PSA inhibitors for contraception: insights from prostate cancer.

Despite multiple contraceptive products on the market, unintended pregnancy rates are at ~48% worldwide [ 1 ]. Highly effective methods, such as intrauterine devices and implants, are not suitable for all women. Moreover, men have only one reversible option - condoms. Although there are growing investments in novel contraceptive research [ 2 – 6 ], no new highly effective non-hormonal contraceptive has reached the market in decades, especially for men [ 7 ]. This underscores the urgent need for novel contraceptives. In an effort to identify contraceptive drugs that can suppress human sperm or ovarian function, Food & Drug Administration (FDA)-approved drug libraries, including ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) and other collections, were investigated. Among the 12,000 compounds screened, Disulfiram, originally used to treat alcohol use disorder, has emerged as a promising candidate for male contraception due to its ability to inhibit sperm motility [ 8 ]. From a repurposing library containing 5,440 drugs, Ruxolitinib, approved for the treatment of myelofibrosis and polycythemia vera, was identified as one of the anti-mullerian hormone (AMH) receptor agonists and effectively prevented folliculogenesis (see Glossary) in mice [ 9 ]. While these drugs show promise, they currently exhibit low potency for contraception, indicating a need for further structural optimization to enhance their contraceptive efficacy. Prostate-specific antigen (PSA) is a serine protease produced by prostate epithelial cells and has been used as a critical biomarker for prostate cancer detection since 1987 [ 10 , 11 ]. However, in a lesser-known but well-established role, PSA plays a central role in semen liquefaction by hydrolyzing gel-forming proteins - semenogelins (SEMGs), resulting in liquefied semen (See Box 1 ) [ 12 , 13 ]. This liquefaction process enables sperm to become motile and acquire fertilization capability in the female reproductive tract ( Figure I ). Mutations of the KLK3 , low levels of PSA, and the presence of anti-PSA antibodies cause semen hyperviscosity and infertility in humans [ 12 , 14 – 17 ] (See Box 1 ), highlighting an indispensable role of PSA in reproduction. Recent preclinical and clinical investigations have explored contraceptive strategies targeting sperm motility and migration as potential approaches for female on-demand contraception [ 3 , 18 , 19 ]. As PSA is produced in men and the semen liquefaction process naturally takes place in women’s vaginas, inhibition of PSA activity presents an opportunity for a novel non-hormonal contraceptive target for both men and women. PSA activity has been shown to promote the prostate cancer microenvironment, which has led to the development of PSA inhibitors for therapeutic use [ 20 , 21 ]. Inhibition of PSA activity can be achieved by multiple pathways: 1) inactivation of PSA enzymatic action, 2) suppression of PSA production, and 3) specific elimination of PSA-expressing cells. Until recently, inhibition of PSA activity, with small molecule and peptide inhibitors, miRNAs, prodrugs, antibodies, gene therapies, and vaccines, has not been considered a viable contraceptive method [ 22 , 23 ]. These existing PSA inhibitors offer a unique opportunity for repurposing in contraceptive innovation, bridging a critical gap in reproductive health. In contraceptive development, ensuring specificity is crucial, as birth control products are used regularly, often daily, by healthy individuals [ 24 ]. Any side effects arising from off-target issues are intolerable and could lead to long-term health problems. As PSA is a chymotrypsin-like serine protease that shares active sites with other proteases [ 25 ], designing drugs that specifically target PSA can be challenging. Recent studies highlight two viable approaches that can be pursued to address specificity issues: 1) the screening of large chemical libraries to be used for female contraceptives and 2) optimizing PSA-specific proteins for antibody development for male and female contraceptives. This Opinion examines preclinical and clinical PSA inhibitors developed for prostate cancer treatment, emphasizing shared biological mechanisms relevant to contraception, such as PSA inhibition, suppressing PSA production and secretion, and target-killing of PSA-expressing cells. The potential of these PSA inhibitors as contraceptive strategies ( Figure 1 ) is evaluated, along with two viable approaches ( Figure 2 ) to identify novel PSA inhibitors for both male and female contraceptives.

Small molecule inhibitors are drug-like chemicals that interact and reduce the biological activity of proteins. PSA inhibitors often disrupt histidine and/or serine residues near the catalytic pocket of PSA, resulting in a competitive inhibition against the SEMG-binding site, blocking the hydrolysis of SEMGs. As PSA has been associated with prostate cancer progression and metastasis [ 20 ], a PSA inhibitor screening was performed on nearly 50,000 small drug-like molecules by measuring the hydrolysis of a fluorescent-SEMG peptide (HSSKLQ). The triazole-based chemical, called B1, was found to be the most promising PSA inhibitor with an IC 50 of 0.5 μM ( Table 1 ) [ 26 ]. However, B1 also inhibits another serine protease enzyme, chymotrypsin. Recent structure-activity relationship analysis of B1 by replacing triazole and benzoxazole units with pyrazole and benzimidazole, a compound called CDD-3290, improves potency and specificity for PSA ( K i of 0.215 μM) [ 27 ]. In addition to small-molecule inhibitors, both aldehydes and boronic acids act as potent serine protease inhibitors by mimicking the tetrahedral transition state of peptide-bond hydrolysis. LeBeau et al. showed that peptide aldehyde and boronic acid (PBA)-containing compound benzyloxycarbonyl-Ser-Ser-Lys-Leu-(boro)Leu or Z-SSKL(boro)L, results in a greater inhibitory activity to PSA (K i of 0.065 μM) compared to chymotrypsin (K i of 3.9 μM) [ 28 ]. This PBA discovery led to further development of other peptide analogs [ 29 , 30 ]. Despite a superior potency for PSA, PBA compounds might not be suitable for intravaginal female contraceptives as acidic pH (4.0–4.4) in the vagina may destabilize the activity of peptides. As such, CDD-3290 is a promising candidate and should be evaluated for its impact on the inhibition of human semen liquefaction ex vivo. For female contraceptive purposes, CDD-3290 could be formulated as vaginal films, gels, rings, or fast-dissolving inserts ( Figure 1A ) and applied “on-demand” immediately before intercourse. Recent studies show that antibodies targeting sperm motility can potentially be used as female contraceptives [ 5 , 18 , 31 , 32 ]. Anti-PSA antibodies can suppress PSA activity in the semen [ 33 ] ( Table 1 ). Fresh human ejaculates treated with an anti-PSA antibody have increased levels of uncleaved SEMGs, leading to greater semen viscosity compared to vehicle-treated controls [ 33 ]. This study demonstrates that specific inhibition of PSA activity using a neutralizing antibody alone can alter the semen liquefaction process and, ultimately, sperm motility and is a promising candidate for male contraceptives ( Figure 1B ). Likewise, targeted ablation of PSA-expressing cells using antibodies may also limit PSA production and activity. In the prostate cancer field, treatments with anti-PSA antibodies cause prostate cell death and limit PSA secretion, which could possess contraceptive implications. The injection of anti-PSA mouse monoclonal antibody (mAb, AR47.47) stimulates immune activation, triggers cell degranulation , and prolongs the survival of transgenic mice carrying PSA-expressing tumors [ 34 ]. Additionally, intravenous injection of anti-PSA-IgG carrying chemotherapeutic drug (5-fluoro-2’-deoxyuridine) decreases cell proliferation and increases PSA-expressing cancer cell death in mice [ 35 ]. Recently, the crystallized region ( Fc ) tagged with endoplasmic reticulum retention signaling motif of PSA and prostatic acid phosphatase (PAP), called PSA-FcK+PAP-FcK, elicits proper binding properties and PSA antigen recognition [ 36 ]. When injected into mice, PSA-FcK+PAP-FcK dual antigens successfully induce specific antibody responses against both PSA and PAP, suggesting potential applications in prostate cancer immunotherapy. Yet, AR47.47, anti-PSA-IgG-5-fluoro-2’-deoxyuridine, and PSA-FcK have not yet been tested for their contraceptive activity. Micro-RNAs (miRNAs) are 21–25 nucleotides of RNA involved in the regulation of gene expression and can be used for therapeutic purposes in humans [ 37 , 38 ]. Several miRNAs can suppress PSA expression, which reduces PSA secretion into the seminal plasma, ultimately leading to hyperviscous semen. Therefore, miRNAs targeting PSA expression have the potential for male contraceptive development ( Figure 1B and Table 1 ). For example, miRNA-3162–5p suppresses PSA production and catalytic activity in prostate cancer cell lines [ 39 ]. PSA is also post-transcriptionally regulated by miRNA-99 family members [ 40 ]. miRNA-99b expression is decreased in prostate cancer tissues, and exogenous expression of miRNA-99b inhibits prostate cancer cell proliferation through a mammalian target of the rapamycin (mTOR)-induced autophagy [ 41 ]. Levels of miRNA-199b-3p are significantly reduced in prostate cancer tissues compared to those from benign prostate enlargement patients [ 42 ]. Increasing the expression of miRNA-199b-3p also minimizes the proliferation of prostate cancer cells. Although reports suggest that miRNAs modulate PSA production and activate autophagy of prostate cells, none of these miRNAs have been investigated for their contraceptive purposes. Drugs can be designed as inactive compounds (called prodrugs, Figure 1B ) that become active only when they encounter specific proteases, such as PSA, in the prostate tissue. These prodrugs consist of a toxic drug molecule attached to a peptide. When the prodrug reaches the target tissue, PSA cleaves the peptide, activating the drug specifically in that location [ 43 ]. While PSA exists throughout the body, most PSA in the bloodstream is inactive due to its binding with endogenous inhibitors. However, active PSA is concentrated in prostate cells, allowing PSA-activated prodrugs to become activated specifically in the prostate tissue [ 44 ]. Considering that men with prostatectomy are healthy, the killing of healthy prostate cells for male contraceptive purposes would not negatively impact the quality of life. A study shows that a PSA-prodrug containing doxorubicin-conjugated PSA substrate (HSSKLQ) can be cleaved by PSA in prostate cancer cells. However, some concerns were raised about a non-specific activation of one of the prodrugs, L-377,202, while in the bloodstream. A recent study has shown improvement in targeted activation by encapsulating the prodrug in temperature-sensitive liposomes. The liposomes only allow drug release in response to mild hyperthermia at 42°C within 5 minutes and extend the survival rate in mice compared to the control prodrug without the liposomes [ 45 ]. These targeted approaches help deliver prostate-cell-killing drugs while minimizing effects on healthy tissue with potential benefits as a contraceptive target in men ( Figure 1B and Table 1 ). Multiple PSA-prodrugs are currently being investigated for prostate cancer therapy in clinical trials [ 46 ]. To leverage existing data and gain insight into whether PSA-prodrugs have contraceptive effects, the fertility status of men in these clinical trials should be investigated. Gene therapy, such as overexpression of AMH, is being considered for female contraceptive targets and has been proven effective in cats [ 47 ]. Non-viral and viral vectors have been studied for gene-based therapy for PSA as a target for prostate cancer [ 48 , 49 ]. The inhibition of PSA-expressing cell proliferation using gene therapy could reduce both PSA production and secretion, potentially disrupting the semen liquefaction process as a male contraceptive target ( Figure 1B and Table 1 ). A recent study in prostate cancer cells shows that gene therapy using liposomes can encapsulate PSA promoter-driven perforin-expressing vector [ 49 ]. Perforin then creates cellular pores [ 50 ], causing prostate cancer cell death and a decreased tumor volume in mice. A recombinant adenovirus carrying a toxic therapeutic gene, such as thymidine kinase [ 51 ], under the control of a PSA promoter (called Ad-PSA-TK) was developed, allowing for a replication-competent cell-killing process [ 52 ]. In this case, the virus replicates and produces the TK toxin, triggering an immune response and causing cell lysis only in PSA-expressing cells. Treatment of Ad-PSA-TK shows attenuation of growth and tumor lysis of human prostate tumors in mice. CV787 is another adenovirus-mediated gene therapy targeting a PSA promoter. A single tail vein injection of CV787 effectively suppresses human prostate cancer cell growth in mice, and the effect was sustained for at least 6 weeks [ 53 ]. A similar replication-competent PSA adenovirus, CV706, was later developed and tested in a phase I clinical trial [ 54 ]. Thirteen of 20 patients with locally recurrent prostate cancer after radiation therapy showed a 15–81% reduction in PSA levels with intraprostatic delivery of CV706, lasting up to 11.3 months. However, 6 of 20 patients did not observe changes in PSA levels [ 54 ]. Based on these data, gene therapies targeting PSA show modest efficacy in suppressing prostate cell growth in mouse models and humans. Furthermore, the effects of viral gene therapy may be irreversible and not suitable for contraception in younger men. Future development of PSA gene therapy for contraceptive purposes should proceed with caution. Vaccines targeting sperm and egg components have been investigated for their contraceptive potential [ 55 , 56 ]. For prostate cancer treatment, over 175 vaccines have been tested in clinical trials [ 57 ]. Yet, there are only a few vaccines specifically targeting PSA. PROSTVAC is a prostate cancer vaccine using vaccinia and fowlpox viral carriers to stimulate the immune system to attack PSA-expressing cancer cells [ 58 ]. In a phase III clinical trial, 57% of patients vaccinated with PROSTVAC for 4 weeks demonstrated a two-fold increase (or more) in PSA-specific T-cells compared to before vaccination [ 58 ]. However, in a more extensive follow-up study of 1,297 participants, PROSTVAC treatment alone does not affect overall survival compared to placebo [ 59 ]. Moreover, a recent study in 33 non-metastatic castration-resistant prostate cancer patients shows that the combination treatment of androgen receptor antagonist, flutamide, with PROSTVAC does not improve outcomes compared to the flutamide-only group [ 60 ]. Based on these findings, PROSTVAC might not be as effective for male contraception as small molecule inhibitors, antibodies, miRNAs, or prodrugs. Another PSA-specific vaccine is the pVAX/PSA DNA vaccine. An initial study finds that pVAX/PSA DNA activates PSA-specific cytotoxic T-lymphocytes in mice, protecting 40% of mice from tumor growth compared to pVAX alone [ 61 ]. In a phase I clinical trial, the pVAX/PSA DNA vaccine stimulates PSA-specific cellular immune responses in patients with hormone-refractory prostate cancer [ 62 ]. As a result, pVAX/PSA DNA vaccine should be investigated to determine whether it can be used to induce cytotoxicity in PSA-expressing cells and suppress PSA production for male contraceptive purposes ( Figure 1B and Table 1 ). Like prodrugs and gene therapies, fertility status from the clinical trials should be evaluated in men treated with PSA-targeting vaccines.

DNA-encoded chemistry technology (DEC-Tec) relies on developing unique DNA-encoded chemical libraries (DECLs) [ 63 ]. DEC-Tec is used for the identification and development of novel small-molecule drugs for a variety of applications, such as serine/threonine kinase 33 for male contraception [ 64 , 65 ], ephrin receptor kinase inhibitors for cancer and endometriosis [ 66 ], thrombin-specific inhibitors for bleeding disorders [ 67 ], and viral protein M Pro for SARS-CoV-2 [ 68 , 69 ]. DECLs often contain billions of distinct molecular entities, each associated with a specific DNA sequence that functions as an identifier or “barcode” ( Figure 2A ). To identify PSA-binding chemicals, DECLs are added to the chemical reaction containing PSA-tagged protein, such as histidine, which is immobilized by nickel-coated magnetic beads. Chemical molecules that bind to PSA are identified using DNA sequencing technology, while chemicals without PSA interaction are eluted or “washed” off. This interaction permits the simultaneous evaluation of billions of compounds in a single experiment, enabling an efficient exploration of a vast chemical space to find the most potent PSA inhibitor(s) [ 67 , 70 ]. DECLs can also be designed based on reported CDD-3290 inhibitors [ 27 ], generating pyrazole-focused DECLs for further potency and specificity improvement of existing compounds. Molecules identified from DECLs as PSA “tight binders” can then be evaluated for their inhibitory activity against PSA. Monoclonal antibodies (mAbs) have been used as therapeutic agents for over 40 years, yet this approach has not been fully explored for male contraception. As PSA mAbs could be administered to antagonize PSA function systemically, it provides a novel contraceptive method for inhibiting PSA activity in men, in addition to intravaginal administration in women. Antibodies are proteins composed of two different parts, heavy (H) and light (L) chains, linked by disulfide bonds to form a Y-shaped structure ( Figure 2B , blue inset). The antigen-binding fragment ( FAb ) of an antibody is composed of heavy and light variable chains (VH and VL), with the complementarity-determining region ( CDR ) of the chains determining the paratope of mAb, which binds an antigen’s epitope. The paratope is unique to each mAb, enabling the antibody with target specificity and limiting off-target effects. A yeast surface display has recently been used to optimize antibodies for multiple therapeutic targets, such as Sintilimab for Hodgkin lymphoma treatment [ 71 ]. Focusing on the CDR [ 72 , 73 ], yeast surface display can be performed to optimize PSA mAb to enhance PSA affinity, inhibitory activity, and specificity ( Figure 2B ). A yeast surface-display library is generated by mutating the CDR and transformed into a yeast system. The yeast library displays various CDR FAb fragments on the cell surface. Optimal CDR FAbs are selected based on their interaction with PSA antigen, which is tagged with a fluorescent label, such as a hemagglutinin (HA), in combination with an HA antibody. Then, yeast cells displaying optimal CDR FAbs binding to fluorescent-tagged-HA are identified using fluorescence-activated cell sorting (FACS) analysis. The binding potency of identified mutated CDR FAbs can be confirmed. Lastly, chemicals identified from DECL and optimized mAb can be produced and evaluated for their biological actions in inhibiting PSA activity and semen liquefaction in ejaculates ( Figure 2C ). Optimized mAbs should also be tested for prostate tissue distribution in vivo. These two methods could lead to specific suppression of PSA activity and protein in the seminal plasma and could be developed as male and female contraceptives. The inhibition of prostate-specific antigen (PSA) does not affect the hypothalamic-pituitary-gonadal axis, which makes it a potential non-hormonal target for contraception. This offers an advantage over hormonal methods, as it can prevent semen liquefaction on demand when administered locally, eliminating the need for a long “wait period” to suppress ovarian or sperm production cycles. PSA inhibitors used vaginally should be assessed for their impact on vaginal health [ 74 ], especially microbiome and pathogen susceptibility (see Outstanding Questions ). Identified anti-PSA antibodies, miRNAs, prodrugs, gene therapies, and vaccines have the potential to be evaluated for male contraception. As KLK3 is only expressed in humans and non-human primates [ 75 , 76 ], validation studies on pregnancy prevention need to be performed in non-human primate models. Lastly, contraceptive products should be reversible. MiRNAs, antibodies, prodrugs, gene therapy, and vaccines targeting PSA-expressing cells must be investigated for their fertility reversal in vivo . If not, they may not be suitable for men who wish to conceive children in the future. In conclusion, existing research and clinical trials from the prostate cancer field are crucial to unlocking the full potential of PSA inhibitors as contraceptive targets.