PROTAC technology for prostate cancer treatment

Abstract

Prostate cancer (PrCa) is the most prevalent urogenital cancer in men, marked by uncontrolled cellular growth that leads to abnormal enlargement of the prostate gland. The metastatic spread of PrCa is the primary cause of mortality, causing cancer cells disseminate to distant sites, such as bones, the pelvis, and other organs. Key contributors to PrCa progression include genetic mutations, elevated androgen receptor (AR) expression, gene amplification, and the rise of AR splice variants. Although androgen deprivation therapy (ADT) remains the mainstay for early-stage PrCa treatment, its efficacy is rather temporary, as many cases advance to castration-resistant PrCa (CRPC), presenting a significant therapeutic hurdle. This review explores key biomarkers for PrCa and the latest therapeutic strategies for CRPC, with a particular focus on the innovative Proteolysis-targeting chimera (PROTAC) technology. This approach offers a novel means of degrading target proteins, and we discuss how PROTACs hold potential as effective strategies to combat resistance mechanisms in CRPC.

1. Introduction

Prostate cancer (PrCa) is characterized by uncontrolled cellular proliferation, resulting in abnormal growth of the prostate gland and is the most common cancer affecting the male urinary and reproductive systems [1]. The metastatic PrCa cells spreading to distant sites, including bones, the pelvis, lumbar vertebrae, bladder, rectum, and even the brain, are the primary driver of PrCa-related mortality [2]. Notably, nearly 48% of cancer cases in men are attributed to malignancies such as prostate, lung, bronchus, and colorectal cancers, with PrCa presenting about 27% of all diagnoses. In the United States alone, projections for 2024 indicated PrCa contribute to an estimated new cases around 299,010 (14.9% of all new cancer cases) and estimated deaths up to 35,250 from NIH [3].

Several factors contribute to PrCa progression, including genetic alterations, elevated AR levels, AR gene amplification, upregulation of the Cytochrome P450 17A1 (CYP17A1), and the development of AR variants [4]. Among these, AR splice variants (AR-Vs), particularly AR-V7 and AR-V9, lack the ligand-binding domain (LBD), rendering them resistant to therapies targeting this domain, such as enzalutamide and abiraterone. These variants remain active in promoting AR signaling, contributing to tumor growth and survival even in the presence of androgen deprivation or AR-targeted therapies [5, 6]. Notably, the standard treatment for localized PrCa involves ADT, achieved through surgical and medical castration. While ADT can induce remission for approximately two to three years, PrCa often progresses to castration-resistant PrCa (CRPC), a stage that presents challenges due to limited prognosis and significant therapeutic hurdles [7].

This review provides an overview of the key biomarkers in PrCa and the latest therapeutic strategies for CRPC. We place particular emphasis on the emerging Proteolysis-targeting chimera (PROTAC) approach, which has garnered considerable attention for its potential to target previously “undruggable” proteins, thereby offering a promising avenue for advancing CRPC treatment [8]. PROTACs function as bispecific small molecules that facilitate proximity between proteins of interest (POI, the target proteins) and E3 ligases, thereby promoting the catalytic ubiquitination and subsequent target proteins degradation. This review will delve into the various PROTACs developed for PrCa, highlighting their mechanisms of actions and potential clinical applications. Ultimately, we hope to shed light on how these innovative strategies might offer promising therapeutic avenues for CRPC by targeting diverse resistance mechanism.

2. Biomarkers of PrCa

2.1. Androgen receptor (AR)

Without ligand, AR is mainly found in the cytosol and it forms complexes with heat shock proteins (HSPs) [9–11], cytoskeletal elements [12], and other chaperone proteins [10, 13]. This interaction with HSP not only stabilizes the receptor, but also induces conformational changes that prepare the AR for optimal binding when a ligand becomes available [14, 15] (Figure 1).

Figure 1. The key biomarkers and their related signaling pathways in prostate cancer (PrCa).

Androgen stimulates androgen receptor (AR) signalling, resulting in the induction and secretion of prostate-specific antigen (PSMA), while illustrating the enzymatic function of prostate-specific membrane antigen PSMA and its role in folate metabolism and mGluR signalling pathways. AR, androgen receptor; DHT, dihydrotestosterone; HSP, heat shock protein; ARE, androgen response elements; BRD4, bromodomain-containing protein 4; PSMA, prostate-specific membrane antigen; PSA, Prostate-specific antigen; TMPRSS2-ERG, transmembrane protease serine 2:v-ets erythroblastosis virus E26 oncogene homolog; mGluR, metabotropic glutamate receptor; PI3K, Phosphoinositide 3-kinases; AKT, Protein kinase B; mTOR, mammalian target of rapamycin; PCFT, proton-coupled folate transporter; RFC1, reduced folate carrier.

Once it binds to androgens like testosterone or DHT, the AR molecule undergoes a significant structural change [16]. This change prompts the AR molecule to dissociate from HSPs, allowing its interaction with ARA70, Filamin-A, and importin-α co-regulators. As such, these interactions facilitate the AR’s entry into the nucleus, where it predominantly functions as a homodimer. However, AR is also capable of forming heterodimers with the estrogen receptor (ER) isoforms or orphan nuclear receptor testicular receptor 4 (TR4), leading to variations in transcriptional activity [10, 13, 17–22].

AR signalling is further regulated by upstream receptor tyrosine kinases (RTKs). For example, HER-2/neu and G-protein coupled receptor (GPCR) pathways can stimulate AR even in the absence of androgen binding [23–26]. At different serine sites, AR will undergo phosphorylation with or without ligand binding, subsequently affecting its protein stability and transcriptional activation activity [27]. It is reported that androgen binding specifically stimulates several phosphorylation of AR at Ser64/80/93 to protect it from degradation [28]. The phosphorylation of other residues, such as Ser213/506/650, via mitogen-activated protein kinases (MAPK), is pivotal in in controlling AR’s transcriptional activation activity. This process heightens AR’s sensitivity to minimal levels of androgens, estrogens, and anti-androgens by recruiting essential nuclear co-activators [29].

Upon AR nuclear translocation, it recognizes androgen response elements (AREs) in DNA [30]. AR’s recruitment of histone acetyltransferase (HAT), along with various co-regulators and core transcriptional elements, initiates the target genes’ transcription, like PSA [18, 31, 32]. When the ligand bound to AR, it will affect the protein stability and transcriptional output of AR-DNA complexes. For instance, when bound to antagonists, AR continues to move to the nucleus, but exhibits reduced retention at AREs, leading to diminished transcriptional activity [33, 34]. Following the dissociation of the ligand, the nuclear export signal promotes the return of AR to cytosol, either for the preparation of another cycle of ligand binding or for proteasomal degradation, thereby maintaining overall AR activity [18, 35–38].

2.2. Prostate-specific membrane antigen (PSMA)

The FOLH1 gene is known to encode PSMA [39, 40]. In the fields of neurology and metabolism, this protein is referred to as the N-acetyl-l-aspartyl-l-glutamate peptidase I (NAALADase I) [41, 42]. Despite these variations in nomenclature, PSMA acts as a glycoprotein on cell membrane, catalyzing the hydrolysis of folate and carboxypeptides [43, 44] (Figure 1). This enzyme plays a critical role in processing dietary folates, and is primarily distributed in the prostate, kidneys, and duodenum. Interestingly, PSMA is also present in low levels within brain tissue, where it stimulates the activation of metabotropic glutamate receptors (mGluRs) [42].

PSMA comprises a intracellular domain, a transmembrane segment, and a large extracellular domain [43, 45]. By cleaving glutamated folate, PSMA is responsible for releasing glutamate, the predominant dietary form of folate [46] (Figure 1). Through PSMA’s enzymatic action, the generated mono-glutamated folate is then absorbed by transporters such as the folate receptor (FR), reduced folate carrier (RFC1), and proton-coupled folate transporter (PCFT) [47]. Folate uptake is particularly advantageous for rapidly dividing cells, as it supports many cellular processes including the one-carbon metabolism, nucleotide synthesis, and epigenetic methylation, all of which intersect with the methionine cycle [48, 49] (Figure 1). Additionally, under conditions of folate scarcity, the hydrolysis of folate by PSMA might confer a growth advantage [50].

AR and neuroendocrine PrCa (NEPC) modulate the one-carbon metabolic pathway, which is closely intertwined with folate-mediated transfer [51–53]. Within the mammalian nervous system, N-acetyl-aspartyl-glutamate (NAAG) is among the most abundant neuropeptides, particularly present at neuronal synapses [54, 55]. NAAG triggers the activation of mGluR3 and is subsequently broken down by PSMA. By releasing glutamate, PSMA promotes PI3K-AKT-mTOR (phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin) signalling [56]. Firstly, the glutamate release initiates the activation of mGluR I, leading to the phosphorylation of p110β and the activation of PI3K. Activated PI3K, in turn, stimulates the AKT-mTOR pathway, driving protein synthesis and cellular proliferation to facilitate tumorigenesis [57] (Figure 1). Notably, preclinical studies have shown that the PSMA inhibitor 2-PMPA can effectively suppress PI3K signalling, resulting in tumor regression [56]. Moreover, elevated levels of mGluRs have been recognized as a possible adaptive strategy to maintain glutamatergic activity in NEPC context [58].

PSMA-regulated folate metabolism impact a variety of cellular processes including tumor cell proliferation, DNA repair, drug resistance, and resource prioritization in tumor microenvironments [59–61]. As such, PSMA contributes to tumor growth and proliferation by increasing folate availability, which drives anabolic metabolic pathways essential for cellular development and division [59]. Additionally, PSMA-regulated folate metabolism is closely linked to DNA repair mechanisms in cancer [60]. Metabolic intermediates, such as methylene tetrahydrofolate, produced through one-carbon metabolism, are essential for thymidine synthesis and DNA repair [60]. Disruption of this pathway may result in genomic instability and address drug resistance. Moreover, PSMA regulates resource prioritization in the tumor microenvironment. To this end, by enhancing folate utilization, PSMA provides cancer cells with a competitive advantage to sustain high proliferation rates within nutrient-deprived tumor microenvironments [61]. This metabolic adaptation is believed to contribute to the aggressive phenotype of PSMA-positive tumors. Notably, PSMA upregulation corresponds with the increased metabolic demands of tumor cells, allowing them to outcompete surrounding normal cells for access to folate and related resources [61].

2.3. Bromodomain-containing protein 4 (BRD4)

Epigenetic readers, including bromodomain-containing protein 4 (BRD4), have been demonstrated as key associates with transcription factors (TFs) like AR, contributing to the development of aggressive cancers, including PrCa [62–66]. Despite their significance, the detailed mechanisms by which bromodomain inhibitors exert their effects remain largely undefined [64]. Recent research has revealed that changes in DNA accessibility can effectively distinguish PrCa from early-stage malignancies and non-cancerous tissue. The increased genomic accessibility observed in advanced tumors appears to be mediated in part by indirect pathways involving AR interaction with BRDs (BRD4, BRD2 and ATAD2), identified as key facilitators contributing to this enhanced accessibility. Notably, these proteins are overexpressed in CRPC and serve as prognostic tissue markers, underscoring their potential as targets in aggressive PrCa [67].

2.4. Prostate-specific antigen (PSA)

PSA, a serine protease, is secreted by the epithelial cells of the prostate [68]. Although naturally occurring in normal prostatic secretions, PSA is frequently increased in PrCa patients [69, 70]. For over two decades, PSA levels in serum have been served as one of crucial biomarkers in the detection of PrCa [71]. Since 1980s, there has been a significant improvement in PrCa management with the introduction of PSA screening, which has contributed to better survival rates. Despite these advancements, PSA showed various limitations as a biomarker that has prompted the exploration of more accurate diagnostic tools and accelerated the development of PrCa biomarkers [71, 72].

2.5. Transmembrane protease serine 2: v-ets erythroblastosis virus E26 oncogene homolog (TMPRSS2-ERG)

A promising biomarker in PrCa diagnostics is the TMPRSS2-ERG fusion. ETS family transcription factors (such as ERG), in fusion with the androgen-regulated TMPRSS2 gene represent the most frequently observed gene fusions, accounting for approximately 50% of PrCa cases [73, 74]. A study by Laxman et al. first identified the presence of gene fusion events in the patients with PrCa [75]. This discovery has since been supported by additional studies indicating that TMPRSS2-ERG fusions could function as a urinary biomarker. Notably, these gene fusions exhibit high specificity and a strong positive predictive value; however, their sensitivity is relatively low [76, 77].

As most tumors consist of multiple distinct foci, the tumor heterogeneity characteristic of PrCa become a significant limitation of using this gene fusion as a biomarker [76]. Moreover, it remains uncertain about the value of the TMPRSS2-ERG in prognostic. While some studies advocate that this gene fusion correlates with increased cancer aggressiveness, metastasis, and mortality [78, 79], other studies have not found a consistent relationship between this fusion and clinical outcomes, which warrants further in-depth investigations [80].

3. The targeted therapies for PrCa biomarkers

The current treatment landscape for PrCa is progressive and adaptable, where the strategies evolve according to the stage and status of the disease. PrCa is often detected through early screening methods in the initial diagnosis, with treatments typically including surgical removal of the prostate or radiotherapy. These initial treatments eliminate the tumor confined to the prostate; however, recurrence remains a concern, as approximately 30% of PrCa patients were experienced with a return of cancer following these local therapies [81] (Figure 2A).

Figure 2. The current therapy landscape for PrCa.

(A). The available treatment options for different stages of PrCa. (B). Various therapies targeting BRD4, AR, PSMA, and immune check points in PrCa. ADT, Androgen Deprivation Therapy; nmCRPC, non-metastatic castration-resistant PrCa; mCRPC, metastatic castration-resistant PrCa; Lu, Lutetium; AR, androgen receptor; BRD4, bromodomain-containing protein 4; PD-L1, programmed cell death-ligand 1; PD-L2, programmed cell death-ligand 2; CTLA-4, Cytotoxic T-lymphocyte antigen 4.

Disease recurrence is diagnosed through elevated PSA levels, or through imaging techniques that reveal the presence of residual or metastatic disease. When facing biochemical or localized recurrence, physicians may choose to closely monitor PrCa patients without immediately resorting to ADT in an effort to avoid its associated side effects. To this end, ADT is withheld until disease progression reaches a pre-established threshold. Once this threshold is reached, ADT is typically initiated using LHRH antagonists or agonists to decrease androgen levels and inhibit cancer growth. This treatment regimen may be administered intermittently, with treatment holidays to help manage side effects, until evidence of disease progression appears [82].

While ADT can initially achieve disease control, many PrCa patients ultimately become resistant to this therapy, progressing to a more advanced form, the CRPC. This progression marks a turning point in treatment regemin, as CRPC no longer responds to traditional hormone therapies and requires alternative strategies. In cases where PrCa has already metastasized beyond the prostate gland before the initiation of local treatments, a more aggressive approach is necessary. To this end, physicians and patients must then decide on the early use of ADT, either in conjunction with radiotherapy [83] or docetaxel [84].

As patients progress through these treatments, the tumor acquires resistance to therapies targeting AR, a range of additional treatment options becomes relevant. These options include Poly(ADP-ribose) polymerase (PARP) inhibitors, which take advantage of DNA repair deficiencies in cancer, and chemotherapy regimens designed to target rapidly dividing tumor cells [85]. Additionally, PSMA-targeted treatments represent a novel and promising option, focusing on selectively targeting PSMA-expressing PrCa cells [82]. Moreover, immunotherapy represents an expanding avenue for advanced PrCa, by harnessing the patient’s immune system to attack cancer cells [82]. This extensive array of treatment strategies, as illustrated in Figure 2, highlights the complexity and adaptability required in managing PrCa.

3.1. BRD4 targeted inhibitors:

The molecular landscape in PrCa is highly intricate, with various signalling pathways and genetic mutations contributing to disease progression and drug resistance. One promising therapeutic approach in PrCa targets the BRD4, which is implicated in cancer cell survival and proliferation [86, 87]. NEO2734, a novel dual inhibitor, has been developed to target both BET proteins (including BRD4) and CBP/p300, a family of transcriptional coactivators with histone acetyltransferase activity. NEO2734 represents an innovative treatment strategy, especially for the most commonly mutated gene in PrCa, Speckle-type POZ protein (SPOP) [88]. Under normal circumstances, wild-type SPOP is pivotal in regulating cellular protein levels by binding to and promoting the BET protein degradation. However, this regulatory function is lost when SPOP is mutated, leading to the excessive accumulation of BET proteins, and the subsequent dysregulation of gene expression pathways drives cancer growth. This loss of function often results in drug resistance against BET inhibitors (BETi) in SPOP-mutant PrCa patients [88].

Preclinical researches have reported that NEO2734 significantly inhibits the growing of SPOP-mutant PrCa [88]. Furthermore, NEO2734 has shown efficacy against enzalutamide-resistant PrCa cells and patient-derived organoids, indicating its potential use as a treatment strategy for cases that have developed resistance to anti-androgen therapies [89]. Recent research has also explored the function of AR splice variants (AR-Vs) in conferring increased castration resistance in PrCa. It has been observed that NEO2734 can counteract the mechanisms associated with anti-androgen-induced ferroptosis [90]. By inhibiting this process, NEO2734 offers a promising strategy for overcoming resistance that restricts the effectiveness of existing therapies [90]. Currently, NEO2734 is being investigated in clinical trial for CRPC patients (NCT05488548). This trial aims to evaluate the safety, tolerability, and preliminary efficacy of NEO2734 in CRPC patients, offering hope for a novel therapeutic option that targets multiple components of the complex molecular machinery driving PrCa progression.

3.2. PSMA targeted therapy- Lu 177-PSMA-617

177Lu-PSMA-617 or Pluvicto is another promising therapeutic option for targeting androgen receptor variant containing cells, particularly those that also display PSMA. This innovative treatment consists of a molecule targeting to PSMA, conjugated with the 177Lu, a radioactive isotope. The mechanism of action enables 177Lu-PSMA-617 to selectively bind to PSMA-expressing PrCa cells, facilitating the precise delivery of radiation to the tumor without damaging normal tissues. As such, 177Lu emits radiation that directly harms the DNA of cancer cells, ultimately leading to their cell death [91]. This precision-targeted approach has shown highly promising outcomes, particularly in advanced-stage PrCa, as demonstrated in the clinical trial (NCT03511664) [92].

Recent studies have explored molecular biomarkers in patients treated with Pluvicto. One study analysed circulating tumor cells and investigated clinical factors, such as protein expression level of PSA and PSMA, tumor size, and the gene expression levels of AR and AR-V7 [5]. The findings revealed that AR and AR-V7 expression could be used as prognostic markers for in metastatic CRPC patients before starting the Pluvicto therapy. Interestingly, while these biomarkers correlated with tumor load, they did not predict the patient’s response to PSMA-targeted therapies. Notably, AR-V7 was associated with higher AR expression, increased tumor load, and elevated PSMA protein level [5].

Altermnatively, Pathmanandavel et al. [93] reported that among AR-V7-positive mCRPC patients treated with 177Lu-PSMA-617, AR-V7 did not adversely impact overall survival. This observation suggests that 177Lu-PSMA-617 could be a viable therapy for mCRPC patients expressed with AR-V7, alongside established treatments such as taxanes. However, exploring the function of protein expression and the potential value of these markers in determining treatment outcomes is important in further research.

The therapeutic potential of 177Lu-PSMA-617 continues to be explored across multiple ongoing clinical trials. The PSMA fore-trial (NCT04689828) aims to compare the efficacy of 177Lu-PSMA-617 therapies in mCRPC patients targeting the AR. Another active trial investigates pairing Pluvicto with pembrolizumab, an immune checkpoint inhibitor (ICI) (NCT03805594) [94, 95].

3.3. Immunotherapy-Nivolumab and Ipilimumab

ICIs, inclusing nivolumab and ipilimumab, have shown as a potential treatment strategy for advanced PrCa, particularly in AR-V7+ PrCa patients. These inhibitors target immune response regulators, effectively enhancing the immune cells’ ability to eliminate cancer cells [96]. Nivolumab targets to the immune cells by blocking the programmed cell death protein 1 (PD-1) receptor. Under normal conditions, PD-1 binds to physiological ligands PD-L1 and PD-L2 expressed on the cell surface of cancer cells, resulting in the suppression of the immune reaction and allowing the cancer cells to escape immune surveillance. Blocking this interaction, nivolumab reactivates the immune response, allowing it to recognize and attack tumor cells more effectively. In contrast, ipilimumab targets another checkpoint molecule, cytotoxic T-lymphocyte antigen 4 (CTLA-4), a membrane receptor on immune cells that suppresses immune activity. By inhibiting CTLA-4, ipilimumab prevents the inhibitory signaling pathway, keeping the immune cells active and enabling them to target cancer cells [96].

In clinical practice, nivolumab and ipilimumab are frequently used together to create a synergistic immune reaction towards cancer cells. NCT02601014, a clinical trial evaluating this combination in AR-V7+ PrCa patients, demonstrated promising efficacy. However, the response was not consistent across AR-V7+ patients [97].

The efficacy of combining nivolumab and ipilimumab, either alongside or independent of enzalutamide, in AR-V7+ mCRPC patients was investigated in the same Phase II trial. While the combination therapy had an acceptable profile regarding to safety, it demonstrated only limited efficacy in this patient population, even when combined with enzalutamide [98]. Further insights were gained from another Phase II trial NCT02985957. Preliminary findings suggested that some CRPC patients could experience complete responses to the combination of nivolumab and ipilimumab, with four participants achieving complete remission [99]. However, the study did not establish a consistent correlation between treatment efficacy and tumor mutational burden in either blood or tissue samples [99].

In parallel, numerous clinical trials are actively exploring the combination of ICIs with other therapeutic agents. These include trials involving the combination of 177Lu-PSMA (NCT05150236) and stereotactic body radiotherapy (SBRT) (NCT05655715). Another promising immunotherapy for metastatic PrCa is the DNA vaccine, MVI-118, which encodes the LBD of the AR and induces the immune response against AR-overexpressing cancer cells mediated by CD8+ T. In the case of NCT02411786, MVI-118 was found to be safe and effective in activating the immune system in PrCa [100]. Another two clinical trials are currently investigating the MVI-118. One trial explores the use of the pTVG-HP DNA vaccine with or without MVI-118 and pembrolizumab in mCRPC patients, while the other trial examines ADT with or without MVI-118 and/or nivolumab in PrCa patients, according to clinical trials NCT04090528 and NCT04989946, respectively.

3.4. Limitations of current approaches

Despite the development of numerous targeted therapies aimed at prostate cancer (PrCa) biomarkers, these strategies often encounter substantial challenges, which highlight the pressing need for novel therapeutic innovations. To this end, PROTAC technology has emerged as a transformative approach, offering unique advantages that address key limitations associated with conventional methods [8]. Compared to the aforementioned therapeutic modalities, PROTACs overcome several notable constraints, underscoring the potential of this technology to redefine treatment paradigms for PrCa.

Resistance to AR Inhibitors:

Enzalutamide and abiraterone, the current standard AR inhibitors, are effective in prolonging survival [101–104]. However, several problems including AR gene amplification, overexpression of splice variants with constitutive activity (e.g., AR-V7), mutations in AR ligand binding domain, and activation of compensatory signaling pathways, contribute to resistance mechanisms and limit their clinical efficacy [5, 6]. Compared to the inhibitors, PROTACs offer a unique approach by degrading AR proteins, including both splice variants and mutant forms, and may potentially overcome these resistance mechanisms [8].

Toxicity and Side Effects of Chemotherapy:

Chemotherapy remains a standard treatment option for advanced CRPC, including the docetaxel and cabazitaxel for PrCa therapy [105]. However, these agents are reported to exhibit significant toxicities, such as neutropenia, neuropathy, and fatigue, which limit their long-term use [106]. In comparison, PROTACs, in part due to their specificity and targeted mechanism of action, may offer a safer therapeutic alternative with fewer off-target effects.

Mechanistic Limitations of Conventional Therapies:

The mechanism of action of traditional therapies primarily focuses on inhibiting AR signaling [106] rather than eliminating the functional AR, which results in drug resistance. In contrast, PROTACs aim to entirely degrade the AR protein, the key driver of CRPC progression. This degradation likely disrupts its downstream transcriptional activity and reduces the likelihood of reactivation.

Addressing Unmet Needs:

PROTACs are designed to target proteins beyond AR that are involved in CRPC progression, thereby expanding the therapeutic scope and enabling personalized treatment strategies based on specific molecular profiles. However, achieving this goal may be challenging with traditional inhibitors.

In conclusion, the unique therapeutic value of the PROTAC technology lies in its ability to address the key challenges of resistance, toxicity, and limited mechanistic action, which underscores their potential in treating CRPC.

3.5. PROTACs towards AR

Among the innovative therapeutic strategies being explored for PrCa, PROTACs have emerged as promising strategies to degrade target proteins (POI) selectively [8]. One of the leading PROTACs in clinical development is ARV-110, which became the first PROTAC to clinical trials by targeting AR for destruction. By leveraging the ubiquitin-proteasome system (UPS), ARV-110 specifically induces AR degradation [107].

The mechanism by which ARV-110 functions involves forming a ternary complex that induces the AR proximity between POI with an E3 ligase, specifically cereblon (CRBN) in this case [8]. It consists of two distinct components linked by a flexible linker. One side features non-covalently binder that binds to the AR and the other side contains a thalidomide-derived “warhead” that covalently attaches to the CRBN. This binding induces the formation of E3 ligase-ARV-110-AR complex, consisting of the E3 ligase, ARV-110, and the AR protein. By bringing AR and CRBN into close proximity, ARV-110 facilitates the tagging of ubiquitin molecules to AR, ultimately leading to its degradation (Figure 2B). This process results in the complete degradation of the AR, rather than merely inhibiting it, thereby significantly reducing its levels within cancer cells.

What makes ARV-110 particularly innovative is that, unlike traditional AR inhibitors that simply block AR activity, ARV-110 actively triggers the AR degradation. This mechanism is advantageous, especially in cases where PrCa cells have developed resistance to conventional AR therapies. By degrading, rather than merely inhibiting, the AR protein, ARV-110 offers a potential solution to overcoming resistance mechanisms arising from mutations in the LBD of AR, which often reduce the treatment efficacy of standard therapies. Another essential advantage of ARV-110 is its catalytical mechanism. The free PROTAC and E3 ligase complex can be recycled and participate in the next round degradation. This catalytic mechanism renders ARV-110 a highly efficient therapeutic agent, offering superiority over traditional inhibitors in a stoichiometric manner. By exploiting the UPS to degrade AR, ARV-110 therefore represents a new frontier in targeted cancer therapy, potentially offering a more effective treatment method for PrCa patients [8].

4. Proximity strategies

4.1. Membrane Protein Degradation Strategy

Endocytosis play an important role in the macromolecules uptake pathway (Figure 3A). Naturally, the internalized macromolecules will bind to the specific receptors on cell surface and get accumulated in the clathrin-coated pits. After detaching from the membrane, these pits facilitate the enclosure of receptors and corresponding ligands through forming vesicles. Subsequently, the vesicles will merge with early endosomes and then they are directed either to lysosomes or back to the cell surface for recycling [108].

Figure 3. Targeted protein degradation therapies utilize different pathways, such as endocytosis, proteosome, and autophagy pathways.

(A). The endocytosis pathway is exploited to degrade membrane proteins (POIs) using engineered bispecific antibodies. (B). The proteosome pathway is leveraged by PROTACs to ubiquitinate and degrade target POIs. (C). AUTACs and ATTECs are designed to degrade POI or organelles in cells by hijacking the autophagy pathway. Ub, ubiquitin; ASGPR, asialoglycoprotein receptor; CXCR7, C-X-C chemokine receptor type 7; RNF43, ring finger protein 43; POI, protein of interest; KineTAC, cytokine receptor targeting chimera; LYTAC, lysosome-targeting chimera; PROTAB, proteolysis-targeting antibody; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; PROTAC, proteolysis targeting chimera; ATTEC, autophagosome-tethering compound; AUTAC, autophagy-targeting chimera; LC3, microtubule-associated protein light chain 3.

Various strategies have hijacked this endocytosis pathway to degrade membrane proteins. These strategies include LYTACs, which utilize receptors for lysosomal degradation of target proteins such as the mannose-6-phosphate receptor [109, 110]; PROTABs, a bifunctional antibody that hijack RNF43 to ubiquitinate membrane proteins [111]; KineTACs, bispecific antibodies that use cytokine receptors to guide target proteins to lysosomes [112]; and TransTAC, an efficient degrader that functions through leveraging the transferrin receptor to target and eliminate membrane proteins [113]. These emerging technologies present promising strategies for disease-associated membrane protein degradation, revolutionizing the treatment landscape by rendering previously “undruggable” targets amenable to therapeutic intervention.

4.2. PROTACs strategy

Besides endocytosis, a key mechanism for regulating protein levels in eukaryotic cells is ubiquitination, followed by targeted protein degradation. Cellular ubiquitination-induced proteolysis is a pathway in which proteins undergo polyubiquitination, then degradation by the proteasome or lysosome [114–117]. This mechanism is precisely controlled and consists of a cascade of enzymatic activities, including E1, E2, and E3 to facilitate the attachment of ubiquitin to the substrate proteins’ lysine residues [118]. Importantly, the ubiquitination process is reversible, as deubiquitinases (DUBs) cleave polyubiquitin chains from modified proteins, thereby allowing for dynamic regulation of protein stability in cells [119].

Various types of polyubiquitin chains are formed, each serving distinct biological functions, due to the presence of seven lysine residues in ubiquitin protein. For instance, two polyubiquitin chains, K11 and K48, primarily tag proteins and then degrade them, while K63-linked chains typically function as docking sites for protein-protein interactions, facilitating signal transduction events [116, 117]. The specificity of the ubiquitination process is controlled by E3 ligase [120]. However, a limited number of these ligases have clearly defined degron recognition capabilities among the more than 600 E3 ligases [121]. It is a crucial process for removing misfolded or damaged proteins and maintaining intracellular protein balance. Targeted protein degradation (TPD) that selectively degrade the pathogenic proteins is an promising therapeutic strategy.

PROTACs, novel bifunctional molecules, are engineered to exploit the ubiquitin-proteasome system for the degradation of POI [122]. This kind of design allows PROTACs to form a ternary structure between the POI and an E3 ligase, facilitating the targeted protein ubiquitination and degradation (Figure 3B). Notably, an AR targeting PROTAC compound-ARV-110, is currently undergoing clinical trials for treating PrCa by the [8].

4.3. AUTACs strategy

Recent advances in autophagy-based degraders have introduced two notable examples: autophagy-targeting chimeras (AUTACs) and autophagosome-tethering compounds (ATTECs). As reported by Takahashi et al. [123][124], AUTACs represent one of the pioneering approaches in this field. Meanwhile, ATTECs, developed by Li et al. [125, 126], function as molecular glues that mimic autophagy adaptors. This model involves the targeted degradation of specific proteins via autophagy, in which ATTECs facilitate the process by acting similarly to natural autophagy adaptors (Figure 3C). While ATTECs and AUTACs have not yet been employed for AR degradation, these platforms hold significant promise for future applications in CRPC therapy.

5. Controllable PROTACs strategies

5.1. Current PROTACs with different components design:

A PROTAC molecule consists of three essential parts: the E3 ligase ligand, a POI ligand, and a linker that connects these two ligands. Each of these elements is crucial in facilitating the targeted degradation, and their detailed characteristics are discussed in the following sections.

5.1.1. PROTAC with various E3 ligases:

Various E3 ligases, such as β-transducin repeat-containing protein (β-TrCP) [127], von Hippel-Lindau (VHL) [128], MDM2 [129], cereblon (CRBN) [130], and cellular inhibitor of apoptosis protein 1 (cIAP1) [131], have been employed in PROTAC development. The catalytic nature of PROTACs allows them to act repeatedly, making them highly effective in the POI degradation [132, 133].

5.1.2. PROTAC with different linkers:

Figure 4A provides a classification of the most commonly used linkers in according to the structural data in the database [134]. The current linker categories include flexible linkers, rigid linkers, triazole-based linkers, bio-orthogonal clickable linkers, and photo-switchable linkers.

Figure 4. The construct of PROTAC and the mechanism of controllable PROTAC.

A. The construct of PROTAC, which includes E3 ligase ligands, target protein ligands, and the linkers connecting them. The reported types of E3 ligases, linkers, and target proteins in PROTACs are summarized in the indicated boxes. B. The design and working mechanism of controllable PROTACs.

A common strategy in modifying the linkers to enhance the solubility of PROTACs involves incorporating heterocycles (saturated) with a basic center. The examples such as the piperidine or piperazine rings belong to the category of rigid linkers commonly employed in PROTAC design (Figure 4A) [135–137]. The inclusion of piperidine or piperazine not only contributes to the increased rigidity of the PROTAC, which can potentially enhance its activity, but also introduces a protonable amino group that may improve solubility. Piperazine-containing linkers, in particular offer the advantage of enhancing the overall solubility of the molecule. However, data from the PROTAC-DB, an open-access database compiling PROTAC information indicates that the pKa of an acidic or basic center within a molecule can be significantly influenced by neighbouring groups [138, 139] and affecting the properties of the PROTAC.

5.1.3. PROTAC targets of different POIs:

The ideal targets for PROTAC therapy typically possess a set of common characteristics, especially the alterations from their natural state, such as overexpression, mutation, protein aggregation, changes in isoform expression. Additionally, an optimal target should feature accessible surface that allows E3 ligase to bind. Furthermore, the presence of an flexible domain that can be efficiently effectively translocated into proteasome is considered advantageous for facilitating protein degradation [139, 140].

Inflammation, immunity and immuno-oncology.

Two promising candidates, a Bruton’s tyrosine kinase (BTK) degrader (NX-5948)[141] and an IRAK4 degrader (KT-474) [142], are currently being investigated to treat a range of immuno-inflammatory conditions in clinical trials. There is a highly validated therapeutic target-BTK in inflammatory diseases and oncology, with several approved treatments already available [143, 144]. However, the rise of resistance mutation (C481S) in BTK has diminished the efficacy of first-generation BTK inhibitors, driving significant interest in developing PROTAC-based therapies to address this challenge [145–147]. PROTACs offer a key advantage through their event-driven pharmacology, enabling them to catalytically degrade target proteins rather than merely inhibit them. This mechanism holds promise for overcoming resistance mutations like C481S, which reduce the potency of conventional, occupancy-driven inhibitors [148, 149].

Neurology and neurodegeneration.

One breakthrough in PROTAC is the ability to degrade undruggable proteins. These proteins typically cannot be targeted by inhibitors due to a lack of binding sites. The advantage of PROTAC make it possible to address these disease-causing proteins, especially in neurodegenerative diseases, such as tau [150–157], α-synuclein [158, 159], and mutant huntingtin (mHTT) [125, 160–162].

Lu et al. identified Peptide 1, a KEAP1-dependent PROTAC, which was shown to degrade Tau protein, highlighting its potential in the future application [154]. In 2020, Qu et al. introduced another TAT-PBD-PTM based on peptide, that specifically targets and degrades α-synuclein in Parkinson’s disease [159]. Additionally, Dr. Tomoshige designed hybrid small molecules, compounds 1 and 2, which effectively degrade mutant huntingtin (mHTT), demonstrating promise for the treatment of Huntington’s disease [162].

Transcription Factor.

Transcription factors and RNA-binding proteins (RBPs) play critical roles in DNA integrity, synthesis, gene regulation, and diverse RNA-mediated mechanisms [163–165]. Despite their importance, many of these proteins of interests (POIs) without ligand-binding sites have been notoriously challenging to target with traditional drugs [166, 167]. To address these “undruggable” targets, various studies have been conducted to establish innovative approaches. For instance, transcription factors have been targeted using strategies such as TRAFTAC (Transcription Factor Targeting Chimera) [168], TF-PROTAC (Transcription Factor PROTAC) [169], O’PROTAC (Oligonucleotide-PROTAC) [170], and G4-PROTAC (G-quadruplex PROTAC) [171]. Beyond transcription factors, telomeres have been targeted using TeloTAC [172], showcasing the potential to expand to more sophisticated methodologies. Additional advanced strategies include developing Myc-targeting PROTACs using a TNA-DNA bivalent binder [173], MeCP2 degraders that utilize methylated DNA [174], and structurally specific Z-DNA PROTACs for targeting Z-DNA binding proteins [175]. These DNA- or RNA-based PROTACs employ consensus RNA or DNA sequences, which is linked to a ligand of E3 ligase. This innovative approach significantly broadens the possibilities for targeting previously undruggable proteins within cells, offering new avenues for therapeutic intervention.

5.2. Current controllable PROTACs:

Several controllable strategies for PROTACs have been reported, as summarized in Table 1, including stimuli-responsive approaches such as photoactivation, folate targeting, hypoxia sensitivity, X-ray induction, and other external triggers.

Table 1.

The current controllable PROTACs

Caging strategy	PROTAC name	Target protein	E3 ligand	Ref.
Photo-controlled	pc-PROTAC1	dBET1	CRBN	[176]
	Opto-pomalidomide	IKZF1/3	CRBN	[177–180]
	Opto-dBET1	BRD4	CRBN
	Opto-dALK	ALK	CRBN
	Trans-photoPROTAC-1	BRD2	VHL	[181]
	DEACM	ERRα	VHL	[179]
	NPOM	BRD4	CRBN	[179]
	DMNB	BRD4	VHL	[180]
Folate-controlled	folate-PROTAC	BRD3/4	VHL	[182, 183]
Hypoxia-controlled	ha-PROTAC	EGFR	CRBN	[184]
	NTR-PROTAC	EGFR	VHL	[185]
X-ray-controlled	RT-PROTACs	BRD4	VHL	[186]
Stimuli-responsive	sr-PROTAC	BRD4	CRBN	[187]

5.3. Recent Developments in AR Degradation Technology

AR degraders utilizing PROTAC principle has seen significant progress, with a growing number of unique AR degraders being synthesized and tested for their efficacy against PrCa.

CRBN-based PROTACs, particularly ARV-110 as mentioned above, is among the most widely studied PROTACs for AR degradation [8]. One notable study modified the FDA-approved AR inhibitor enzalutamide using various scaffolds in combination with different linkers. The most promising compound from this subseries, PROTAC 35 (compound 1), exhibited potent degradation capabilities, with DC₅₀ around 80 nM in LNCaP cell line. Specificity tests also indicated that compound 1 exhibited a strong preference for the LNCaP PrCa, suggesting its potential for targeted therapy in AR-expressing PrCa [188]. In another study, Akshay D. Takwale et al. developed various CRBN-mediated AR PROTACs by combining the AR antagonist with the CRBN ligand. Compound TD-802 showed the highest degradation efficiency of AR in LNCaP cells [189]. Targeting AR by RU59063 derivatives, Jian-Jia Liang and colleagues developed novel AR PROTACs. Among these, compound A16 (also known as compound 3) emerged as the leading candidate, matching the efficacy of enzalutamide [190]. GaYeong Kim and colleagues synthesized PROTACs using bicalutamide analogs with a PEG linker attached to thalidomide, which serves as the recruiter of CRBN. Among these, compound 13b (compound 4) effectively decreased the expression of AR-regulated genes. Compound 13c (compound 5) demonstrated even greater AR degradation, particularly in targeting AR-V7, making it more effective than the wild-type AR and suggesting its potential for treating AR-V-expressing PrCas [191]. In another significant development, Shaomeng Wang’s team developed ARD-61 (compound 14), a highly potent AR degrader. To address its poor oral bioavailability, they optimized the molecule by incorporating the cereblon ligand, resulting in the development of ARD-2128 (compound 6). This compound exhibited excellent oral bioavailability and showed potent AR degradation, significantly reducing AR, PSA, TMPRSS2, and FKBP5 levels, demonstrating its potential as an orally administered AR-targeted therapy [192]. Finally, Weiguo Xiang et al. developed ARD-2585 (compound 7), which emerged as an efficient degrader of AR. Pharmacokinetic and tissue distribution studies confirmed its promise as an AR degrader, suggesting it could become a highly effective therapy for PrCa, particularly for patients with resistance to conventional AR inhibitors [193].

Alongside CRBN-recruiting PROTACs, VHL-recruiting PROTACs have also been developed to target AR for degradation, with several novel compounds demonstrating promising potential as therapeutic agents in PrCa treatment. Xin Han et al. analysed the correlation between the binding affinity of VHL ligand and the degradation efficacy. Their findings advocated that using ligand with relatively low binding affinity (2–3 μM) could still produce highly effective AR degraders. By optimization, ARD-266 (compound 8) emerged as a standout candidate, achieving greater than 90% AR degradation in 10 nM after further optimization. And ARD-266 could degrade over 95% AR in PrCa within 6 h [194]. Linrong Chen and colleagues reported another degrader, A031 (compound 9), which was optimized from two AR antagonists and several E3 ligands. By comparing the linker composition with different heterocyclic and phenyl rings, they discovered that AR ligand rather than the linker had a substantial effect on the overall activity of the PROTACs [195]. Munoz et al. developed niclosamide-based PROTACs that utilized VHL-032 as the VHL ligand. One compound, Niclo-Click PROTAC 5, achieved an IC₅₀ around 1 μM in LNCaP, but its effectiveness was lower than that of clinical candidates like ARV-110 [196].

Lee et al. introduced MTX-23 (compound 11), a unique PROTAC that has been demonstrated to degrade both AR-FL and AR-V7. Compound 11 showed a DC₅₀ of 0.37 and 2 μM for AR-V7 and AR-FL in immunoblotting studies, respectively. It effectively inhibited cell division and induced programmed cell death in PrCa cells, even in models resistant to enzalutamide and abiraterone [197]. Jemilat Salami et al. synthesized VHL-based PROTACs targeting AR by utilizing enzalutamide. The most potent compound, ARCC-4 (compound 12), displayed a DC₅₀ of 5 nM. It effectively degraded AR in VCaP cells and was able to degrade several clinically relevant AR mutants, demonstrating enhanced efficacy in inducing apoptosis and inhibiting proliferation in CRPC cells [198].

In further optimization efforts, Xin Han’s team developed ARD-69 (compound 13), which exhibited exceptional potency. ARD-69 was optimized by adjusting the linker length and incorporating a pyridine group, enhancing both activity and solubility [137]. Finally, Steven Kregel et al. reported ARD-61 (compound 14), an AR degrader effective against PrCa resistant to standard treatments. ARD-61 triggered PARP cleavage in multiple AR-driven cell lines and exhibited potent activity in the enzalutamide resistant xenograft model. Despite not directly degrading AR-V7, ARD-61 inhibited tumor growth in AR-V7 overexpressing models, suggesting that full-length AR remains crucial for survival in CRPC [199].

Zhang et al. developed a novel PROTAC, named BWA-522, which is designed to target to the N-terminal transcriptional domain of AR (AR-NTD) [200]. By binding to this domain, BWA-522 induces the degradation of both AR-FL and the AR-V7 variant in PrCa cell lines. BWA-522 exhibits promising therapeutic value in both in vitro and in vivo studies, with a sub-micromolar DC₅₀ value in VCaP cells and a 76% inhibition of tumor growth in LNCaP xenograft mice studies, respectively [200].

ARD-2051, another potent PROTAC degrader of AR, was developed by Han et. al in 2023 [201]. It exhibits a 0.6 nM DC₅₀ and also effectively inhibits AR-regulated genes, thereby suppressing cancer cell growth. Moreover, the oral bioavailability studies indicate favorable pharmacokinetic properties of ARD-2051, making it a potential agent for PrCa [201].

Moreover, ARD-1676 developed by Xiang et al., exhibits highly efficient AR degradation, with DC₅₀ values of 0.1 and 1.1 nM in VCaP and LNCaP cells, respectively [202].

In conclusion, the achieved advancements in AR-targeted PROTACs provide novel strategies to overcome the limitations of inhibitors such as enzalutamide, paving the way for enhanced treatment strategies for PrCa.

5.4. Recent Developments in Non-AR targets

While initially developed to degrade the AR in PrCa due to its critical role in disease progression, the application of PROTACs has broadened to include non-AR proteins implicated in PrCa, such as BET [203, 204], BCL6 [205], FAK [206], and CDKs [207].

Bromodomain and Extra-Terminal (BET) proteins, key epigenetic regulators, play a central role in gene transcription [208]. The degradation of BET proteins has been shown to inhibit the PrCa cell growth, motivating the development of PROTACs targeting BET proteins. For example, the degrader MZ1 selectively degrades BRD4, disrupting its interaction with acetylated chromatin [203]. This disruption suppresses MYC-driven gene transcription, ultimately inhibiting cancer cell proliferation. Furthermore, MZ1 has demonstrated potent anti-proliferative effects in prostate cancer models [203].

PROTACs targeting B-cell Lymphoma 6 (BCL6), a transcriptional repressor, have been primarily investigated in lymphomas [205]. For instance, a PROTAC called BI-3802 induces the proteasomal degradation of BCL6, leading to reactivation of tumor-suppressor genes. While BI-3802 primarily studied in lymphomas, its mechanism of action offers valuable insights into the potential for targeting BCL6 pathways in PrCa.

Focal Adhesion Kinase (FAK), a non-receptor tyrosine kinase involved in cell adhesion and survival signaling pathways, is specifically degraded by PROTACs termed PROTAC-FAK-01 [206]. In prostate cancer models, PROTAC-FAK-01 disrupts focal adhesion complexes, effectively suppressing cell migration and proliferation.

Cyclin-Dependent Kinases (CDKs), such as CDK4/6, serve as key regulator of the cell cycle [209]. PROTACs targeting specific CDKs have been investigated to induce cancer cell apoptosis [207]. Jiang et al. developed dTAG-CDK4/6, a PROTAC that degrades these kinases, with significant tumor-suppressive activity observed in prostate cancer xenograft models [207].

These developments highlight the versatility of the PROTAC technology in targeting a broad range of proteins beyond AR in prostate cancer, potentially addressing various oncogenic pathways involved in the disease.

5.5. Recent Clinical Progress of PROTACs

Table 3 presents an overview of recent clinical progress in PROTACs, highlighting key details such as disease indications, developmental phases, preliminary outcomes, and their corresponding challenges and implications [8, 107, 141, 142, 200–202].

Table 3.

Summary of clinical progress of PROTACs and inhibitors

PROTAC	Target	Indication	Phase of Development	Preliminary Outcomes	Challenges and Implications
Enzalutamide [102]	AR	mCRPC	FDA approved	Improved overall survival and radiographic progression-free survival in Phase 3 trials; PREVAIL trial showed a median survival of 32.4 months vs. 30.2 months (placebo).	A standard treatment across prostate cancer stages; resistance development and side effects (fatigue, hypertension, seizures) limit long-term efficacy.
Bavdegalutamide (ARV-110) [8, 107]	AR	mCRPC	Phase I/II	Antitumor activity observed, particularly in patients with AR ligand-binding domain mutations T878 and H875. Demonstrated a manageable tolerability profile in clinical trials.	Companion diagnostics remain essential for identifying patients harboring specific AR mutations. Addressing resistance mechanisms and ensuring therapeutic specificity are critical for success.
NX-5948 [141]	BTK	B-cell malignancies, autoimmune diseases	Phase I	Responses observed within 8 weeks; treatment was well-tolerated, with common adverse events including purpura, thrombocytopenia, and neutropenia.	Ensuring selective BTK degradation is vital to minimize off-target effects. Determining optimal dosing regimens and managing immune-related adverse events remain key challenges.
KT-474 [142]	IRAK4	Autoimmune, inflammatory diseases	Phase I	Over 95% reduction in IRAK4 levels and up to 97% reduction in pro-inflammatory cytokines observed in healthy volunteers. Maintained a favorable safety profile, with only mild to moderate adverse events reported.	Translating findings from healthy volunteers to patients with autoimmune diseases poses challenges. Long-term studies are needed to confirm safety and efficacy for therapeutic potential.
BWA-522 [200]	AR-NTD	Prostate Cancer	Preclinical	Demonstrated efficacy in degrading AR-FL and AR-V7, inducing apoptosis in cell lines and achieving significant tumor growth inhibition in xenograft models.	Offers a strategy for targeting resistant AR variants. Challenges include ensuring the translation of preclinical success into clinical efficacy and overcoming resistance mechanisms.
ARD-2051 [201]	AR	Advanced Prostate Cancer	Preclinical	High degradation potency with Dmax >90%, suppression of AR-regulated genes, and inhibition of cancer cell growth were observed in preclinical models. Demonstrated good oral bioavailability	Developing companion diagnostics for AR mutations and refining oral dosing strategies is critical for therapeutic benefit in patients.
ARD-1676 [202]	AR and AR mutants	AR-positive Prostate Cancer	Preclinical	DC50 values ranging from 0.1–1.1 nM in cell lines achieved >90% degradation. Oral administration effectively reduced AR protein levels in tumor tissues and inhibited tumor growth without toxicity.	Exceptional pharmacokinetics and activity against AR mutants suggest significant therapeutic potential. Addressing off-target effects and achieving robust clinical trial outcomes remain key hurdles.

6. Perspective and future directions

This section explores the key mechanisms driving AR signalling in CRPC and discusses the future directions for PrCa treatment by harnessing PROTAC strategy. We focused on the mechanisms of AR amplification and overexpression, AR splice variants, mutations leading to receptor promiscuity, posttranslational modifications, interactions with coactivators and corepressors, and intratumoral steroid hormone synthesis. Figure 5 highlights the diverse AR-dependent pathways, showcasing therapeutic targets that could potentially be leveraged to enhance CRPC treatment strategies.

Figure 5. The potential molecular mechanisms underlying CRPC.

The scheme illustrates seven potential mechanisms of CRPC, including aberrant activation of AR, AR amplification, intra-tumoral steroid synthesis, AR splicing variants, AR promiscuity, post-translational modifications of AR, and coregulator modification.

AR Amplification and Overexpression:

AR amplification, characterized by increased AR gene count surpassing the normal diploid level, is recognized as a prominent characteristic of CRPC and has been identified in approximately 20–31% of cases [210–212]. Fluorescence in situ hybridization analysis has shown that AR amplification is uncommon in the early-stage of PrCa but becomes more prevalent as the disease advances to castration-resistant stages, correlating with increased AR mRNA expression. For example, an increasement of two-fold AR mRNA levels is discovered in CRPC tumors, highlighting the essential function of AR amplification in enabling tumor adaptation to androgen-depleted conditions [212, 213]. Chen et al. further validated that CRPC cells maintain elevated AR protein levels, emphasizing the function of AR amplification and overexpression in sustaining AR signalling, even in low androgen conditions [214]. In response to these findings, degrader-based therapies have emerged as promising strategies for addressing CRPC driven by AR amplification and overexpression. A notable example is ARV-110, a PROTAC targeting AR, has shown potential in clinical trials by degrading AR while mitigating the effects of overactive AR signalling pathways in CRPC [8].

Posttranslational modifications:

Moreover, posttranslational modifications showcase a pivotal role in the stabilization of AR protein in CRPC, thereby contributing to the receptor’s sustained activity. These modifications, for example, phosphorylation, acetylation, ubiquitination, and SUMOylation-are crucial regulators which modulate AR’s stability, promote its translocation into nucleus, and fine-tune its gene expression function [215]. Collectively, these posttranslational changes ensure prolonged AR signalling, further complicating the management of CRPC and highlighting the urgent need for targeted therapeutic strategies [215]. The interaction with heat shock proteins (HSPs), particularly the HSP family, acts as a critical molecular chaperone, safeguarding AR from degradation while preserving its active conformation [216]. This protective mechanism allows PrCa cells to remain highly responsive to even minimal levels of circulating hormones, enabling sustained proliferative growth in an androgen-deficient environment. Consequently, the involvement of HSPs in maintaining AR stability further exacerbates the challenge of targeting AR signalling in CRPC, necessitating more advanced therapeutic approaches to disrupt this protective interaction.

AR Splice Variants:

Another critical adaptation mechanism in CRPC is the emergence of AR splice variants, which generate truncated AR protein. Among these, AR-V1 and AR-V7 are particularly notable for their role in therapy resistance. Lacking the ligand-binding domain (LBD) but retaining the N-terminal and DNA-binding domains, AR-V1 and V7 function as constitutively active transcription factors independent of androgen binding. This ligand-independent activity enables the splice variants to drive AR signalling continuously, contributing to therapeutic resistance and tumor progression in CRPC [217, 218]. This ligand-independent activity enables AR splice variants to drive downstream pathways even without circulating hormones. Their expression plays a particularly significant role in driving resistance to enzalutamide and abiraterone [219, 220]. These therapies aim to decrease the activity of AR by inhibiting the LBD domain; however, the AR variants circumvent this blockade, as they lack the LBD. Consequently, AR splice variants contribute to both primary resistance, which occurs in 20–40% of patients, and secondary resistance, which almost invariably develops over time in nearly all treated patients. This highlights the pressing need for therapeutic strategies that can effectively target these variants in CRPC [221–223]. This resistance mechanism underscores the urgent need for therapeutic strategies capable of targeting AR splice variants or their downstream effectors to overcome treatment failures in CRPC. Recently, the development of degraders (Figure 5) that target both AR and AR-V7 [197, 223] has opened promising new avenues for CRPC treatment, offering a broader range of therapeutic options. As these AR-V7-targeting strategies continue to advance, they warrant increased focus and exploration within the PrCa research field, as they hold considerable potential for addressing resistance mechanisms.

In summary, AR amplification, stabilization, splice variants, and other factors have been demonstrated to be critical in maintaining persistent AR signalling in CRPC, underscoring critical pathways that can be exploited for the development of future therapies. A deeper study of these regulatory mechanisms will pave the way for more precise and potent PROTAC strategies, tailored to target these distinct alterations in AR signalling (Figure 5). These advancements offer the potential for enhanced therapeutic efficacy and better treatment to patients with PrCa.

Table 2.

Summary of AR-PROTAC

E3	PROTAC name	Ref.
CRBN	ARV-110	[8]
	PROTAC 35	[188]
	TD-802	[189]
	A16	[190]
	Compound 13b/13c	[191]
	ARD-2128	[192]
	ARD-2585	[193]
	BWA-522	[200]
	ARD-2051	[201]
	ARD-1676	[202]
VHL	ARD-266	[194]
	A031	[195]
	Niclo-Click PROTAC 5	[196]
	MTX-23	[197]
	ARCC-4	[198]
	ARD-69	[137]
	ARD-61	[199]