An emerging target for male contraception.

An inhibitor of a nonhormonal target is identified using a DNA-encoded chemical library.

Strategies for developing retinoic acid receptor alpha-selective antagonists as novel agents for male contraception.

Reported here are the synthesis and in vitro evaluation of a series of 26 retinoic acid analogs based on dihydronaphthalene and chromene scaffolds using a transactivation assay. Chromene amide analog 21 was the most potent and selective retinoic acid receptor α antagonist identified from this series. In vitro evaluation indicated that 21 has favorable physicochemical properties and a favorable pharmacokinetic PK profile in vivo with significant oral bioavailability, metabolic stability, and testes exposure. Compound 21 was evaluated for its effects on spermatogenesis and disruption of fertility in a mouse model. Oral administration of compound 21 at low doses showed reproducibly characteristic albeit modest effects on spermatogenesis, but no effects on fertility were observed in mating studies. The inhibition of spermatogenesis could not be enhanced by raising the dose and lengthening the duration of dosing. Thus, 21 may not be a good candidate to pursue further for effects on male fertility.

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding.

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2-/- and Spdya-/- phenotypes.

Investigation of selective retinoic acid receptor alpha antagonist ER-50891 and related analogs for male contraception.

Retinoic acid receptor alpha (RARα) antagonist ER-50891 and 15 analogs were prepared and tested in vitro for potency and selectivity at RARα, RARß, and RARγ using transactivation assays. Minor modifications to the parent molecule such as the introduction of a C4 tolyl group in place of the C4 phenyl group on the quinoline moiety slightly increased the RARα selectivity but larger substituents significantly decreased the potency. Replacement of the pyrrole moiety of ER-50891 with triazole, amides, or a double bond produced inactive compounds. ER-50891 was found to be stable in male mouse liver microsomes and was tested in male mice to assess its effects on spermatogenesis. Characteristic, albeit modest and transient, effects on spermatogenesis were observed.

Enhancing the Visibility of Women in the ACS Division of Medicinal Chemistry (ACS MEDI).

On the occasion of the 2023 International Women's Day on March 8, 2023, we want to celebrate and highlight the contributions of many women volunteers in the American Chemical Society Division of Medicinal Chemistry (ACS MEDI).

Enhancing the Visibility of Women in the ACS Division of Medicinal Chemistry (ACS MEDI).

On the occasion of the 2023 International Women's Day on March 8, 2023, we want to celebrate and highlight the contributions of many women volunteers in the American Chemical Society Division of Medicinal Chemistry (ACS MEDI).

Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice.

Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series' therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.

The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We tested the effectiveness of the indazole carboxylic acid H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells, and it significantly inhibited both cAMP- and epidermal growth factor-induced proliferation of ADPKD cells. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and decreased hyperphosphorylated retinoblastoma levels. H2-GMZ treatment also decreased ErbB2, Akt, and cyclin-dependent kinase 4, consistent with inhibition of heat shock protein 90, and it decreased levels of the cystic fibrosis transmembrane conductance regulator Cl- channel protein. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Experiments using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox: Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is a renal neoplastic disorder characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. This study shows that the lonidamine derivative H2-GMZ inhibits Cl- secretion, cell proliferation, and cyst growth, suggesting that it might have therapeutic value for the treatment of ADPKD.

1,4-Dihydropyridinebutyrolactone-derived ring-opened ester and amide analogs targeting BET bromodomains.

Based on a previously reported 1,4-dihydropyridinebutyrolactone virtual screening hit, nine lactone ring-opened ester and seven amide analogs were prepared. The analogs were designed to provide interactions with residues at the entrance of the ZA loop of the testis-specific bromodomain (ZA) channel to enhance the affinity and selectivity for the bromodomain and extra-terminal (BET) subfamily of bromodomains. Compound testing by AlphaScreen showed that neither the affinity nor the selectivity of the ester and lactam analogs was improved for BRD4-1 and the first bromodomain of the testis-specific bromodomain (BRDT-1). The esters retained affinity comparable to the parent compound, whereas the affinity for the amide analogs was reduced 10-fold. A representative benzyl ester analog was found to retain high selectivity for BET bromodomains as shown by a BROMOscan. X-ray analysis of the allyl ester analog in complex with BRD4-1 and BRDT-1 revealed that the ester side chain is located next to the ZA loop and solvent exposed.

Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na+, K+, or Cl- levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.

Bivalent BET Bromodomain Inhibitors Confer Increased Potency and Selectivity for BRDT via Protein Conformational Plasticity.

Bromodomain and extraterminal domain (BET) proteins are important regulators of gene transcription and chromatin remodeling. BET family members BRD4 and BRDT are validated targets for cancer and male contraceptive drug development, respectively. Due to the high structural similarity of the acetyl-lysine binding sites, most reported inhibitors lack intra-BET selectivity. We surmised that protein-protein interactions induced by bivalent inhibitors may differ between BRD4 and BRDT, conferring an altered selectivity profile. Starting from nonselective monovalent inhibitors, we developed cell-active bivalent BET inhibitors with increased activity and selectivity for BRDT. X-ray crystallographic and solution studies revealed unique structural states of BRDT and BRD4 upon interaction with bivalent inhibitors. Varying spacer lengths and symmetric vs unsymmetric connections resulted in the same dimeric states, whereas different chemotypes induced different dimers. The findings indicate that the increased intra-BET selectivity of bivalent inhibitors is due to the differential plasticity of BET bromodomains upon inhibitor-induced dimerization.

Discovery and Characterization of Multiple Classes of Human CatSper Blockers.

The cation channel of sperm (CatSper) is a validated target for nonhormonal male contraception, but it lacks selective blockers, hindering studies to establish its role in both motility and capacitation. Via an innovative calcium uptake assay utilizing human sperm we discovered novel inhibitors of CatSper function from a high-throughput screening campaign of 72,000 compounds. Preliminary SAR was established for seven hit series. HTS hits or their more potent analogs blocked potassium-induced depolarization and noncompetitively inhibited progesterone-induced CatSper activation. CatSper channel blockade was confirmed by patch clamp electrophysiology and these compounds inhibited progesterone- and prostaglandin E1-induced hyperactivated sperm motility. One of the hit compounds is a potent CatSper inhibitor with high selectivity for CatSper over hCav1.2, hNav1.5, moderate selectivity over hSlo3 and hERG, and low cytotoxicity and is therefore the most promising inhibitor identified in this study. These new CatSper blockers serve as useful starting points for chemical probe development and drug discovery efforts.

Cytotoxicity of phenylpironetin analogs and the metabolic fate of pironetin and phenylpironetin.

To improve pironetin's metabolic stability we prepared four analogs by replacing its C12-14 segment with an aryl group. The antiproliferative activity of phenyl analog 4 was reduced two-fold and dihydroxy-4-fluorophenyl analog 5 was slightly more effective against OVCAR5 and A2780 ovarian cancer cell lines compared with the parent compound pironetin (1). The activity of 4-fluorophenyl analog 6 was reduced 3-fold in both cell lines. The activity of 7-O-methyl analog 7 was reduced 36-fold in OVCAR5 cells and 47-fold and A2780 cells, compared with pironetin. Phenylpironetin (4) was rapidly metabolized by mouse and human liver microsomes. We identified 17 human metabolites for phenyl analog 4 and 14 human metabolites for pironetin. Metabolism occurred at the C12-13 moiety, the α,ß-unsaturated lactone and the side chains of the molecules (C6-C11 segments). The significant extent of oxidative metabolism suggests that it may not be possible to attain a metabolically stable pironetin analog by structural modifications of the parent compound.

Steroidal Antagonists of Progesterone- and Prostaglandin E1-Induced Activation of the Cation Channel of Sperm.

The cation channel of sperm (CatSper) is the principal entry point for calcium in human spermatozoa and its proper function is essential for successful fertilization. As CatSper is potently activated by progesterone, we evaluated a range of steroids to define the structure-activity relationships for channel activation and found that CatSper is activated by a broad range of steroids with diverse structural modifications. By testing steroids that failed to elicit calcium influx as inhibitors of channel activation, we discovered that medroxyprogesterone acetate, levonorgestrel, and aldosterone inhibited calcium influx produced by progesterone, prostaglandin E1, and the fungal natural product l-sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K+ and pH. In contrast to these steroid antagonists, we demonstrated for the first time that the T-type calcium channel blocker ML218 acts similarly to mibefradil, blocking CatSper channels activated by both ligands and alkalinization/depolarization. These T-type calcium channel blockers produced an insurmountable blockade of CatSper, whereas the three steroids produced antagonism that was surmountable by increasing concentrations of each activator, indicating that the steroids selectively antagonize ligand-induced activation of CatSper rather than blocking channel function. Both the channel blockers and the steroid antagonists markedly reduced hyperactivated motility of human sperm assessed by computer-aided sperm analysis, consistent with inhibition of CatSper activation. Unlike the channel blockers mibefradil and ML218, which reduced total and progressive motility, medroxyprogesterone acetate, levonorgestrel, and aldosterone had little effect on these motility parameters, indicating that these steroids are selective inhibitors of hyperactivated sperm motility. SIGNIFICANCE STATEMENT: The steroids medroxyprogesterone acetate, levonorgestrel, and aldosterone selectively antagonize progesterone- and prostaglandin E1-induced calcium influx through the CatSper cation channel in human sperm. In contrast to T-type calcium channel blockers that prevent all modes of CatSper activation, these steroid CatSper antagonists preferentially reduce hyperactivated sperm motility, which is required for fertilization. The discovery of competitive antagonists of ligand-induced CatSper activation provides starting points for future discovery of male contraceptive agents acting by this unique mechanism.

Dihydropyridine Lactam Analogs Targeting BET Bromodomains.

Inhibitors of Bromodomain and Extra Terminal (BET) proteins are investigated for various therapeutic indications, but selectivity for BRD2, BRD3, BRD4, BRDT and their respective tandem bromodomains BD1 and BD2 remains suboptimal. Here we report selectivity-focused structural modifications of previously reported dihydropyridine lactam 6 by changing linker length and linker type of the lactam side chain in efforts to engage the unique arginine 54 (R54) residue in BRDT-BD1 to achieve BRDT-selective affinity. We found that the analogs were highly selective for BET bromodomains, and generally more selective for the first (BD1) and second (BD2) bromodomains of BRD4 rather than for those of BRDT. Based on AlphaScreen and BromoScan results and on crystallographic data for analog 10 j, we concluded that the lack of selectivity for BRDT is most likely due to the high flexibility of the protein and the unfavorable trajectory of the lactam side chain that do not allow interaction with R54. A 15-fold preference for BD2 over BD1 in BRDT was observed for analogs 10 h and 10 m, which was supported by protein-based 19 F NMR experiments with a BRDT tandem bromodomain protein construct.

TSSK3, a novel target for male contraception, is required for spermiogenesis.

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

Tetrahydroindazole inhibitors of CDK2/cyclin complexes.

Over 50 tetrahydroindazoles were synthesized after 7-bromo-3,6,6-trimethyl-1-(pyridin-2-yl)-5,6,7,7a-tetrahydro-1H-indazol-4(3aH)-one (3) was identified as a hit compound in a high throughput screen for inhibition of CDK2 in complex with cyclin A. The activity of the most promising analogues was evaluated by inhibition of CDK2 enzyme complexes with various cyclins. Analogues 53 and 59 showed 3-fold better binding affinity for CDK2 and 2- to 10-fold improved inhibitory activity against CDK2/cyclin A1, E, and O compared to screening hit 3. The data from the enzyme and binding assays indicate that the binding of the analogues to a CDK2/cyclin complex is favored over binding to free CDK2. Computational analysis was used to predict a potential binding site at the CDK2/cyclin E1 interface.