structure Panel of D hydroxyflutamide: Cartoon model of (10 a )crystal bound structure to the T877A of a mutantSARM 14 AR bound LBD (PDBto the ID: LBD 2AX6 (PDB [25 ID:]). Panel5CJ6 [26]).D: Cartoon model of a crystal structure of a SARM 14 bound to the LBD (PDB ID: 5CJ6 [26]).

ModernModern structuralstructural biology biology allowed allowed many many AR AR agonist agonist co-crystal co‐crystal structures structures to be solved, to be solved,such as 13suchand as14 13(Figure and 214B,D), (Figure which 2B,D), are shown which as recentare shown representative as recent examples representative [ 24,26]. examplesUnfortunately, [24,26]. no Unfortunately, antiandrogen–wtAR no antiandrogen–wtAR co-crystal structure existsco‐crystal yet, limitingstructure the exists potential yet, limitingof structure-based the potential antiandrogen of structure design.‐based antiandrogen Antiandrogen design. resistance Antiandrogen is frequently resistance caused by is frequentlymutations incaused AR-LBD by mutations (mtAR) in in such AR a‐LBD manner (mtAR) that the in such steric a bulk manner of the that antiandrogen the steric bulk (see ofW741L the antiandrogen for bicalutamide (see (11 W741L) or F876L for forbicalutamide3 or 4), which (11 destabilizes) or F876L AR,for 3 is accomodatedor 4), which destabilizesby mutation AR, to a smalleris accomodated amino acid by [25 mutation]. These antiandrogen–mtARto a smaller amino complexes acid [25]. activate These antiandrogen–mtARthe AR axis, leading complexes to a type ofactivate mutational the AR resistance axis, leading referred to a to type an agonistof mutational switch resistanceor escape referred mutation to [ 27an]. agonist Co-crystal switch structures or escape of mutation antiandrogens [27]. Co bound‐crystal to structures their agonist of antiandrogensswitch mutation bound have beento their published, agonist and switch the T877A mutation and hydroxyflutamidehave been published, (10) co-crystal and the T877Astructure and serves hydroxyflutamide as a representative (10) co escape‐crystal mutation structure (Figure serves2C) as [ 25a ].representative escape mutation (Figure 2C) [25].

3. Need for AR‐Directed Therapy Beyond Canonical Antiandrogens As described above, the discovery of antiandrogens that compete for binding with testosterone (1) and DHT (2) to the HBP of the LBD continues to be productive, but anti‐ androgen resistance via agonist switch and AR truncation (e.g., AR‐V7) mutations limits

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3. Need for AR-Directed Therapy Beyond Canonical Antiandrogens As described above, the discovery of antiandrogens that compete for binding with testosterone (1) and DHT (2) to the HBP of the LBD continues to be productive, but antiandrogen resistance via agonist switch and AR truncation (e.g., AR-V7) mutations limits their duration of efficacy. Correspondingly, innovative new approaches to provide hormone therapy in CRPC are needed. This review seeks to briefly outline a variety of novel small molecule ligands with AR antagonist activity and/or noncanonical AR binding sites with the goal of stimulating interest in further discovery of noncanonical ligands that may overcome known mechanisms of FDA-approved endocrine therapies.

3.1. LBD-Targeted SARDs An emerging alternative approach to relying on competitive binding of the HBP to prevent endogenous androgen action is to downregulate (decrease expression or destroy (destabilize or proteolyze)) the AR, thereby eliminating AR-mediated signaling [28]. Se- lective AR downregulators or degraders (SARDs) have been discovered, which bind to the LBD (discussed here) or the NTD (vide infra). In general, direct binding to AR is preferred to provide AR specificity, and degradation (protein destruction) is preferred to downregulation. ASC-J9 (15) was an early downregulator that was not demonstrated to bind to AR but lowered AR levels and produced antiproliferative activity in C4-2 and DU145 (AR-negative) PCa cells with artificially expressed F876L [29], an escape mutant conferring 3 or 4 resistance. Another small molecule downregulator is AZD3514 (16), which was demonstrated to bind to AR LBD and downregulate AR. In two phase I clinical trials, Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWmoderate anti-tumor activity was observed in CRPC patients as judged by significant6 PSAof 21 declines, but the drug was not well tolerated. Although development was discontinued, 16 provided a rationale for future development of SARD compounds [30].

Galeterone (17) [31] is a steroidal antiandrogen developed for the treatment of CRPC. Galeterone (17)[31] is a steroidal antiandrogen developed for the treatment of CRPC. It possesses a unique combination of three separate biochemical mechanisms of action, It possesses a unique combination of three separate biochemical mechanisms of action, namely, acting as an antagonist that binds competitively to AR LBD, inhibiting CYP17A1 namely, acting as an antagonist that binds competitively to AR LBD, inhibiting CYP17A1 with selectivity for 17,20‐lyase activity over 17α‐hydroxylase activity of CYP17A1, and with selectivity for 17,20-lyase activity over 17α-hydroxylase activity of CYP17A1, and acting as an AR degrader that markedly enhances AR degradation [32–34]. acting as an AR degrader that markedly enhances AR degradation [32–34]. Galeterone (17) (also known as TOK‐001 or VN/124‐1) was developed by Tokai Phar‐ Galeterone (17) (also known as TOK-001 or VN/124-1) was developed by Tokai maceuticals and tested in a phase III clinical trial (ARMOR3‐SV) in 2016 for AR‐V7‐posi‐ Pharmaceuticals and tested in a phase III clinical trial (ARMOR3-SV) in 2016 for AR-V7- tive mCRPC [32,35]. The studies revealed that 17 has poor oral bioavailability, which un‐ positive mCRPC [32,35]. The studies revealed that 17 has poor oral bioavailability, which doubtedlyundoubtedly contributed contributed to toits its clinical clinical failure failure in in phase phase III. III. The The multi multi-targeted‐targeted lyase lyase inhibitor inhibitor and direct AR antagonist/SARD 17 is a novel approach that is still in clinical development by the University of Maryland, Baltimore (NCT04098081), with a next‐generation lead 18 still in preclinical development [36].

3.2. Proteolysis‐Targeting Chimeras (PROTACs) as Nonsteroidal AR Degraders PROTACs targeted to AR are heterobifunctional small molecules that consist of a central linker and two warheads, one warhead is based on a known AR ligand to bind AR and the other recruits an E3 ligase, as shown in Figure 3. Although activity is through HBP binding, PROTACs provide targeted AR protein degradation at very high potency, unlike traditional canonical antiandrogens. PROTACs bring E3 ubiquitin ligase enzyme to AR to form a ternary complex, which results in very efficient ubiquitination of AR, causing AR degradation by the proteasome (Figure 3).

Figure 3. Mechanism of action of AR‐directed proteolysis‐targeting chimeras (PROTACs). The AR

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Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW and direct AR antagonist/SARD 17 is a novel approach that is still in7 of clinical 21 development by the University of Maryland, Baltimore (NCT04098081), with a next-generation lead 18

still in preclinical development [36].

3.2. Proteolysis-Targeting Chimeras (PROTACs) as Nonsteroidal AR Degraders 3.2. Proteolysis‐Targeting Chimeras (PROTACs) as Nonsteroidal AR Degraders PROTACs targeted to AR are heterobifunctional small molecules that consist of a PROTACs targeted to AR are heterobifunctional small molecules that consist of a central linker and two warheads, one warhead is based on a known AR ligand to bind AR central linker andand two the warheads, other recruits one warhead an E3 ligase, is based as shown on a known in Figure AR3 ligand. Although to bind activity AR is through HBP and the other recruitsbinding, an E3 PROTACs ligase, as shown provide in targeted Figure 3. AR Although protein degradationactivity is through at very HBP high potency, unlike binding, PROTACstraditional provide targeted canonical AR antiandrogens. protein degradation PROTACs at very bring high E3 ubiquitinpotency, unlike ligase enzyme to AR to traditional canonicalform antiandrogens. a ternary complex, PROTACs which bring results E3 inubiquitin very efficient ligase enzyme ubiquitination to AR to of AR, causing AR form a ternary complex,degradation which by results the proteasome in very efficient (Figure ubiquitination3). of AR, causing AR degradation by the proteasome (Figure 3).

Figure 3. Mechanism of action of AR‐directed proteolysis‐targeting chimeras (PROTACs). The AR Figure 3. Mechanism of action of AR-directed proteolysis-targeting chimeras (PROTACs). The AR ligand warhead binds AR ligand warhead binds AR and the E3 ligase warhead binds to E3 ligase, forming a ternary com‐ and the E3plex. ligase The warhead ternary complexes binds to E3 allow ligase, for forming efficient a ternary catalysis complex. of the transfer The ternary of ubiquitin complexes (Ub) allow to lysine for efficient catalysis of the transfer(NH2) ofresidues ubiquitin on (Ub)the AR, to lysine targeting (NH AR2) residues to be destroyed on the AR, by the targeting proteosome. AR to be destroyed by the proteosome.

Since first proposedSince by firstCrews proposed et al. [37], by PROTAC Crews et technology al. [37], PROTAC has employed technology several has employed sev- AR ligands to targeteral ARthe ligandsAR. These to target AR ligands the AR. were These based AR ligands on known were antagonists based on known or antagonists or SARMs but resultedSARMs in AR but degradation resulted in rather AR degradation than antagonizing rather than the antagonizingAR axis by compet the AR‐ axis by compet- itive binding alone. A variety of E3 ligases and linkers have been employed and tested. itive binding alone. A variety of E3 ligases and linkers have been employed and tested. Correspondingly, substantial effort is required to optimize PROTACs by synthesizing the Correspondingly, substantial effort is required to optimize PROTACs by synthesizing the various possible combinations of AR ligands, linkers, and E3 ligases, as summarized below. various possible combinations of AR ligands, linkers, and E3 ligases, as summarized be‐ Nonsteroidal AR ligands offer greater nuclear receptor selectivity as well as being low. relatively amenable to synthetic modification. Examples of nonsteroidal AR warheads Nonsteroidal AR ligands offer greater nuclear receptor selectivity as well as being include 19-23, as shown in Figure4. Research found that 19 is derived from the SARM relatively amenable to synthetic modification. Examples of nonsteroidal AR warheads in‐ enobosarm (12)[38], whereas 21 is derived from enzalutamide (3). clude 19‐23, as shown in Figure 4. Research found that 19 is derived from the SARM eno‐ Early PROTACs possessed peptide-based linkers, but non-peptide-based linkers are bosarm (12) [38], whereas 21 is derived from enzalutamide (3). preferred, or various lengths of polymers such as polyethylene glycol (PEG)-based (e.g., Early PROTACs possessed peptide‐based linkers, but non‐peptide‐based linkers are see in 24) to connect the warheads. The complexity of PROTAC design has increased as preferred, or various lengths of polymers such as polyethylene glycol (PEG)‐based (e.g., more complex linker segments are now commonly employed (see black atoms in ARD-266 see in 24) to connect(25) the and warheads. ARCC-4 (26)). The complexity of PROTAC design has increased as more complex linker segmentsA wide variety are now of E3commonly ligase warheads employed has (see been black explored. atoms A in representative ARD‐ sample of 266 (25) and ARCCfour‐4 ( different26)). E3 ligases are shown in AR-targeted PROTACs in Figure4, namely, MDM2 A wide varietyligands of E3 based ligase on warheads nutlin, cIAP1 has been ligands explored. based onA representative bestatin esters, sample Von Hippel–Lindau of (VHL) four different E3 ligasesligands are based shown on in the AR hydroxyproline‐targeted PROTACs structure in Figure found 4, namely, in HIF1a, MDM2 and cereblon (CRBN) ligands based onligands nutlin, based cIAP1 on ligands thalidomide based (Figure on bestatin4)[39 ].esters, Von Hippel–Lindau (VHL) ligands based on the hydroxyproline structure found in HIF1a, and cereblon (CRBN) ligands based on thalidomide (Figure 4) [39].

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Figure 4. ExemplaryFigure AR-directed 4. Exemplary PROTACs AR‐directed and examples PROTACs of AR and ligands examples and E3of AR ligase ligands ligands and [39 E3– 44ligase]. ligands [39,40–44]. Many AR-degrading PROTACs exist, however, ARCC-4 (26) is shown here as an exemplaryMany SARDAR‐degrading [40,45,46 PROTACs]. ARCC-4 exist, (26) in however, Figure4 utilizesARCC‐43 (26as) the is shown AR ligand here and as an VHL ex‐ emplaryas the ubiquitin SARD [40,45,46]. ligase, thereby ARCC targeting‐4 (26) in AR Figure to the 4 proteaseutilizes 3 foras the degradation. AR ligand ARCC-4 and VHL (26 as) wasthe ubiquitin able to degrade ligase, ARthereby in VCaP targeting cells (resistanceAR to the protease mechanisms for degradation. include AR amplification, ARCC‐4 (26) AR-V7was able expression, to degrade and AR TMPRSS2–ERG in VCaP cells (resistance (a fusion ofmechanisms TMPRSS2 (transmembraneinclude AR amplification, protease, ARserine‐V7 2)expression, to ETS-related and TMPRSS2–ERG gene (ERG)) translocation) (a fusion of TMPRSS2 at 5 nM with (transmembrane maximal degradation protease, serineof 98%. 2) ARCC-4to ETS‐related (26) was gene able (ERG)) to degrade translocation) wtAR and at 5 anM variety with maximal of escape degradation mutants with of 98%.nanomolar ARCC potencies‐4 (26) was including able to degrade F876L (confers wtAR and resistance a variety to of3 andescape4)[ 47mutants], W741L with (confers nano‐ molarresistance potencies to bicalutamide including ( F876L11)) [48 (confers], M896V resistance (confers resistanceto 3 and 4 to) [47],11)[ 49W741L], T877A (confers (confers re‐ sistanceresistance to to bicalutamide flutamide (9 (),11 glucocorticoids,)) [48], M896V (confers and progesterone) resistance to [50 11],) H874Y [49], T877A (confers (confers resis- resistancetance to 9, to glucocorticoids, flutamide (9), andglucocorticoids, progesterone) and [50 progesterone),51], and L702H [50], (glucocorticoids) H874Y (confers [ 52re].‐ Assistance illustrated to 9, glucocorticoids, by 26, PROTACs and tend progesterone) to be large [50,51], compounds and L702H that (glucocorticoids) do not comply with [52]. Asstandard illustrated rules by for 26 oral, PROTACs bioavailability, tend and to be thus large attaining compounds this broad that scope do not of antiandrogencomply with standardactivity in rules vivo formay oral be bioavailability, difficult. Another and thus disadvantage attaining this with broad PROTACs, scope of assuming antiandrogen full activityoral bioavailability, in vivo may is be the difficult. limitation Another of requiring disadvantage an LBD with (canonical PROTACs, AR ligandsassuming have full been oral employed thus far) to destroy the AR, indicating that AR-SVs may not be destroyed by

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these PROTACs. Lastly, PROTACs do not eliminate the possibility of PR- or GR-mediated AR-axis signaling, referred to as AR bypass resistance. A reportedly orally bioavailable SARD whose structure is undisclosed, ARV110, developed by Arvinas, was initiated in phase I trials in 2019 in a late-stage mCRPC patient population, and recently released an interim data update (https://ir.arvinas.com/news-releases/news-release-details/arvinas- releases-interim-clinical-data-further-demonstrating; accessed January 2021). Dose esca- lation of ARV110 produced PSA responses at 280 mg, and at 420 mg attained sufficient exposure to have antitumor effects as judged by analogy to preclinical models. Two of five patients (40%) with H874 or T877 mutations had PSA reductions >50% compared to 2 of 15 wtAR patients, supporting testing of a molecularly defined late-line patient population. Additionally, Arvinas is considering testing in a less pre-treated population. This data established the proof-of-concept that it is possible to have clinical efficacy with an orally active AR-directed PROTAC; however, the high dose (420 mg) to achieve efficacious expo- sures in contrast to the low nanomolar preclinical potencies suggests that pharmacokinetic (PK) properties could be improved.

3.3. Summary of Hormone Binding Pocket (HPB)-Targeted Antiandrogens Two generations of (competitive) antiandrogens have been approved, and with each new approval, the scope of antiandrogen activities was expanded and/or antiandrogen potencies improved. For example, flutamide (9) was less potent than bicalutamide (11), and both were susceptible to resistance due to AR overexpression. Enzalutamide (3) and apalutamide (4) could overcome AR overexpression and demonstrated the novel ability to inhibit nuclear translocation of the AR; however, all these antiandrogens were susceptible to escape mutations [47,53]. Darolutamide also inhibited translocation and overcame AR overexpression, but additionally was a more potent inhibitor than first- and second-generation antiandrogens and was a pan-antagonist of escape mutations [47,53]. Similarly, PROTACs are also able to overcome AR overexpression and have demonstrated perhaps the broadest scope of pan-antagonism of HBP-dependent antiandrogens; however, PROTACs are in early stages of clinical testing with limited efficacy reported thus far. Despite the progressively broadening scope of antiandrogen activities, made possible with HBP-directed antiandrogens, none of these approaches are able to address the problem of inhibiting AR-SV-dependent PCa growth.

4. Noncompetitive Antiandrogens Developing nonconventional AR-modulating agents targeting noncanonical binding sites is likely to be a promising future approach to develop multiple and synergistic strategies [54]. LBD and DBD crystal structures facilitated the discovery of solvent-exposed regions on the surface of AR that are suitable for the design of non-competitive ligands. Ligands of the NTD have also been reported, despite the intrinsically disordered nature of the NTD. All noncompetitive ligands thus far have been inhibitors and are expected to have unique properties when compared to the FDA-approved competitive inhibitors. Noncompetitive antagonists are reported for the following noncanonical sites: (1) activating function (AF)-2 and (2) binding function-3 (BF-3) sites that reside in the LBD, DBD sites include (3) the DNA recognition helix [55] and (4) the DBD/dimerization region (not discussed here, but reported) [56,57], and (5) AF-1 inhibitors that bind to and inhibit the NTD.

4.1. Noncanonical (Noncompetitive) LBD Ligands The LBD consists of the C-terminal residues from 676 to 919 and, in addition to the internal HBP, also possesses the AF-2 function, which forms an external binding surface for interactions with co-regulatory proteins recruited upon agonist binding. The noncanonical LBD inhibitors have had weak antiandrogen potencies but nonetheless provide novel ways to modulate the AR axis by directly blocking (AF-2 inhibitors) or modulating (BF-3 inhibitors) the protein–protein interactions that occur between AF-2 and Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 21

for interactions with co‐regulatory proteins recruited upon agonist binding. The non‐ Int. J. Mol. Sci. 2021, 22, 2124 canonical LBD inhibitors have had weak antiandrogen potencies but nonetheless provide9 of 20 novel ways to modulate the AR axis by directly blocking (AF‐2 inhibitors) or modulating (BF‐3 inhibitors) the protein–protein interactions that occur between AF‐2 and its cofac‐ tors [58]. Direct AF‐2 inhibitors mimic the LXXLL or F/WXXLF consensus‐binding motifs its cofactors [58]. Direct AF-2 inhibitors mimic the LXXLL or F/WXXLF consensus-binding of cofactors and thereby bind competitively with cofactors for occupancy of the AF‐2 site. motifs of cofactors and thereby bind competitively with cofactors for occupancy of the Initially peptides were discovered and tested as AF‐2 inhibitors. Phage display studies AF-2 site. Initially peptides were discovered and tested as AF-2 inhibitors. Phage display revealed that mtAR and wtAR bound to similar peptidomimetic inhibitors of AF‐2, studies revealed that mtAR and wtAR bound to similar peptidomimetic inhibitors of AF-2, including BUD31 (structure not shown), which contains an Fxx(F/H/L/W/Y)Y motif including BUD31 (structure not shown), which contains an Fxx(F/H/L/W/Y)Y motif clustercluster with with tyrosine tyrosine (Y) (Y) in in their their respective respective position position [23 [23].]. Thus, Thus, structural structural analyses analyses of of the the AR–LBD–BUD31AR–LBD–BUD31 complex complex revealed revealed thethe formation of an an extra extra hydrogen hydrogen bond bond between between the thespecific specific Y residue Y residue of the of the peptide peptide and and AF‐ AF-2.2. A combination A combination of peptide of peptide screening screening and andX‐ray X-raystructure structure analysis analysis may may serve serve as asa anew new strategy strategy for developingdeveloping ARAR antagonists antagonists that that simultaneouslysimultaneously stop stop both both wild-type wild‐type and and mutated mutated AR AR function. function. IMB-A6IMB‐A6 (31 (31),), an an exemplary exemplary small small molecule molecule inhibitor inhibitor of of the the AF-2 AF‐2 site, site, was was discovered discovered onon virtual virtual screening screening for for ligands ligands thatthat bindbind to the AF AF-2‐2 site. site. IMB IMB-A6‐A6 (31 (31) demonstrated) demonstrated the theability ability to toinhibit inhibit binding binding of co of‐ co-factorfactor PELP PELP-1‐1 (proline (proline-glutamic‐glutamic acid acid-‐ and and leucine leucine-rich‐rich pep‐ peptide)tide) at at 10 10 μµMM range range (noncompetitive (noncompetitive with HBP ligands)ligands) [ 59[59].]. Another Another novel novel allosteric allosteric pocketpocket on on the the LBD, LBD, one one that that is is potentially potentially amenable amenable to to pharmacological pharmacological manipulation, manipulation, is is namednamed binding binding function-3 function‐3 (BF-3). (BF‐3). The The BF-3 BF‐3 pocket pocket is is adjacent adjacent to to the the AF-2 AF‐2 pocket, pocket, and and smallsmall molecule molecule ligands ligands of of BF-3 BF‐3 regulate regulate which which co-regulatorsco‐regulators bindbind toto AF-2,AF‐2, resulting in a amodulation of AR activity similarsimilar toto SARMSARM butbut not not through through HBP. HBP. An An exemplary exemplary BF-3 BF‐3 bindingbinding compound, compound, MEPB MEPB (32 ),(32 was), was shown shown unequivocally unequivocally by X-ray by X crystallography‐ray crystallography to bind to thebind BF-3 the domain BF‐3 domain [60]. MEPB [60]. MEPB (32) was (32 recently) was recently tested tested in a model in a model of spinobulbar of spinobulbar muscular mus‐ atrophycular atrophy (SBMA), (SBMA), a neurodegenerative a neurodegenerative disorder disorder in males in characterized males characterized by expanded by expanded length CAGlength trinucleotide CAG trinucleotide repeat (long repeat poly-Q (long tract). poly In‐Q SBMA, tract). the In longSBMA, poly-Q the long tract resultspoly‐Q in tract toxic re‐ neurodegenerativesults in toxic neurodegenerative effects upon androgen effects stimulation upon androgen of defective stimulation AR. MEPB of defective (32) caused AR. increasedMEPB (32 recruitment) caused increased of co-repressors recruitment such of as co nuclear‐repressors receptor such corepressor as nuclear (NCoR) receptor to core the ‐ AF-2pressor domain (NCoR) and to ameliorated the AF‐2 domain neuronal and loss, ameliorated neurogenic neuronal atrophy, loss, and neurogenic testicular atrophy atrophy, inand a mouse testicular model atrophy of SMBA, in a mouse validating model AF-2 of SMBA, modulation validating as a AF potent‐2 modulation androgen-sparing as a potent strategyandrogen for‐sparing SBMA therapy strategy [61 for]. SBMA therapy [61].

4.2.4.2. Noncanonical Noncanonical DBD DBD Antiandrogens antiandrogens AA theoretical theoretical advantage advantage of of targeting targeting the the DBDDBD isis that potent inhibition of of the the DBD DBD to toDNA DNA interaction interaction would would effectively effectively abrogate abrogate transcriptional transcriptional activation activation and and would would be be un‐ unaffectedaffected by by LBD LBD point point mutations mutations (e.g., escape mutants) oror LBD LBD truncations truncations (e.g., (e.g., AR-SVs). AR‐SVs). AA theoretical theoretical disadvantage disadvantage is is that that the the DBD DBD is is the the most most conserved conserved domain domain between between steroid steroid receptors,receptors, and and thus thus obtaining obtaining steroid steroid receptor receptor selectivity selectivity might might be be challenging. challenging. Characterizing DBD by X-ray crystallography provided structures that facilitated the discovery of surface-exposed regions of the AR that might serve as a noncanonical ligand binding site. In silico virtual screening of residues S579 to K610 identified candidate molecules that were effective at abolishing transcription in LNCaP cells [55]. Screening of the candidates demonstrated activity for a series of compounds consisting of a system of Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 21

Characterizing DBD by X‐ray crystallography provided structures that facilitated the discovery of surface‐exposed regions of the AR that might serve as a noncanonical ligand Int. J. Mol. Sci. 2021, 22, 2124 binding site. In silico virtual screening of residues S579 to K610 identified candidate10 mol of 20‐ ecules that were effective at abolishing transcription in LNCaP cells [55]. Screening of the candidates demonstrated activity for a series of compounds consisting of a system of three rings directly connected by single bonds, with the middle ring being heterocyclic and most three rings directly connected by single bonds, with the middle ring being heterocyclic and often a thiazole. Synthetic expansion explored substituted aryl A‐rings with a thiazole B‐ most often a thiazole. Synthetic expansion explored substituted aryl A-rings with a thiazole ring and a morpholino C‐ring. A lead that emerged from this effort was VPC14449 (33), B-ring and a morpholino C-ring. A lead that emerged from this effort was VPC14449 (33), which suppressed PSA expression comparable to enzalutamide (3) (0.17 M vs. 0.12 M) which suppressed PSA expression comparable to enzalutamide (3) (0.17 µM vs. 0.12 µM) in LNCaP cells [55]. in LNCaP cells [55].

InIn an artificial artificial inin vitro vitro transcriptionaltranscriptional activation activation assay, assay, 33 33andand 3 again3 again displayed displayed sim‐ similarilar potency potency (0.340 (0.340 M vs.µM 0.314 vs.  0.314M) [62].µM) To [validate62]. To the validate DBD binding the DBD site, binding the researchers site, the researchersprepared point prepared mutations point mutationsY594D and Y594D Q592D, and which Q592D, were which found were to foundbe inhibited to be inhibited by 3 but bynot3 bybut 33 not, supporting by 33, supporting the proposed the proposed binding site. binding VPC14449 site. VPC14449 (33) fully inhibited (33) fully wtAR inhibited at 5 wtARM but at only 5 µM partially but only affected partially estrogen affected receptor estrogen at 15 receptor M and atdid 15 notµM affect and glucocorticord did not affect glucocorticordreceptor or progesterone receptor or receptor progesterone at <25 μ receptorM, demonstrating at <25 µM, surprising demonstrating nuclear surprising receptor nuclearselectivity receptor (similar selectivity to 3). On (similar the basis to 3 ).of On this the in basis vitro of characterization, this in vitro characterization, the researchers the researchersdeemed 33 deemedto be a useful33 to be prototypical a useful prototypical DBD inhibitor DBD and inhibitor was tested and was in models tested in of models CPRC of[63] CPRC such [ 63LNCaP] such (T877A), LNCaP (T877A), C4‐2 (T877A), C4-2 (T877A), MR49F and MR49F MR49C and MR49C (F876L_T877A), (F876L_T877A), and 22RV1 and 22RV1(H874C (H874C and overexpressed and overexpressed AR‐V7). AR-V7).

4.3. Noncanonical NTD Antiandrogensantiandrogens Two separate groups groups are are developing developing antiandrogens antiandrogens that that bind bind to the to AF the‐1 AF-1 region. region. Mu‐ Mutationaltational analysis analysis has has revealed revealed two two regions regions in in the the NTD, NTD, encoded encoded by by amino amino acid residues 141-338141‐338 (transcription(transcription activation unit (Tau)-1)(Tau)‐1) andand 380-529380‐529 (Tau-5),(Tau‐5), toto bebe essentialessential forfor thisthis transcriptionaltranscriptional activation, activation, being being termed termed as as the the AF AF-1‐1 [64]. [64 ].AR AR has has the the capacity capacity to use to usedif‐ differentferent regions regions in in the the N N-terminal‐terminal domain domain (residues (residues 1 1-450)‐450) as as transcription transcription activation unitsunits (TAUs)(TAUs) [[65].65]. TheThe inhibitorsinhibitors belowbelow havehave beenbeen reportedreported asas blockingblocking tau-5tau‐5 (EPI-506(EPI‐506 ((3434)))) [[66]66] and tau-1tau‐1 (UT-155(UT‐155 ((3535)))) [[67].67]. DeletionsDeletions ofof thethe AF-1AF‐1 regionregion renderedrendered ARAR transcriptionallytranscriptionally inactiveinactive [[68].68]. In contrast, deletion of LBD caused constitutiveconstitutive activation, suggesting that, inin thethe absenceabsence ofof boundbound androgen,androgen, thethe LBDLBD exertsexerts anan inhibitoryinhibitory effecteffect onon AR.AR. ThisThis helpshelps toto rationalizerationalize thethe selectiveselective advantageadvantage ofof expressingexpressing AR-SVsAR‐SVs inin tumortumor cellscells subjectedsubjected toto LBD competitivecompetitive antagonists.antagonists. Collectively, Collectively, this provides a rationalerationale forfor inhibitinginhibiting thethe functionfunction of AF-1AF‐1 inin heavily pre-treatedpre‐treated CRPCCRPC populationspopulations thatthat areare expressingexpressing highhigh levelslevels of a heterogenous mixture of wtAR, AR-SV, and escape mutations. Unlike DBD and LBD, of a heterogenous mixture of wtAR, AR‐SV, and escape mutations. Unlike DBD and LBD, which are intrinsically ordered (globular) domains, the NTD is intrinsically disordered, which are intrinsically ordered (globular) domains, the NTD is intrinsically disordered, confounding efforts to use structure-based drug design [69]. Binding to coregulatory confounding efforts to use structure‐based drug design [69]. Binding to coregulatory bind‐ binding partners is postulated to induce folding of the NTD into α-helical conformations ing partners is postulated to induce folding of the NTD into ‐helical conformations in in order to maintain affinity, and similarly, small molecule ligands may also induce or order to maintain affinity, and similarly, small molecule ligands may also induce or stabi‐ stabilize secondary or tertiary structure within the otherwise disordered domain [69]. lize secondary or tertiary structure within the otherwise disordered domain [69]. 4.4. AF-1 Inhibitors Derived from Natural Products 4.4. AF‐1 inhibitors derived from natural products A new generation of androgen receptor antagonists including 34 was discovered in 2010 in vitro assays. Unlike bicalutamide (11), these antagonists had noncompetitive activity as judged by the lack of a right shift with increasing concentrations of an agonist such as R1881 and were not able to displace fluoromone from the HBP (i.e., a fluorescent hormone that binds to the HBP) [70], supporting a noncanonical binding site. Preclinical investigation revealed that 38 was the active component of a mixture of hydroxyl group Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 12 of 21

A new generation of androgen receptor antagonists including 34 was discovered in 2010 in vitro assays. Unlike bicalutamide (11), these antagonists had noncompetitive ac‐ tivity as judged by the lack of a right shift with increasing concentrations of an agonist Int. J. Mol. Sci. 2021, 22, 2124 such as R1881 and were not able to displace fluoromone from the HBP (i.e., a fluorescent11 of 20 hormone that binds to the HBP) [70], supporting a noncanonical binding site. Preclinical investigation revealed that 38 was the active component of a mixture of hydroxyl group epimers, irreversibly bound to the N‐terminal domain of the AR via the 20S chlorohydrin [71],epimers, and was irreversibly able to inhibit bound androgen to the N-terminal‐dependent domain genes of in the cells AR expressing via the 20 onlyS chlorohy- AR‐SV (v567es).drin [71], The and irreversibility was able to inhibit of the androgen-dependent interaction was postulated genes in cellsas a basis expressing for inhibiting only AR-SV the (v567es). The irreversibility of the interaction was postulated as a basis for inhibiting the intrinsically disordered NTD. In VCaP tumors overexpressing full‐length (FL) AR and intrinsically disordered NTD. In VCaP tumors overexpressing full-length (FL) AR and expressing AR‐V7, 38 inhibited AR‐V7‐dependent genes, i.e., genes shown to be uniquely expressing AR-V7, 38 inhibited AR-V7-dependent genes, i.e., genes shown to be uniquely upregulated by AR‐V7 and not AR‐FL [72]. Although micromolar levels were required to upregulated by AR-V7 and not AR-FL [72]. Although micromolar levels were required observe effects in vitro, the triacetate prodrug 34 was tested in patients that had failed to observe effects in vitro, the triacetate prodrug 34 was tested in patients that had failed enzalutamide (3) or abiraterone therapy, finding only a reduction of PSA by <30% for a enzalutamide (3) or abiraterone therapy, finding only a reduction of PSA by <30% for a short period of time. Further, very high doses of up to 3.6 g were required. Due to the poor short period of time. Further, very high doses of up to 3.6 g were required. Due to the poor pharmacokinetics, the trial was stopped. This was interpreted as a proof‐of‐principle but pharmacokinetics, the trial was stopped. This was interpreted as a proof-of-principle but insufficient potency and drug exposure (i.e., rapid metabolism) to inhibit the hetero‐ insufficient potency and drug exposure (i.e., rapid metabolism) to inhibit the heterogenous genous and resistant ARs present in this difficult‐to‐treat patient population, and new and resistant ARs present in this difficult-to-treat patient population, and new agents were agents were prepared. prepared. Compounds from this template with 20 20-fold‐fold greater greater potency potency and and improved improved pharma pharma-‐ cokinetics have have been been reported reported [73]. [73]. For For example, example, EPI EPI-7170‐7170 (39 (39),), a a semi semi-synthetic‐synthetic sulfona sulfon-‐ mideamide NTD NTD antiandrogen antiandrogen derived derived from from the the bisphenol bisphenol A (37 A) ( nucleus,37) nucleus, was wasreported reported to pro to‐ videprovide synergistic synergistic activity activity in inAR AR-V7‐V7 PCa PCa when when combined combined with with the the canonical canonical antiandrogen antiandrogen 3.. A A very very recent recent new new drug, drug, EPI EPI-7386‐7386 (structure (structure unknown), has been characterized preclini-preclin‐ icallycally in in VCaP VCaP and and several several other other xenografts. xenografts. EPI-7386 EPI‐7386 demonstrated demonstrated a significant a significant inhibition inhibi‐ tionof VCaP of VCaP tumors tumors in castrated in castrated mice mice with awith tumor a tumor growth growth inhibition inhibition of close of toclose 100%. to 100%. ESSA ESSAPharma Pharma reported reported the compound the compound to have to entered have clinicalentered trials clinical (NCT04421222) trials (NCT04421222) for PCa. The for PCa.reported The reported irreversibly irreversibly binding NTDbinding inhibitor NTD inhibitor mechanism mechanism of the EPI of the series, EPI series, if preclinical if pre‐ clinicalactivities activities translate translate to the clinic to the and clinic sufficient and sufficient bioavailability bioavailability is possible, is possible, suggests suggests broad broadscope scope AR antagonism AR antagonism in prostate in prostate cancers cancers expressing expressing escape escape and/or and/or truncation truncation mutant; mu‐ tant;however, however, poor pharmacokineticspoor pharmacokinetics of this of template this template suggest suggest that AR that overexpression AR overexpression may be maydifficult be difficult to overcome. to overcome. Phase I dosePhase escalation I dose escalation trials are trials expected are expected to end in to 2022. end in 2022.

Sadar et al. have also reported chemically unrelated natural products that interact Sadar et al. have also reported chemically unrelated natural products that interact with with the NTD. For example, sintokamide A (40) was one of the first natural products re‐ the NTD. For example, sintokamide A (40) was one of the first natural products reported ported to block the NTD transactivation of the AR in prostate cancer cells [74]. Early pre‐ to block the NTD transactivation of the AR in prostate cancer cells [74]. Early preclinical clinical SAR studies of this template were reported recently [75], including 41. Another SAR studies of this template were reported recently [75], including 41. Another agent, niphatenone B (42), was isolated from the marine sponge Niphates digitalis that represents a novel structural class of AR antagonist. Research found that 41 binds covalently to the AF-1 region of the AR NTD and blocks the proliferation of prostate cancer cells that are dependent on functional AR, and many analogs have been prepared and examined [76]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 13 of 21

agent, niphatenone B (42), was isolated from the marine sponge Niphates digitalis that rep‐ resents a novel structural class of AR antagonist. Research found that 41 binds covalently Int. J. Mol. Sci. 2021, 22, 2124 to the AF‐1 region of the AR NTD and blocks the proliferation of prostate cancer cells12 ofthat 20 are dependent on functional AR, and many analogs have been prepared and examined [76].

4.5. Propanamide AF‐1 inhibitors 4.5. Propanamide AF-1 Inhibitors A series of NTD‐binding compounds emerged from propanamide structure–activity relationshipA series studies. of NTD-binding Unlike the compounds structurally emerged similar SARM, from propanamide enobosarm ( structure–activity12), or antiandro‐ relationshipgen bicalutamide studies. (11 Unlike), these thewere structurally full‐antagonists similar with SARM, a tertiary enobosarm amine ( or12 ),nitrogenous or antiandro- B‐ genring bicalutamidethat were discovered (11), these to werebe SARDs. full-antagonists Leads inhibit with and a tertiary degrade amine a broad or nitrogenousscope of ex‐ B-ringpressed that ARs were to include discovered wtAR, to beall SARDs.point mutations Leads inhibit tested, and and degrade all AR‐SVs a broad tested. scope These of expressedmolecules ARsbind to to include the LBD wtAR, and NTD all point of AR, mutations but are believed tested, and to target all AR-SVs AR for tested. degradation These moleculesvia the NTD bind binding to the site LBD [67,77], and NTD suggesting of AR, but the are ability believed to overcome to target not AR only for degradation point muta‐ viation the resistance, NTD binding including site [enzalutamide67,77], suggesting (3) [78] the resistance, ability to overcomebut also resistance not only conferred point muta- by tion resistance, including enzalutamide (3)[78] resistance, but also resistance conferred AR‐SV, which is pan‐resistant among FDA‐approved agents. The initial SARD in this se‐ by AR-SV, which is pan-resistant among FDA-approved agents. The initial SARD in this ries, UT‐69 (43) (78 nM LBD binding; 48 nM inhibition of wtAR), demonstrated poor met‐ series, UT-69 (43) (78 nM LBD binding; 48 nM inhibition of wtAR), demonstrated poor abolic stability due to de‐methylation of the tertiary amine and hydroxylation of the biaryl metabolic stability due to de-methylation of the tertiary amine and hydroxylation of the B‐ring. biaryl B-ring.

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TheseThese metabolic liabilities liabilities were were eliminated eliminated by by cyclicizing cyclicizing the the amine amine into into a series a series of ofindoles indoles and and indolines indolines [78], [78 exemplified], exemplified by UT by‐ UT-155155 (35) ( (26735) (267 nM nMLBD LBD binding; binding; 85 nM 85 inhi nM‐ inhibitionbition of wtAR). of wtAR). Despite Despite having having reduced reduced AR inhibitory AR inhibitory potency, potency, 35 was35 foundwas to found possess to possessimproved improved pharmacokinetic pharmacokinetic properties properties such that such in vivo that activityin vivo wasactivity observed. was observed. Subcuta‐ Subcutaneouslyneously administered administered 35 reduced35 reduced tumor tumorweights weights and degraded and degraded full‐length full-length AR and AR andAR‐ AR-SVSV (AR (AR-V7)‐V7) in 22RV1 in 22RV1 cells, cells, unlike unlike 3, and3, and further further suppressed suppressed PSA PSA in LNCaP in LNCaP (T877A) (T877A) tu‐ tumorsmors [67]. [67 ]. BiophysicalBiophysical interactioninteraction experimentsexperiments usingusing NTD-onlyNTD‐only peptidespeptides co-incubatedco‐incubated withwith thesethese SARDsSARDs andand monitoredmonitored byby NMR,NMR, fluorescencefluorescence polarization,polarization, and the likelike [[67,77,78],67,77,78], havehave providedprovided consistentconsistent evidence that these compounds bind to an NTD binding site. Unfortunately,Unfortunately, thethe tertiarytertiary amine,amine, indole,indole, andand indolineindoline templatestemplates publishedpublished thusthus farfar hadhad lessless thanthan exemplaryexemplary pharmacokinetics.pharmacokinetics. InIn orderorder to to improve improve oral oral bioavailability bioavailability and and reveal reveal the the pharmacodynamics pharmacodynamics possibilities possibili‐ accessibleties accessible with with an NTD-directed an NTD‐directed SARD, SARD, the same the same research research group group developed developed and reported and re‐ theported second the generationsecond generation of these of SARDs, these SARDs, which nearly which maintained nearly maintained potency butpotency dramatically but dra‐ improvedmatically improvedin vivo efficacies in vivo [ 77efficacies]. A series [77]. of pyrazol-1-yl-propanamideA series of pyrazol‐1‐yl‐propanamide compounds com was‐ preparedpounds was by introducingprepared by the introducing pyrazole moiety the pyrazole as the moiety B-ring in as the the common B‐ring in A-ring–linkage– the common A‐ B-ringring–linkage–B nonsteroidal‐ring antiandrogens nonsteroidal generalantiandrogens pharmacophore. general pharmacophore. The pyrazoles are The exemplified pyrazoles byare UT-34 exemplified (45) (low by affinity UT‐34 for(45 LBD) (low (>10 affinityµM in for purified LBD (>10 LBD); μM 199 in nM purified inhibition LBD); of 199 wtAR), nM whichinhibition was of the wtAR), first well-characterized which was the first orally well‐characterized bioavailable SARDorally bioavailable from this group SARD [77 from,79]. Unlikethis group previous [77,79]. SARDs, Unlike45 previoushas low binding SARDs, affinity 45 has tolow LBD binding but maintained affinity to AF-1 LBD binding but main as‐ supportedtained AF‐ by1 binding steady-state as supported fluorescence by steady emission‐state spectra. fluorescence Like previous emission SARDs, spectra.45 Likerequires pre‐ thevious tau-5 SARDs, region 45 of requires AF-1 for the promoting tau‐5 region AR of degradation AF‐1 for promoting through the AR ubiquitin degradation proteasome through pathwaythe ubiquitin to degrade proteasome both AR-FLpathway and to AR-SVs degrade [ 77both]. AR‐FL and AR‐SVs [77]. SARSAR studiesstudies of of these these pyrazol-1-yl-propanamides pyrazol‐1‐yl‐propanamides successfully successfully improved improved potency potency while retainingwhile retaining oral bioavailability. oral bioavailability. NTD SARDs NTD SARDs were screened were screenedin vitro infor vitro high for potency high potency inhibi-

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Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEWinhibition of wtAR (IC50 in the low nanomolar range, preferably below 50 nM) and15 high of 21 efficacytion of wtAR (>70%) (IC degradationin the low of nanomolar both AR‐FL range, and preferably AR SV. Similar below to 50 the nM) indoles and high discussed efficacy inhibition of wtAR50 (IC50 in the low nanomolar range, preferably below 50 nM) and high above,efficacy(>70%) these degradation(>70%) studies degradation demonstrated of both of AR-FL both that AR and ‐theFL AR strongerand SV. AR Similar SV.the Similarelectron to the indolesto‐withdrawing the indoles discussed discussedmoiety above, is oninhibitionthese the studiespyrazole of wtAR demonstrated ring, (IC the50 in more the that lowpotent the nanomolar stronger the AR inhibitory the range, electron-withdrawing preferably activity, belowwith the 50 moiety potencynM) and is onorder high the above, these studies demonstrated that the stronger the electron‐withdrawing moiety is2 inhibitionefficacypyrazole2 (>70%) ring, of wtAR the degradation more (IC50 potentin the of low theboth ARnanomolar 2AR inhibitory‐FL and range, AR activity, SV. preferably Similar with2 the tobelow potency the indoles 50 ordernM) discussed2and of 46 high (R ofon 46 the (R pyrazole = NO2; IC ring,50 of the36 nM)more > 47potent (R = theCN; AR 45 inhibitorynM) > 48 ( Ractivity, = CF3; 71with nM) the > 49potency (R = Cl; order 136 above, these2 studies demonstrated2 that the stronger the2 electron‐withdrawing2 moiety is nM)efficacy= NO > 245; 2(>70%) IC (R50 =of F; degradation 36 199 nM) nM), > 47 but of (R SARD both= CN; AR2 efficacy 45‐FL nM) and did > AR 48not (SV.R necessary =Similar2 CF3; 71 correlateto nM) the indoles > 49with (R thesediscussed2= Cl; AR 136‐ onof 46the (R pyrazole = NO2 2; IC ring,50 of the 36 nM)more > potent47 (R = the CN; AR 45 inhibitorynM) > 48 ( Ractivity, = CF3; 71with nM) the > 49potency (R = Cl; order 136 FLabove,nM) inhibitory > 45these (R studies =activities F; 199 demonstrated nM), (IC50 but). Disubstitution SARD that efficacy the stronger didof the not pyrazole necessarythe electron ring correlate‐ withdrawingwas tolerated with these moiety without AR-FL is nM) > 45 (R2 = F; 199 nM), but SARD efficacy did not necessary correlate with these AR‐ of 46 (R2 = NO2; IC50 of 36 nM) > 47 (R2 = CN; 45 nM) > 48 (R2 = CF3; 71 nM) > 49 (R2 = Cl; 136 lossoninhibitory the of ARpyrazole inhibitory activities ring, (IC potencythe50 ).more Disubstitution and potent possibly the AR ofenhanced the inhibitory pyrazole bioavailability, activity, ring was with tolerated as theexemplified potency without orderby loss 50 FL inhibitory2 activities (IC50). Disubstitution of the pyrazole ring was tolerated without ofnM)of 46 AR >(R 45 inhibitory2 = ( NOR =2 ;F; IC potency19950 of nM), 36 nM) and but possibly> SARD 47 (R2 =efficacy enhanced CN; 45 did nM) bioavailability, not > 48 necessary (R2 = CF as3 ;correlate 71 exemplified nM) > with 49 (R by these2 = 50 Cl; (R AR 136=‐ (Rloss1 = of F, AR R2 =inhibitory Br). Research potency showed and possiblythat 50 retained enhanced high bioavailability, potency AR inhibitionas exemplified (IC50 byof 18450 nM)FLF, Rinhibitory 2and>= 45 Br). was(R2 Research = among activitiesF; 199 thenM), showed (IC highest but50). that DisubstitutionSARD efficacy50 retainedefficacy degraders ofdid high the not potency ofpyrazole necessarythe pyrazole AR ring inhibitioncorrelate was template tolerated with (IC with50 theseof without greater 84 AR nM)‐ (R1 = F, R2 = Br). Research showed that 50 retained high potency AR inhibition (IC50 of 84 FLlossand inhibitory of was AR among inhibitory activities the highest potency (IC50). efficacy andDisubstitution possibly degraders enhanced of of the the pyrazole bioavailability, pyrazole ring template was as exemplifiedtolerated with greater without by than 50 thannM) and70% wasdegradation among the of highestboth AR efficacy FL and degraders AR SV at of1 and the 10pyrazole μM. By template comparison, with greater45 was loss(R70%1 = of degradationF, AR R2 =inhibitory Br). Research of both potency ARshowed FLand and possiblythat AR 50 SV retained enhanced at 1 and high 10bioavailability, µpotencyM. By comparison, AR asinhibition exemplified45 (ICwas50 by aof less 5084 athan less 70% potent degradation wtAR inhibitor of both (199 AR nM) FL andbut ARwas SV a full at 1efficacy and 10 μdegraderM. By comparison, of AR‐FL and 45 ARwas‐ (RnM)potent1 = andF, wtARR was2 = Br). among inhibitor Research the (199 highest showed nM) butefficacy that was 50 adegraders retained full efficacy high of the degrader potency pyrazole ofAR AR-FLtemplate inhibition and with AR-SV.(IC greater50 of We84 SV.a less We potent have wtARlisted theinhibitor compounds (199 nM) that but are was in clinical a full efficacy and preclinical degrader development of AR‐FL and in ARTa‐‐ nM)thanhave and70% listed was degradation the among compounds the of highestboth that AR areefficacy FL in and clinical degraders AR SV and at preclinical of1 andthe pyrazole10 μ developmentM. By template comparison, in with Table greater451. was bleSV. 1. We have listed the compounds that are in clinical and preclinical development in Ta‐ thana less 70% potent degradation wtAR inhibitor of both (199 AR nM) FL and but ARwas SV a full at 1 efficacy and 10 μdegraderM. By comparison, of AR‐FL and 45 ARwas‐ ble 1. TableTableaSV. less 1. WeSummary 1.potent Summaryhave wtARlisted of compounds of thecompoundsinhibitor compounds in (199 preclinical in preclinical nM) that but andare wasand clinicalin clinical clinicala full development. efficacy development.and preclinical degrader development of AR‐FL and in AR Ta‐ SV.ble 1.We have listed the compounds that are in clinical and preclinical development in Ta‐ Structure/Cmpd ID Table 1. Summary of compounds in preclinical and clinical development. ble 1. InIn Vitro Vitro Data Data InIn VivoVivo oror ClinicalClinical DataData (Target(Target Activity) Structure/Cmpd ID Table 1. Summary of compounds in preclinical and clinical development. In Vitro Data In Vivo or Clinical Data (Target Activity) Structure/Cmpd ID Table 1. Summary of compounds in preclinical and clinical development. In Vitro Data  In Ina phase Vivo Ior clinical Clinical trial Data in CRPC (Target Activity) • In a phase I clinical trial in CRPC Structure/Cmpd ID  Binds AR LBD patients, moderate antitumor effects were • Binds ARIn LBD Vitro Data  patients,In Ina phase Vivo moderate orI clinical Clinical antitumor trial Data in CRPC effects (Target Activity)  Downregulates AR seen as decreased PSA levels. Trial was  • DownregulatesBinds AR LBD AR patients,were moderate seen as decreasedantitumor PSAeffects levels. were discontinued. In a phase I clinical trial in CRPC  Downregulates AR seen asTrial decreased was discontinued. PSA levels. Trial was  Binds AR LBD patients, moderate antitumor effects were (LBD directed SARD) discontinued.In a phase I clinical trial in CRPC  Downregulates AR seen as decreased PSA levels. Trial was (LBD directed SARD)  Binds AR LBD patients, moderate antitumor effects were (LBD directed SARD) discontinued.  Downregulates AR seen as decreased PSA levels. Trial was (LBD directed SARD) discontinued. In a phase III clinical trial in AR‐  Acts as an LBD SARD V7‐positive mCRPC patients, poor oral (LBD directed SARD)  Acts as a CYP17A1 inhibitor  In a phase III clinical trial in AR‐  Acts as an LBD SARD b•ioavailabilityIn a phase contributed III clinical trialto poor in AR-V7- effi‐  • Acts as a an competitive LBD SARD antagonist V7‐positive mCRPC patients, poor oral  Acts as a CYP17A1 inhibitor cacy. positiveTrialIn a phasewas mCRPC discontinued. III clinical patients, trial poorin AR oral‐  • Acts as aan CYP17A1 LBD SARD inhibitor bioavailability contributed to poor effi‐  Acts as a competitive antagonist V7 ‐positivebioavailability mCRPC patients, contributed poor to oral poor  • Acts as a competitiveCYP17A1 inhibitor antagonist cacy. TrialIn a phasewas discontinued. III clinical trial in AR‐ (multiple includes LBD SARD)  Acts as an LBD SARD bioavailabilityefficacy. Trial contributed was discontinued. to poor effi‐  Acts as a competitive antagonist V7‐positive mCRPC patients, poor oral  Acts as a CYP17A1 inhibitor  (multiple includes LBD SARD) bcacy.ioavailability TrialIn a phasewas contributeddiscontinued. I clinical trial to poor of ARV110 effi‐  DegradedActs as a competitive AR in VCaP antagonist cells at 5 nM (structure not shown) in late‐stage (multiple includes LBD SARD) cacy. TrialIn a wasphase discontinued. I clinical trial of ARV110 (multiple includes LBD SARD) with 98% maximal effect mCRPC patients, PSA responses were  Degraded AR in VCaP cells at 5 nM (structure not shown) in late‐stage (multiple includes LBD SARD)  Degraded wtAR and multiple escape seen atIn high a phase dose Iincluding clinical trial 2 of of 5 ARV110escape with 98% maximal effect mCRPC• In a patients, phase I clinicalPSA responses trial of ARV110were mutants Degraded AR in VCaP cells at 5 nM mutants(structure and not 2 shown) of 15 with in late wtAR.‐stage Trial is  • Degraded ARwtAR in VCaPand multiple cells at 5escape nM seen at(structureIn high a phase dose not I includingclinical shown) trial 2 in of of late-stage 5 ARV110 escape (PROTAC) with 98% maximal effect ongoing.mCRPC patients, PSA responses were mutantswithDegraded 98% maximal AR in VCaP effect cells at 5 nM (structuremutantsmCRPC and not 2 shown) patients,of 15 with in late PSAwtAR.‐stage responses Trial is  • Degraded wtAR and multiple escape seen at high dose including 2 of 5 escape (PROTAC) with 98%Degraded maximal wtAR effect and multiple es- mCRPCongoing.were patients, seen at PSA high responses dose including were 2 mutants Degradedcape mutants wtAR and multiple escape seenmutants atof high 5 and escape dose 2 of mutants 15including with andwtAR. 2 2of of 5Trial 15escape with is (PROTAC) mutants mutantsongoing.wtAR. and Trial 2 of is15 ongoing. with wtAR. Trial is  Blocked PELP‐1 binding to the AF‐2 (PROTAC)(PROTAC) ongoing. N.R. binding site at 10 μM  Blocked PELP‐1 binding to the AF‐2 N.R. binding site at 10 μM  Blocked PELP‐1 binding to the AF‐2 (AF‐2 interaction inhibitor) N.R. b indingBlocked site at 10PELP μM‐1 binding to the AF‐2 • Blocked PELP-1 binding to the AF-2 N.R. (AF‐2 interaction inhibitor) binding site at 10 μM  Improved symptomsN.R. in spino‐ binding site at 10 µM bulbar muscular atrophy (SBMA) mouse (AF‐2 interaction inhibitor)  Improved symptoms in spino‐ model, including ameliorated neuronal (AF‐2 interaction inhibitor) bulbar muscular atrophy (SBMA) mouse  Binds to BF‐3 binding site as deter‐ loss, neurogenicImproved atrophy,symptoms and in testicularspino‐ model, including ameliorated neuronal (AF-2 interaction inhibitor) mined by X‐ray crystallography atrophy.b ulbar muscular atrophy (SBMA) mouse  Binds to BF‐3 binding site as deter‐ loss, neurogenicImproved atrophy,symptoms and in testicularspino‐ model, In including SBMA model, ameliorated increased neuronal NCoR mined by X‐ray crystallography batrophy.ulbar muscular atrophy (SBMA) mouse  Binds to BF‐3 binding site as deter‐ recruitmentloss, neurogenic to AF atrophy,‐2 binding and site. testicular model, In including SBMA model, ameliorated increased neuronal NCoR mined by X‐ray crystallography atrophy. No clinical data. Binds to BF‐3 binding site as deter‐ loss,recruitment neurogenic to AF atrophy,‐2 binding and site. testicular (BF‐3 inhibitor)  In SBMA model, increased NCoR mined by X‐ray crystallography atrophy. No clinical data. (BF‐3 inhibitor) recruitment In SBMA to AF model,‐2 binding increased site. NCoR  No clinical data. (BF‐3 inhibitor) recruitment to AF‐2 binding site.  No clinical data. (BF‐3 inhibitor)

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 15 of 21

inhibition of wtAR (IC50 in the low nanomolar range, preferably below 50 nM) and high efficacy (>70%) degradation of both AR‐FL and AR SV. Similar to the indoles discussed above, these studies demonstrated that the stronger the electron‐withdrawing moiety is on the pyrazole ring, the more potent the AR inhibitory activity, with the potency order of 46 (R2 = NO2; IC50 of 36 nM) > 47 (R2 = CN; 45 nM) > 48 (R2 = CF3; 71 nM) > 49 (R2 = Cl; 136 nM) > 45 (R2 = F; 199 nM), but SARD efficacy did not necessary correlate with these AR‐ FL inhibitory activities (IC50). Disubstitution of the pyrazole ring was tolerated without loss of AR inhibitory potency and possibly enhanced bioavailability, as exemplified by 50 (R1 = F, R2 = Br). Research showed that 50 retained high potency AR inhibition (IC50 of 84 nM) and was among the highest efficacy degraders of the pyrazole template with greater than 70% degradation of both AR FL and AR SV at 1 and 10 μM. By comparison, 45 was a less potent wtAR inhibitor (199 nM) but was a full efficacy degrader of AR‐FL and AR‐ SV. We have listed the compounds that are in clinical and preclinical development in Ta‐ ble 1.

Table 1. Summary of compounds in preclinical and clinical development.

Structure/Cmpd ID In Vitro Data In Vivo or Clinical Data (Target Activity)

 In a phase I clinical trial in CRPC  Binds AR LBD patients, moderate antitumor effects were  Downregulates AR seen as decreased PSA levels. Trial was discontinued.

(LBD directed SARD)

 In a phase III clinical trial in AR‐  Acts as an LBD SARD V7‐positive mCRPC patients, poor oral  Acts as a CYP17A1 inhibitor bioavailability contributed to poor effi‐  Acts as a competitive antagonist cacy. Trial was discontinued.

(multiple includes LBD SARD)  In a phase I clinical trial of ARV110  Degraded AR in VCaP cells at 5 nM (structure not shown) in late‐stage with 98% maximal effect mCRPC patients, PSA responses were  Degraded wtAR and multiple escape seen at high dose including 2 of 5 escape mutants mutants and 2 of 15 with wtAR. Trial is (PROTAC) ongoing. Int. J. Mol. Sci. 2021, 22, 2124 15 of 20

 Blocked PELP‐1 binding to the AF‐2 Table 1. Cont. N.R. binding site at 10 μM Structure/Cmpd ID In Vitro Data In Vivo or Clinical Data (AF‐2(Target interaction Activity) inhibitor)

 Improved symptoms in spino‐ • Improved symptoms in spinobulbar bulbar muscular atrophy (SBMA) mouse muscular atrophy (SBMA) mouse model, including ameliorated neuronal model, including ameliorated neu-  • Binds to BF BF-3‐3 binding binding site site as as deter deter-‐ loss, neurogenicronal loss, neurogenic atrophy, and atrophy, testicular and minedmined by X‐ray by crystallography X-ray crystallography atrophy.testicular atrophy.  Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW • InIn SBMA SBMA model, model, increased increased NCoR NCoR16 of re- 21 recruitmentcruitment to AF to‐ AF-22 binding binding site. site. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 21 • NoNo clinical clinical data. data. Int. J. Mol. Sci. 2021(BF, 22‐3, inhibitor)x FOR PEER REVIEW 16 of 21 Int. J. Mol. Sci. 2021(BF-3, 22, inhibitor)x FOR PEER REVIEW 16 of 21  Suppressed PSA in LNCaP cells com‐ • parably Suppressed to enzalutamide PSAPSA in in ( LNCaP 3LNCaP) cells cells com- com‐ parably to enzalutamide (3) parably Inhibited to enzalutamide transcriptional (3) activation • InhibitedSuppressed transcriptional PSA in LNCaP activation cells com‐ comparably to 3 parably comparablySuppressedInhibited to enzalutamide transcriptional PSA to 3 in ( 3LNCaP) activation cells com ‐ comparably Unlike to 3 ,3 inhibition was abrogated by parably • UnlikeInhibited to enzalutamide 3, transcriptional inhibition (3) was activation abrogated No clinical data. DBDcomparably mutationsUnlike to 3 ,3 inhibition was abrogated by No clinical data.  byInhibited DBD mutations transcriptional activation No clinical data. comparablyDBD • mutationsSuppressedUnlike to 3 ,3 inhibition androgen androgen-dependent was‐dependent abrogated by No clinical data. genesDBD mutations ingenesUnlikeSuppressed CRPC in 3, CRPC model inhibition androgen model cell waslines‐ celldependent abrogated such lines as such by No clinical data. (DBD inhibitor) DBDLNCaP,genes mutations inasSuppressed C4CRPC LNCaP,‐2, AD1, model C4-2, androgen MR49F, cell AD1, lines ‐22rv1, MR49F,dependent such and 22rv1,as D567 and D567 (DBD inhibitor) genesLNCaP, inSuppressed C4CRPC‐2, AD1, model androgen MR49F, cell lines ‐22rv1,dependent such and as D567

(DBD inhibitor) genesLNCaP, in C4CRPC‐2, AD1, model MR49F, cell lines 22rv1, such and as D567

(DBD inhibitor) LNCaP, C4‐2, AD1, MR49F, 22rv1, and D567  For 38: in VCaP tumors, sup‐  For 38: irreversibly bound to NTD, pressed For AR 38:‐V7 in‐ dependentVCaP tumors, genes. sup ‐ • For 38: in VCaP tumors, suppressed b indingFor is 38noncompetitive: irreversibly bound to NTD, pressed For AR 3438:‐V7: in‐ dependentaVCaP phase tumors, I clinical genes. sup trial ‐ in AR-V7-dependent genes. b •indingFor is 38 38:noncompetitive: inhibited irreversibly androgen bound‐dependent to NTD, patients For that 34 :previously in a phase failedI clinical enzalutam trial in ‐  For 38: irreversibly bound to NTD, pressed• ForFor AR 34: 38:‐V7 inin‐ dependent aVCaP phase tumors, I clinical genes. sup trial‐ in genes inbinding cells with is noncompetitive only AR‐SV (v567es; i.e., ide or abiraterone treatment, <30% de‐ b indingFor is 38noncompetitive: irreversiblyinhibited androgen bound to‐dependent NTD, pressedpatients patientsFor ARthat 34‐V7 :previously in that‐dependent a phase previously failedI clinical genes. failed enzalutam trial enza- in ‐ • For 38: inhibited androgen- bnogenes inding AR in ForFL) iscells 38noncompetitive: withinhibited only androgenAR ‐SV (v567es;‐dependent i.e., creasedidepatients orlutamide Forabiraterone PSAthat 34 :atpreviously in orvery a phase abirateronetreatment, large Ifailed clinicaldoses <30% enzalutam treatment, up trial deto ‐in3.6 ‐ dependent genes in cells with  nogenes AR inFor FL) cells 38 : inhibitedwith only androgen AR‐SV (v567es;‐dependent i.e., patientsg.idecreased or<30% abiraterone PSAthat decreased previouslyat very treatment, large PSA failed doses at <30% veryenzalutam up deto large ‐3.6 ‐ only AR-SV (v567es; i.e., no AR FL) genes no AR in FL) cells with only AR‐SV (v567es; i.e., ideg.creased ordoses abirateroneTrial PSA updiscontinued. at to very 3.6 treatment, large g. doses <30% up de to‐ 3.6 (AF‐1 (Tau‐5) inhibitor) no AR FL) creasedg.• TrialTrial PSA discontinued. discontinued. at very large doses up to 3.6  Inhibited wtAR transcriptional activa‐ (AF‐1 (Tau‐5) inhibitor) g. Trial discontinued. tion (85Inhibited nM) wtAR transcriptional activa‐ (AF(AF-1‐1 (Tau (Tau-5)‐5) inhibitor)inhibitor)  TrialSQ administration discontinued. decreased tumor tion (85Reversibly nM) bound to purified LBD (AF‐1 (Tau‐5) inhibitor)  Inhibited wtAR transcriptional activa‐weights and degraded AR FL and AR‐V7 • Inhibited wtAR transcriptional acti-  SQ administration decreased tumor (267tion (85nM)InhibitedReversibly nM) wtAR bound transcriptional to purified LBD activa ‐ vation (85 nM) intratumor.weights SQ and administration degraded AR decreased FL and AR tumor‐V7 tion(267 (85nM)CompletelyReversibly nM) bound degraded to purified AR FL andLBD AR ‐ • SQ administration decreased tumor • Reversibly bound to purified LBD weightsintratumor. SQNo and administrationoral degraded bioavailability. AR decreased FL and AR tumor‐V7 V7(267 (100%/100%) nM)ReversiblyCompletely bound degraded to purified AR FL andLBD AR ‐ weights and degraded AR FL and (AF‐1 (Tau‐1) inhibitor) (267 nM) weightsintratumor. No and clinicaloral degraded bioavailability. data. AR FL and AR‐V7 V7 (100%/100%)NTDCompletely binding degraded via multiple AR FLbiophysical and AR‐ AR-V7 intratumor. (267• nM)Completely degraded AR FL and  No clinical data. (AF‐1 (Tau‐1) inhibitor) measurements intratumor.• NoNo oral oral bioavailability. bioavailability. V7 (100%/100%)AR-V7CompletelyNTD binding (100%/100%) degraded via multiple AR FLbiophysical and AR‐ • NoNo clinical oralclinical bioavailability. data. data. (AF‐1 (Tau‐1) inhibitor) V7measurements • (100%/100%)NTDInhibited binding wtAR via transcriptional multiple biophysi-biophysical activa‐  No clinical data. (AF(AF-1‐1 (Tau (Tau-1)‐1) inhibitor) tionmeasurements (199calNTDInhibited nM) measurements binding wtAR via transcriptional multiple biophysical activa‐  Unprecedented full regression with measurementstion (199WeakInhibited nM) reversible wtAR transcriptionalpurified LBD binding activa‐ PO administrationUnprecedented in intact full regression rats with with (>10tion μ(199M)InhibitedWeak nM) reversible wtAR transcriptional purified LBD binding activa‐ MR49FPO administrationUnprecedented LNCaP and in enzalutamide intact full regression rats with‐re‐ with • Inhibited wtAR transcriptional acti- tion(>10 μ(199M)CompletelyWeak nM) reversible degraded purified AR LBD FL and binding AR‐  sistantMR49FPO administrationUnprecedented VCaP LNCaP xenografts. and in enzalutamide intact full regression rats with‐re‐ with vation (199 nM) • Unprecedented full regression with V7(>10 (100%/100%) μM)WeakCompletely reversible degraded purified AR LBD FL and binding AR‐ POsistantMR49F administrationNo VCaP LNCaP clinical xenografts. and data. in enzalutamide intact rats with‐re‐ • Weak reversible purified LBD bind- PO administration in intact rats with (AF‐1 (Tau‐1) inhibitor) V7 (100%/100%)NTDCompletely binding degraded AR FL and AR‐ sistant No VCaP clinical xenografts. data. (>10 μM)ing (>10 µM) MR49FMR49F LNCaP LNCaP and enzalutamide and enzalutamide-‐re‐ (AF‐1 (Tau‐1) inhibitor)  NTD binding V7 • (100%/100%)Completely degraded degraded AR AR FL FL and and AR‐ sistant resistant NoVCaP clinical xenografts. VCaP data. xenografts.  80% tumor growth inhibition with (AF‐1 (Tau‐1) inhibitor) V7 (100%/100%)AR-V7InhibitedNTD binding (100%/100%) wtAR transcriptional activa‐• NoNo clinical clinical data. data. PO administration80% tumor growth in intact inhibition rats with with xen‐ (AF‐1 (Tau‐1) inhibitor) tion • (84NTDInhibited nM) binding wtAR transcriptional activa‐ ograftsPO administration80% derived tumor from growth in theintact enzalutamide inhibition rats with with xen‐re ‐‐ (AF-1 (Tau-1) inhibitor) tion (84HighInhibited nM) efficacy wtAR degradation transcriptional of AR activa‐FL ‐ sistantograftsPO administration80% (Enzderived tumor‐R) VCaP from growth in thecellintact enzalutamide line.inhibition rats with with xen‐re ‐‐ andtion AR(84InhibitedHigh ‐nM)V7 (70%/70%)efficacy wtAR degradation transcriptional of AR activa‐FL ‐ POsistantografts administrationNo (Enzderived clinical‐R) VCaP from data. in theintactcell enzalutamideline. rats with xen‐re‐‐ tionand AR(84High ‐nM)V7 (70%/70%)efficacy degradation of AR‐FL (AF‐1 (Tau‐1) inhibitor) ograftssistant No (Enzderived clinical‐R) VCaPfrom data. thecell enzalutamide line. ‐re‐ and ARHigh‐V7 (70%/70%)efficacy degradation of AR‐FL (AF‐1 (Tau‐1) inhibitor) sistant No(Enz clinical‐R) VCaP data. cell line. 5. Future Antiandrogenand AR‐V7 (70%/70%) Design and Screening Paradigms (AF‐1 (Tau‐1) inhibitor)  No clinical data. (AF‐1 (Tau‐1) inhibitor)5. FutureThus Antiandrogen far, all approved Design antiandrogens and Screening are competitive Paradigms HBP ligands that function by denying5. FutureThus access Antiandrogen far, all of approved endogenous Design antiandrogens androgens and Screening areto the competitive Paradigms HBP pocket. HBP As ligands discussed that functionabove, this by 5.approachdenying FutureThus access Antiandrogen seemsfar, all oflimited approved endogenous inDesign view antiandrogens androgensofand the Screening extreme areto the complexitycompetitive Paradigms HBP pocket. and HBP highly As ligands discussed regulated that functionabove, nature this byof ARdenyingapproach biologyThus access seemsfar, and all ofthelimited approved endogenous known in view resistanceantiandrogens androgensof the mechanismsextreme areto the competitivecomplexity HBP that thesepocket. and HBP agents highly As ligands discussed elicit. regulated that AR function above,agonist nature this acby of‐ denyingtivityARapproach biology requires access seems and a of functionalthelimited endogenous known in viewLBD resistance androgensofto thebind mechanismsextreme agonist to the complexity and HBP that induce pocket.these and the agents highlyN/CAs discussed globalelicit. regulated ARconformation, above,agonist nature this ac of‐ approachhomodimerize,tivityAR biology requires seems and a andfunctionalthelimited knowntranslocate in view LBDresistance ofto thebindthe mechanismsextreme nucleus agonist complexity toand recruit that induce these coregulatory and the agents highlyN/C globalelicit. regulated proteins ARconformation, agonist natureto either ac of‐ ARAFtivityhomodimerize,‐ 1biology orrequires AF ‐and2, anda andthefunctional this knowntranslocate is further LBDresistance toto modulated bindthe mechanisms nucleus agonist by toand growth recruit that induce these regulatory coregulatory the agents N/C kinaseglobalelicit. proteins AR conformation,signaling agonist to either cas ac‐ tivitycades.homodimerize,AF‐1 orrequires The AF complexity‐2, aand andfunctional this translocate providesis further LBD totomultiple modulated bindthe nucleus agonist points by toand togrowth recruit interfere induce regulatory coregulatory the with N/C AR globalkinase activity proteins conformation,signaling without to either cas re‐ homodimerize,sortingcades.AF‐1 or The to AF blocking complexity‐2, and and this HBPtranslocate providesis binding, further tomultiple modulated andthe nucleusearly points attempts by to togrowth recruit interfere to regulatory explorecoregulatory with nonAR kinase ‐activitycompetitive proteins signaling without to directeither cas re‐ AFactingsortingcades.‐1 or ARThe toAF blocking inhibitorscomplexity‐2, and this HBP that providesis binding,bindfurther to multiple modulated noveland early sites points attemptsofby actiontogrowth interfere toare regulatoryexplore discussed with nonAR kinase above;‐activitycompetitive signaling however, without direct cas reno‐ cades.nonactingsorting‐competitive TheAR to blockinginhibitorscomplexity antagonist HBP that provides binding,bind has to been multiple noveland successfully early sites points attemptsof actionto trialed. interfere toare Further,explore discussed with nonARsome above;‐activitycompetitive LBD (competitivehowever, without direct reno‐ sortingactingnon‐competitive AR to blockinginhibitors antagonist HBP that binding,bind has to been noveland successfully early sites attemptsof action trialed. toare exploreFurther, discussed non some ‐above;competitive LBD however,(competitive direct no‐ actingnon‐competitive AR inhibitors antagonist that bind has to been novel successfully sites of action trialed. are Further, discussed some above; LBD however,(competitive no non‐competitive antagonist has been successfully trialed. Further, some LBD (competitive

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 16 of 21

 Suppressed PSA in LNCaP cells com‐ parably to enzalutamide (3)  Inhibited transcriptional activation comparably to 3  Unlike 3, inhibition was abrogated by No clinical data. DBD mutations  Suppressed androgen‐dependent genes in CRPC model cell lines such as (DBD inhibitor) LNCaP, C4‐2, AD1, MR49F, 22rv1, and D567

 For 38: in VCaP tumors, sup‐  For 38: irreversibly bound to NTD, pressed AR‐V7‐dependent genes. binding is noncompetitive  For 34: in a phase I clinical trial in  For 38: inhibited androgen‐dependent patients that previously failed enzalutam‐ genes in cells with only AR‐SV (v567es; i.e., ide or abiraterone treatment, <30% de‐ no AR FL) creased PSA at very large doses up to 3.6 g.  Trial discontinued. (AF‐1 (Tau‐5) inhibitor)  Inhibited wtAR transcriptional activa‐ tion (85 nM)  SQ administration decreased tumor  Reversibly bound to purified LBD weights and degraded AR FL and AR‐V7 (267 nM) intratumor.  Completely degraded AR FL and AR‐  No oral bioavailability. V7 (100%/100%)  No clinical data. (AF‐1 (Tau‐1) inhibitor)  NTD binding via multiple biophysical measurements Int. J. Mol. Sci. 2021, 22, 2124 16 of 20  Inhibited wtAR transcriptional activa‐ tion (199 nM)  Unprecedented full regression with  Weak reversible purified LBD binding PO administration in intact rats with (>10 μM) Table 1. Cont. MR49F LNCaP and enzalutamide‐re‐  Completely degraded AR FL and AR‐ sistant VCaP xenografts. Structure/Cmpd ID V7 (100%/100%)In Vitro Data No In clinical Vivo or data. Clinical Data (AF(Target‐1 (Tau‐ Activity)1) inhibitor)  NTD binding

• 80%80% tumor tumor growthgrowth inhibition with  Inhibited wtAR transcriptional activa‐ • Inhibited wtAR transcriptional acti- PO administrationPO administration in intact in rats intact with ratsxen‐ tion (84 nM) vation (84 nM) ograftswith derived xenografts from the derived enzalutamide from the‐re‐  High efficacy degradation of AR‐FL • High efficacy degradation of AR-FL sistantenzalutamide-resistant (Enz‐R) VCaP cell line. (Enz-R) and AR‐V7 (70%/70%) and AR-V7 (70%/70%)  VCaPNo clinical cell line. data. • No clinical data. (AF‐1 (Tau‐1) inhibitor) (AF-1 (Tau-1) inhibitor) 5. Future Antiandrogen Design and Screening Paradigms Thus far, all approved antiandrogens are competitive HBP ligands that function by denying5. Future access Antiandrogen of endogenous Design androgens and Screening to the Paradigms HBP pocket. As discussed above, this approachThus seems far, all limited approved in view antiandrogens of the extreme are complexity competitive and HBP highly ligands regulated that function nature byof ARdenying biology access and the of endogenous known resistance androgens mechanisms to the HBP that these pocket. agents As discussed elicit. AR above,agonist this ac‐ tivityapproach requires seems a functional limited in LBD view to of bind the extremeagonist and complexity induce the and N/C highly global regulated conformation, nature homodimerize,of AR biology and and the translocate known resistance to the nucleus mechanisms to recruit that coregulatory these agents proteins elicit. AR to agonist either AFactivity‐1 or requiresAF‐2, and a functional this is further LBD tomodulated bind agonist by growth and induce regulatory the N/C kinase global signaling conformation, cas‐ cades.homodimerize, The complexity and translocate provides multiple to the nucleus points toto recruitinterfere coregulatory with AR activity proteins without to either re‐ sortingAF-1 or to AF-2, blocking and this HBP is further binding, modulated and early by attempts growth regulatoryto explore kinase non‐competitive signaling cascades. direct‐ actingThe complexity AR inhibitors provides that bind multiple to novel points sites to interfereof action withare discussed AR activity above; without however, resorting no nonto blocking‐competitive HBP antagonist binding, andhas been early successfully attempts to trialed. explore Further, non-competitive some LBD (competitive direct-acting AR inhibitors that bind to novel sites of action are discussed above; however, no non- competitive antagonist has been successfully trialed. Further, some LBD (competitive ligands) or NTD-binding AR ligands also have the ability to degrade AR-FL or AR-SV, providing additional advantages in AR axis suppression over traditional canonical antian- drogens. The field of noncanonical inhibitors and SARDs have exerted AR antagonism profiles commensurate in scope to the function of the site blocked [80], but are thus far limited by binding affinity and/or absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. The molecular structure details of full-length AR in its global agonist and antagonist conformations are not available, and AR biology, in many ways, is still poorly understood. This complexity and still emerging molecular basis of AR biology presents an opportunity for medicinal chemists willing to target nonconventional binding sites. In conclusion, the field of AR-targeted therapeutics to treat advanced PCa is entering a very exciting decade and the patients will see more mechanistically advanced drugs in the market that will provide them extended benefits.

Funding: This work was supported by a research grant from National Cancer Institute (NCI) 1R01CA229164 to RN. Conflicts of Interest: R.N. is a consultant of Oncternal, Inc.

Abbreviations ADT: androgen deprivation therapy; ADMET: absorption, distribution, metabolism, excretion, and toxicity; AF-1: activating function-1; AF-2: activating function-2; AR: androgen receptor; AR-FL: full-length androgen receptor; ARE: androgen responsive element; AR-SV: splice variant androgen receptor; BF-3: binding function-3; CRBN: cereblon; CRPC: castration-resistant prostate cancer; CYP17A1: cytochrome P450 17A1, also called steroid 17α-hydroxylase and 17,20-lyase; DBD: DNA binding domain; DES: diethylstilbestrol; DHT: 5α-dihydrotestosterone; DNA: deoxyribonucleic acid; FL: full-length; GR: glucocorticoid receptor; FDA: Food and Drug Administration; HBP: hor- mone binding pocket; LBD: ligand binding domain; mCRPC: metastatic castration-resistant prostate cancer; MDVR: enzalutamide (MDV3100)-resistant; mtAR: mutant androgen receptor; nmCRPC: nonmetastatic castration-resistant prostate cancer; NMR: nuclear magnetic resonance; NCoR: nu- Int. J. Mol. Sci. 2021, 22, 2124 17 of 20

clear receptor corepressor; NTD: N-terminal domain; OS: overall survival; PCa: prostate cancer; PDB: Protein Database; PEG: polyethylene glycol; PELP-1: proline-, glutamic acid-, and leucine-rich peptide-1; PK: pharmacokinetic; PR: progesterone receptor; PROTAC: proteolysis-targeting chimera; PSA: prostate-specific antigen; SAR: structure–activity relationship; SARD: selective androgen recep- tor degrader (or downregulator); SARM: selective androgen receptor modulator; SBMA: spinobulbar muscular atrophy; TAU: transcription activation unit; Tau-1: transcription activation unit-1; Tau-2: transcription activation unit-2; TMPRSS2–ERG: a fusion of TMPRSS2 (transmembrane protease, serine 2) to ETS-related gene (ERG); VHL: Von Hippel–Lindau; Ub: ubiquitin; wtAR: wild-type androgen receptor; C4-2: prostate cancer cell line derived from LNCaP; C4-2B: prostate cancer cell line derived from LNCaP; CWR22Rv1 (22Rv1): AR- and AR splice variant-positive prostate cancer cells; DU145: AR-negative prostate cancer cells; LNCaP: lymph node metastasis AR-positive prostate cancer cell line; MR49C: enzalutamide-resistant LNCaP; MR49F: enzalutamide-resistant LNCaP; VCaP: AR-positive prostate cancer cell line.

References 1. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2020. CA Cancer J. Clin. 2020, 70, 7–30. [CrossRef] 2. Shah, R.B.; Mehra, R.; Chinnaiyan, A.M.; Shen, R.; Ghosh, D.; Zhou, M.; Macvicar, G.R.; Varambally, S.; Harwood, J.; Bismar, T.A.; et al. Androgen-independent prostate cancer is a heterogeneous group of diseases: Lessons from a rapid autopsy program. Cancer Res. 2004, 64, 9209–9216. [CrossRef] 3. Brinkmann, A.O.; Faber, P.W.; van Rooij, H.C.; Kuiper, G.G.; Ris, C.; Klaassen, P.; van der Korput, J.A.; Voorhorst, M.M.; van Laar, J.H.; Mulder, E.; et al. The human androgen receptor: Domain structure, genomic organization and regulation of expression. J. Steroid Biochem. 1989, 34, 307–310. [CrossRef] 4. Taylor, B.S.; Schultz, N.; Hieronymus, H.; Gopalan, A.; Xiao, Y.; Carver, B.S.; Arora, V.K.; Kaushik, P.; Cerami, E.; Reva, B.; et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 2010, 18, 11–22. [CrossRef][PubMed] 5. Huggins, C. Effect of Orchiectomy and Irradiation on Cancer of the Prostate. Ann. Surg. 1942, 115, 1192–1200. [CrossRef] [PubMed] 6. Bluemn, E.G.; Coleman, I.M.; Lucas, J.M.; Coleman, R.T.; Hernandez-Lopez, S.; Tharakan, R.; Bianchi-Frias, D.; Dumpit, R.F.; Kaipainen, A.; Corella, A.N.; et al. Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling. Cancer Cell 2017, 32, 474–489.e6. [CrossRef][PubMed] 7. Epstein, J.I.; Amin, M.B.; Beltran, H.; Lotan, T.L.; Mosquera, J.M.; Reuter, V.E.; Robinson, B.D.; Troncoso, P.; Rubin, M.A. Proposed morphologic classification of prostate cancer with neuroendocrine differentiation. Am. J. Surg. Pathol. 2014, 38, 756–767. [CrossRef][PubMed] 8. Wadosky, K.M.; Koochekpour, S. Androgen receptor splice variants and prostate cancer: From bench to bedside. Oncotarget 2017, 8, 18550–18576. [CrossRef][PubMed] 9. Brand, L.J.; Dehm, S.M. Androgen receptor gene rearrangements: New perspectives on prostate cancer progression. Curr. Drug Targets 2013, 14, 441–449. [CrossRef] 10. Xu, J.; Qiu, Y. Role of androgen receptor splice variants in prostate cancer metastasis. Asian J. Urol. 2016, 3, 177–184. [CrossRef] 11. Melnyk, J.E.; Steri, V.; Nguyen, H.G.; Hann, B.; Feng, F.Y.; Shokat, K.M. The splicing modulator sulfonamide indisulam reduces AR-V7 in prostate cancer cells. Bioorg. Med. Chem. 2020, 28, 115712. [CrossRef] 12. Armstrong, A.J.; Luo, J.; Nanus, D.M.; Giannakakou, P.; Szmulewitz, R.Z.; Danila, D.C.; Healy, P.; Anand, M.; Berry, W.R.; Zhang, T.; et al. Prospective Multicenter Study of Circulating Tumor Cell AR-V7 and Taxane Versus Hormonal Treatment Outcomes in Metastatic Castration-Resistant Prostate Cancer. Jco. Precis. Oncol. 2020, 4, 1285–1301. [CrossRef] 13. Liu, C.; Armstrong, C.; Zhu, Y.; Lou, W.; Gao, A.C. Niclosamide enhances abiraterone treatment via inhibition of androgen receptor variants in castration resistant prostate cancer. Oncotarget 2016, 7, 32210–32220. [CrossRef][PubMed] 14. Liu, C.; Lou, W.; Zhu, Y.; Nadiminty, N.; Schwartz, C.T.; Evans, C.P.; Gao, A.C. Niclosamide inhibits androgen receptor variants expression and overcomes enzalutamide resistance in castration-resistant prostate cancer. Clin. Cancer Res. 2014, 20, 3198–3210. [CrossRef][PubMed] 15. Mainwaring, W.I. The separation of androgen receptor and 5 alpha-reductase activities in subcellular fractions of rat prostate. Biochem. Biophys. Res. Commun. 1970, 40, 192–198. [CrossRef] 16. Sack, J.S.; Kish, K.F.; Wang, C.; Attar, R.M.; Kiefer, S.E.; An, Y.; Wu, G.Y.; Scheffler, J.E.; Salvati, M.E.; Krystek, S.R., Jr.; et al. Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone. Proc. Natl. Acad. Sci. USA 2001, 98, 4904–4909. [CrossRef] 17. Jacobo, E.; Schmidt, J.D.; Weinstein, S.H.; Flocks, R.H. Comparison of flutamide (SCH-13521) and diethylstilbestrol in untreated advanced prostatic cancer. Urology 1976, 8, 231–233. [CrossRef] 18. Furr, B.J.; Valcaccia, B.; Curry, B.; Woodburn, J.R.; Chesterson, G.; Tucker, H. ICI 176,334: A novel non-steroidal, peripherally selective antiandrogen. J. Endocrinol. 1987, 113, R7–R9. [CrossRef] Int. J. Mol. Sci. 2021, 22, 2124 18 of 20

19. Dalton, J.T.; Mukherjee, A.; Zhu, Z.; Kirkovsky, L.; Miller, D.D. Discovery of nonsteroidal androgens. Biochem. Biophys. Res. Commun. 1998, 244, 1–4. [CrossRef] 20. Yin, D.; Gao, W.; Kearbey, J.D.; Xu, H.; Chung, K.; He, Y.; Marhefka, C.A.; Veverka, K.A.; Miller, D.D.; Dalton, J.T. Pharmacody- namics of selective androgen receptor modulators. J. Pharm. Exp. 2003, 304, 1334–1340. [CrossRef] 21. Marhefka, C.A.; Gao, W.; Chung, K.; Kim, J.; He, Y.; Yin, D.; Bohl, C.; Dalton, J.T.; Miller, D.D. Design, synthesis, and biological characterization of metabolically stable selective androgen receptor modulators. J. Med. Chem. 2004, 47, 993–998. [CrossRef] 22. Solomon, Z.J.; Mirabal, J.R.; Mazur, D.J.; Kohn, T.P.; Lipshultz, L.I.; Pastuszak, A.W. Selective Androgen Receptor Modulators: Current Knowledge and Clinical Applications. Sex. Med. Rev. 2019, 7, 84–94. [CrossRef] 23. Hsu, C.L.; Liu, J.S.; Wu, P.L.; Guan, H.H.; Chen, Y.L.; Lin, A.C.; Ting, H.J.; Pang, S.T.; Yeh, S.D.; Ma, W.L.; et al. Identification of a new androgen receptor (AR) co-regulator BUD31 and related peptides to suppress wild-type and mutated AR-mediated prostate cancer growth via peptide screening and X-ray structure analysis. Mol. Oncol. 2014, 8, 1575–1587. [CrossRef][PubMed] 24. Aikawa, K.; Asano, M.; Ono, K.; Habuka, N.; Yano, J.; Wilson, K.; Fujita, H.; Kandori, H.; Hara, T.; Morimoto, M.; et al. Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl) benzonitrile derivative 2f as a clinical candidate. Bioorg. Med. Chem. 2017, 25, 3330–3349. [CrossRef] 25. Bohl, C.E.; Miller, D.D.; Chen, J.; Bell, C.E.; Dalton, J.T. Structural basis for accommodation of nonsteroidal ligands in the androgen receptor. J. Biol. Chem. 2005, 280, 37747–37754. [CrossRef][PubMed] 26. Saeed, A.; Vaught, G.M.; Gavardinas, K.; Matthews, D.; Green, J.E.; Losada, P.G.; Bullock, H.A.; Calvert, N.A.; Patel, N.J.; Sweetana, S.A.; et al. 2-Chloro-4-[[(1R,2R)-2-hydroxy-2-methyl-cyclopentyl]amino]-3-methyl-benzonitrile: A Transdermal Selective Androgen Receptor Modulator (SARM) for Muscle Atrophy. J. Med. Chem. 2016, 59, 750–755. [CrossRef][PubMed] 27. Zhang, Z.; Connolly, P.J.; Lim, H.K.; Pande, V.; Meerpoel, L.; Teleha, C.; Branch, J.R.; Ondrus, J.; Hickson, I.; Bush, T.; et al. Discovery of JNJ-63576253: A Clinical Stage Androgen Receptor Antagonist for F877L Mutant and Wild-Type Castration-Resistant Prostate Cancer (mCRPC). J. Med. Chem. 2021, 64, 909–924. [CrossRef][PubMed] 28. Ge, R.; Xu, X.; Xu, P.; Li, L.; Li, Z.; Bian, J. Degradation of Androgen Receptor through Small Molecules for Prostate Cancer. Curr. Cancer Drug Targets 2018, 18, 652–667. [CrossRef] 29. Wang, R.; Lin, W.; Lin, C.; Li, L.; Sun, Y.; Chang, C. ASC-J9((R)) suppresses castration resistant prostate cancer progression via degrading the enzalutamide-induced androgen receptor mutant AR-F876L. Cancer Lett. 2016, 379, 154–160. [CrossRef] 30. Omlin, A.; Jones, R.J.; van der Noll, R.; Satoh, T.; Niwakawa, M.; Smith, S.A.; Graham, J.; Ong, M.; Finkelman, R.D.; Schellens, J.H.; et al. AZD3514, an oral selective androgen receptor down-regulator in patients with castration-resistant prostate cancer—Results of two parallel first-in-human phase I studies. Investig. New Drugs 2015, 33, 679–690. [CrossRef][PubMed] 31. Latysheva, A.S.; Zolottsev, V.A.; Veselovsky, A.V.; Scherbakov, K.A.; Morozevich, G.E.; Pokrovsky, V.S.; Novikov, R.A.; Timofeev, V.P.; Tkachev, Y.V.; Misharin, A.Y. New steroidal oxazolines, benzoxazoles and benzimidazoles related to abiraterone and galeterone. Steroids 2020, 153, 108534. [CrossRef] 32. Njar, V.C.; Brodie, A.M. Discovery and development of Galeterone (TOK-001 or VN/124–1) for the treatment of all stages of prostate cancer. J. Med. Chem. 2015, 58, 2077–2087. [CrossRef][PubMed] 33. Alyamani, M.; Li, Z.; Berk, M.; Li, J.; Tang, J.; Upadhyay, S.; Auchus, R.J.; Sharifi, N. Steroidogenic Metabolism of Galeterone Reveals a Diversity of Biochemical Activities. Cell Chem. Biol. 2017, 24, 825–832.e6. [CrossRef] 34. Yu, Z.; Cai, C.; Gao, S.; Simon, N.I.; Shen, H.C.; Balk, S.P. Galeterone prevents androgen receptor binding to chromatin and enhances degradation of mutant androgen receptor. Clin. Cancer Res. 2014, 20, 4075–4085. [CrossRef] 35. Kwegyir-Afful, A.K.; Ramalingam, S.; Purushottamachar, P.; Ramamurthy, V.P.; Njar, V.C. Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo. Oncotarget 2015, 6, 27440–27460. [CrossRef] 36. Kwegyir-Afful, A.K.; Ramalingam, S.; Ramamurthy, V.P.; Purushottamachar, P.; Murigi, F.N.; Vasaitis, T.S.; Huang, W.; Kane, M.A.; Zhang, Y.; Ambulos, N.; et al. Galeterone and The Next Generation Galeterone Analogs, VNPP414 and VNPP433–3beta Exert Potent Therapeutic Effects in Castration-/Drug-Resistant Prostate Cancer Preclinical Models In Vitro and In Vivo. Cancers (Basel.) 2019, 11, 1637. [CrossRef][PubMed] 37. Sakamoto, K.M.; Kim, K.B.; Kumagai, A.; Mercurio, F.; Crews, C.M.; Deshaies, R.J. Protacs: Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 2001, 98, 8554–8559. [CrossRef][PubMed] 38. Schneekloth, A.R.; Pucheault, M.; Tae, H.S.; Crews, C.M. Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorg. Med. Chem. Lett. 2008, 18, 5904–5908. [CrossRef] 39. Lohbeck, J.; Miller, A.K. Practical synthesis of a phthalimide-based Cereblon ligand to enable PROTAC development. Bioorg. Med. Chem. Lett. 2016, 26, 5260–5262. [CrossRef] 40. Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang, C.Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; et al. Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer. J. Med. Chem. 2019, 62, 941–964. [CrossRef][PubMed] 41. Guo, C.; Linton, A.; Kephart, S.; Ornelas, M.; Pairish, M.; Gonzalez, J.; Greasley, S.; Nagata, A.; Burke, B.J.; Edwards, M.; et al. Discovery of aryloxy tetramethylcyclobutanes as novel androgen receptor antagonists. J. Med. Chem. 2011, 54, 7693–7704. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 2124 19 of 20

42. Zhao, L.; Han, X.; Lu, J.; McEachern, D.; Wang, S. A highly potent PROTAC androgen receptor (AR) degrader ARD-61 effectively inhibits AR-positive breast cancer cell growth in vitro and tumor growth in vivo. Neoplasia 2020, 22, 522–532. [CrossRef] 43. Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.; Hattori, T.; Koyama, R.; Sano, O.; Imaeda, Y.; Nara, H.; et al. Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands. J. Med. Chem. 2018, 61, 543–575. [CrossRef] 44. Scott, D.E.; Rooney, T.P.C.; Bayle, E.D.; Mirza, T.; Willems, H.M.G.; Clarke, J.H.; Andrews, S.P.; Skidmore, J. Systematic Investigation of the Permeability of Androgen Receptor PROTACs. ACS Med. Chem. Lett. 2020, 11, 1539–1547. [CrossRef] [PubMed] 45. Flanagan, J.J.; Neklesa, T.K. Targeting Nuclear Receptors with PROTAC degraders. Mol. Cell. Endocrinol. 2019, 493, 110452. [CrossRef] 46. Beretta, G.L.; Zaffaroni, N. Androgen Receptor-Directed Molecular Conjugates for Targeting Prostate Cancer. Front. Chem. 2019, 7, 369. [CrossRef] 47. Balbas, M.D.; Evans, M.J.; Hosfield, D.J.; Wongvipat, J.; Arora, V.K.; Watson, P.A.; Chen, Y.; Greene, G.L.; Shen, Y.; Sawyers, C.L. Overcoming mutation-based resistance to antiandrogens with rational drug design. Elife 2013, 2, e00499. [CrossRef] 48. Hara, T.; Miyazaki, J.; Araki, H.; Yamaoka, M.; Kanzaki, N.; Kusaka, M.; Miyamoto, M. Novel mutations of androgen receptor: A possible mechanism of bicalutamide withdrawal syndrome. Cancer Res. 2003, 63, 149–153. [PubMed] 49. Liu, H.; An, X.; Li, S.; Wang, Y.; Li, J.; Liu, H. Interaction mechanism exploration of R-bicalutamide/S-1 with WT/W741L AR using molecular dynamics simulations. Mol. Biosyst. 2015, 11, 3347–3354. [CrossRef] 50. Fenton, M.A.; Shuster, T.D.; Fertig, A.M.; Taplin, M.E.; Kolvenbag, G.; Bubley, G.J.; Balk, S.P. Functional characterization of mutant androgen receptors from androgen-independent prostate cancer. Clin. Cancer Res. 1997, 3, 1383–1388. 51. Tan, J.; Hamil, Y.S.K.G.; Gregory, C.W.; Zang, D.Y.; Sar, M.; Gumerlock, P.H.; White, R.W.D.; Pretlow, T.G.; Harris, S.E.; Wilson, E.M.; et al. Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. Mol. Endocrinol. 1997, 11, 450–459. [CrossRef] 52. Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. Nat. Med. 2000, 6, 703–706. [CrossRef] 53. Korpal, M.; Korn, J.M.; Gao, X.; Rakiec, D.P.; Ruddy, D.A.; Doshi, S.; Yuan, J.; Kovats, S.G.; Kim, S.; Cooke, V.G.; et al. An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). Cancer Discov. 2013, 3, 1030–1043. [CrossRef][PubMed] 54. Fujii, S.; Kagechika, H. Androgen receptor modulators: A review of recent patents and reports (2012–2018). Expert Opin. Pat. 2019, 29, 439–453. [CrossRef][PubMed] 55. Li, H.; Ban, F.; Dalal, K.; Leblanc, E.; Frewin, K.; Ma, D.; Adomat, H.; Rennie, P.S.; Cherkasov, A. Discovery of small-molecule inhibitors selectively targeting the DNA-binding domain of the human androgen receptor. J. Med. Chem. 2014, 57, 6458–6467. [CrossRef] 56. Nadal, M.; Prekovic, S.; Gallastegui, N.; Helsen, C.; Abella, M.; Zielinska, K.; Gay, M.; Vilaseca, M.; Taules, M.; Houtsmuller, A.B.; et al. Structure of the homodimeric androgen receptor ligand-binding domain. Nat. Commun. 2017, 8, 14388. [CrossRef] 57. Dalal, K.; Ban, F.; Li, H.; Morin, H.; Roshan-Moniri, M.; Tam, K.J.; Shepherd, A.; Sharma, A.; Peacock, J.; Carlson, M.L.; et al. Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer. Cancer Lett. 2018, 437, 35–43. [CrossRef][PubMed] 58. Munuganti, R.S.; Leblanc, E.; Axerio-Cilies, P.; Labriere, C.; Frewin, K.; Singh, K.; Hassona, M.D.; Lack, N.A.; Li, H.; Ban, F.; et al. Targeting the binding function 3 (BF3) site of the androgen receptor through virtual screening. 2. development of 2-((2-phenoxyethyl) thio)-1H-benzimidazole derivatives. J. Med. Chem. 2013, 56, 1136–1148. [CrossRef][PubMed] 59. Liu, Y.; Wu, M.; Wang, T.; Xie, Y.; Cui, X.; He, L.; He, Y.; Li, X.; Liu, M.; Hu, L.; et al. Structural Based Screening of Antiandrogen Targeting Activation Function-2 Binding Site. Front. Pharm. 2018, 9, 1419. [CrossRef][PubMed] 60. Lack, N.A.; Axerio-Cilies, P.; Tavassoli, P.; Han, F.Q.; Chan, K.H.; Feau, C.; LeBlanc, E.; Guns, E.T.; Guy, R.K.; Rennie, P.S.; et al. Targeting the binding function 3 (BF3) site of the human androgen receptor through virtual screening. J. Med. Chem. 2011, 54, 8563–8573. [CrossRef][PubMed] 61. Badders, N.M.; Korff, A.; Miranda, H.C.; Vuppala, P.K.; Smith, R.B.; Winborn, B.J.; Quemin, E.R.; Sopher, B.L.; Dearman, J.; Messing, J.; et al. Selective modulation of the androgen receptor AF2 domain rescues degeneration in spinal bulbar muscular atrophy. Nat. Med. 2018, 24, 427–437. [CrossRef][PubMed] 62. Dalal, K.; Roshan-Moniri, M.; Sharma, A.; Li, H.; Ban, F.; Hassona, M.D.; Hsing, M.; Singh, K.; LeBlanc, E.; Dehm, S.; et al. Selectively targeting the DNA-binding domain of the androgen receptor as a prospective therapy for prostate cancer. J. Biol. Chem. 2014, 289, 26417–26429. [CrossRef][PubMed] 63. Dalal, K.; Che, M.; Que, N.S.; Sharma, A.; Yang, R.; Lallous, N.; Borgmann, H.; Ozistanbullu, D.; Tse, R.; Ban, F.; et al. Bypassing Drug Resistance Mechanisms of Prostate Cancer with Small Molecules that Target Androgen Receptor-Chromatin Interactions. Mol. Cancer 2017, 16, 2281–2291. [CrossRef][PubMed] 64. Messner, E.A.; Steele, T.M.; Tsamouri, M.M.; Hejazi, N.; Gao, A.C.; Mudryj, M.; Ghosh, P.M. The Androgen Receptor in Prostate Cancer: Effect of Structure, Ligands and Spliced Variants on Therapy. Biomedicines 2020, 8, 422. [CrossRef] 65. Jenster, G.; van der Korput, H.A.; Trapman, J.; Brinkmann, A.O. Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J. Biol. Chem. 1995, 270, 7341–7346. [CrossRef] Int. J. Mol. Sci. 2021, 22, 2124 20 of 20

66. De Mol, E.; Fenwick, R.B.; Phang, C.T.; Buzon, V.; Szulc, E.; de la Fuente, A.; Escobedo, A.; Garcia, J.; Bertoncini, C.W.; Estebanez- Perpina, E.; et al. EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor. ACS Chem. Biol. 2016, 11, 2499–2505. [CrossRef] 67. Ponnusamy, S.; Coss, C.C.; Thiyagarajan, T.; Watts, K.; Hwang, D.J.; He, Y.; Selth, L.A.; McEwan, I.J.; Duke, C.B.; Pagadala, J.; et al. Novel Selective Agents for the Degradation of Androgen Receptor Variants to Treat Castration-Resistant Prostate Cancer. Cancer Res. 2017, 77, 6282–6298. [CrossRef] 68. Jenster, G.; van der Korput, H.A.; van Vroonhoven, C.; van der Kwast, T.H.; Trapman, J.; Brinkmann, A.O. Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol. Endocrinol. 1991, 5, 1396–1404. [CrossRef] 69. McEwan, I.J. Intrinsic disorder in the androgen receptor: Identification, characterisation and drugability. Mol. Biosyst. 2012, 8, 82–90. [CrossRef] 70. Andersen, R.J.; Mawji, N.R.; Wang, J.; Wang, G.; Haile, S.; Myung, J.K.; Watt, K.; Tam, T.; Yang, Y.C.; Banuelos, C.A.; et al. Regression of castrate-recurrent prostate cancer by a small-molecule inhibitor of the amino-terminus domain of the androgen receptor. Cancer Cell 2010, 17, 535–546. [CrossRef] 71. Myung, J.K.; Banuelos, C.A.; Fernandez, J.G.; Mawji, N.R.; Wang, J.; Tien, A.H.; Yang, Y.C.; Tavakoli, I.; Haile, S.; Watt, K.; et al. An androgen receptor N-terminal domain antagonist for treating prostate cancer. J. Clin. Investig. 2013, 123, 2948–2960. [CrossRef] [PubMed] 72. Hu, R.; Lu, C.; Mostaghel, E.A.; Yegnasubramanian, S.; Gurel, M.; Tannahill, C.; Edwards, J.; Isaacs, W.B.; Nelson, P.S.; Bluemn, E.; et al. Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer. Cancer Res. 2012, 72, 3457–3462. [CrossRef] 73. Hirayama, Y.; Tam, T.; Jian, K.; Andersen, R.J.; Sadar, M.D. Combination therapy with androgen receptor N-terminal domain antagonist EPI-7170 and enzalutamide yields synergistic activity in AR-V7-positive prostate cancer. Mol. Oncol. 2020, 14, 2455–2470. [CrossRef] 74. Banuelos, C.A.; Tavakoli, I.; Tien, A.H.; Caley, D.P.; Mawji, N.R.; Li, Z.; Wang, J.; Yang, Y.C.; Imamura, Y.; Yan, L.; et al. Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain. J. Biol. Chem. 2016, 291, 22231–22243. [CrossRef][PubMed] 75. Yan, L.; Banuelos, C.A.; Mawji, N.R.; Patrick, B.O.; Sadar, M.D.; Andersen, R.J. Structure-Activity Relationships for the Marine Natural Product Sintokamides: Androgen Receptor N-Terminus Antagonists of Interest for Treatment of Metastatic Castration- Resistant Prostate Cancer. J. Nat. Prod. 2020.[CrossRef] 76. Banuelos, C.A.; Lal, A.; Tien, A.H.; Shah, N.; Yang, Y.C.; Mawji, N.R.; Meimetis, L.G.; Park, J.; Kunzhong, J.; Andersen, R.J.; et al. Characterization of niphatenones that inhibit androgen receptor N-terminal domain. PLoS ONE 2014, 9, e107991. [CrossRef] 77. Ponnusamy, S.; He, Y.; Hwang, D.J.; Thiyagarajan, T.; Houtman, R.; Bocharova, V.; Sumpter, B.G.; Fernandez, E.; Johnson, D.L.; Du, Z.; et al. Orally-Bioavailable Androgen Receptor Degrader, A Potential Next-Generation Therapeutic for Enzalutamide-Resistant Prostate Cancer. Clin. Cancer Res. 2019, 25, 6764–6780. [CrossRef] 78. Hwang, D.J.; He, Y.; Ponnusamy, S.; Mohler, M.L.; Thiyagarajan, T.; McEwan, I.J.; Narayanan, R.; Miller, D.D. New Generation of Selective Androgen Receptor Degraders: Our Initial Design, Synthesis, and Biological Evaluation of New Compounds with Enzalutamide-Resistant Prostate Cancer Activity. J. Med. Chem. 2019, 62, 491–511. [CrossRef][PubMed] 79. He, Y.; Hwang, D.J.; Ponnusamy, S.; Thiyagarajan, T.; Mohler, M.L.; Narayanan, R.; Miller, D.D. Pyrazol-1-yl-propanamides as SARD and Pan-Antagonists for the Treatment of Enzalutamide-Resistant Prostate Cancer. J. Med. Chem. 2020, 63, 12642–12665. [CrossRef] 80. Haendler, B.; Cleve, A. Recent developments in antiandrogens and selective androgen receptor modulators. Mol. Cell. Endocrinol. 2012, 352, 79–91. [CrossRef]