The retinoic acid receptor alpha (RARα) has emerged as a compelling genetically and pharmacologically validated target for nonhormonal male contraception due to its essential role in spermatogenesis. In the present study, a search for specific inhibitors of RARα utilized systematic linker bioisosterism, hydrophobic core modification, and iterative structure-activity relationship refinement, identified the acid 9, a pyrrole-linked analog that potently inhibits RARα (IC50 = 1.2 nM) with >300-fold selectivity over RARβ and RARγ. Sprague-Dawley rat studies with the sodium salt of 9, YCT-529, showed good oral bioavailability and dose-proportional pharmacokinetics without drug accumulation after 28 days of dosing. Once-daily oral administration (0.75 mg/kg for 28 days) reversibly suppressed epididymal sperm counts and fertility in rats, with a no-observed-adverse-effect level at 30 mg/kg (highest dose tested), affording a ≥40-fold therapeutic window. These findings validated aromatic linker substitution as a powerful design strategy for identifying RARα antagonists and led to the clinical advancement of YCT-529 as a nonhormonal male contraceptive.

The first bromodomain of the BET protein BRDT (BRDT-BD1) possesses a unique Arg54 residue at the terminus of the ZA channel, absent in other BET family members. We explored this structural uniqueness with 23 analogs of the BET/kinase inhibitor SG3-179, each bearing an amino acid side chain to enable potential interactions between the positively charged arginine group and the negatively charged carboxylate groups. In an AlphaScreen assay, serine analog 13 showed 35-fold selectivity for BRDT-T over BRD4-T. The BRDT-BD1 cocrystal structure with glutamic acid analog 14 showed no interaction with Arg54, suggesting that the observed preference may be related to differences in the structured water molecules. Compound 13 displayed exceptional in vitro metabolic stability but had limited cellular permeability in MDCK-MDR1 cells. Compounds 13 and 14 are among the best BRDT-BD1-preferring inhibitors reported to date and demonstrate a significant step toward identifying highly selective BRDT inhibitors for male contraception.

BACKGROUND:Pharmacological treatments for fibrocalcific aortic valve stenosis (FCAVS) have been elusive for >50 years. Here, we tested the hypothesis that reactivation of oxidized sGC (soluble guanylate cyclase), the primary receptor for nitric oxide, with ataciguat is a safe and efficacious strategy to slow progression of FCAVS. METHODS:We used quantitative real-time reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry to characterize sGC signaling and the biological effects of ataciguat on signaling cascades related to nitric oxide, calcification, and fibrosis in excised human aortic valve tissue, aortic valve interstitial cells, and mouse aortic valves. We then conducted randomized, placebo-controlled phase I (14-day safety/tolerance) and phase II (6-month efficacy) trials in patients with moderate aortic valve stenosis. RESULTS:In excised human tissue, we found robust losses in sGC signaling despite upregulation of sGC subunits. In vitro, ataciguat increased sGC signaling and reduced BMP2 (bone morphogenetic protein 2) signaling in aortic valve interstitial cells. In mice with established FCAVS, treatment with ataciguat attenuated BMP signaling and slowed progression of valve calcification and dysfunction. In a phase I, randomized, placebo-controlled trial, treatment with ataciguat for 2 weeks was safe and well tolerated in patients with moderate FCAVS (https://www.clinicaltrials.gov; Unique identifier: NCT02049203). In a separate phase II, randomized, placebo-controlled trial, treatment with ataciguat for 6 months slowed the progression of aortic valve calcification and tended to slow the progression of valvular and ventricular dysfunction in patients with moderate FCAVS (https://www.clinicaltrials.gov; Unique identifier: NCT02481258). CONCLUSIONS:Collectively, this study highlights the therapeutic potential of the targeted restoration of the diseased/inactive form of sGC for treatment of FCAVS. REGISTRATION:URL: https://www.clinicaltrials.gov; Unique identifier: NCT02049203. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02481258.

Starting from dihydropyridopyrimidine benzyl ester 1, we pursued a macrocyclization strategy by linking its two aryl rings, hypothesizing that decreasing the conformational flexibility of the ester side chain would increase bromodomain and extra-terminal (BET) protein affinity and selectivity. We prepared 14 analogs and tested them in our fluorescent polarization (FP) assay for BRDT-1 and BRD4-1 affinity. Based on their Ki values, we selected compounds 6b (BRDT-1 Ki = 1.05 μM and BRD4-1 Ki = 0.68 μM) and 6d (BRDT-1 Ki = 0.86 μM and BRD4-1 Ki = 0.70 μM) for further testing. Differential Scanning fluorimetry (DSF) experiments with the BD1 and BD2 proteins of BRD4 and BRDT showed that the most significant increases in the melting temperatures occurred for BRDT-2 for both compounds (13 °C for 6b and 8.9 °C for 6d). Preferential binding to the second bromodomain of BRDT-2 was further confirmed by protein-observed fluorine NMR with the tandem bromodomain of BRDT. A BROMOscan showed that both compounds are pan-BET-BD2 selective (KD = 33-160 nM). A bromoMAX assay with 32 bromodomains verified BET bromodomain selectivity for 6d. The co-crystal structure between macrocyclic analogs 6b and 6d and BRD4-1 shows that the two molecules adopt almost identical conformations despite different spacer lengths. We posit that the increased BD2 selectivity could result from pi-stacking (and additional H-bonds) between the inhibitors and a His residue that is conserved across BET-BD-2 but is absent in BET-BD-1. Compound 6d inhibited MM.1S cancer cell growth with an IC50 of 2.6 μM. The study exemplifies how constraining conformational flexibility can impart target selectivity. The results indicate that the macrocyclization strategy achieved an increase in pan-BD1-affinity and comparable pan-BD2-affinity compared to lead compound 1.

Protein kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2β) and two catalytic subunits (CK2α and CK2α′). CK2 controls several cellular processes, including proliferation, inflammation, and cell death. However, CK2α and CK2α′ possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α′ has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α′ in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α′ presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α′ due to their high structural homology, especially in the targeted ATP-binding site. Using computational analyses, we found a potential type IV ("D" pocket) allosteric site that contained different residues between CK2α and CK2α′ and was distal from the ATP-binding pocket featured in both kinases. We decided to look for allosteric modulators that might interact in a biased fashion with the type IV pocket on both CK2α and CK2α′. We screened a commercial library containing ∼29,000 allosteric-kinase-inhibitor-like compounds using a CK2α′ activity-dependent ADP-Glo Kinase assay. Obtained hits were counter-screened against CK2α using the ADP-Glo Kinase assay, revealing two CK2α′-biased compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

Protein Kinase CK2 is a holoenzyme composed of two regulatory subunits (CK2β) and two catalytic subunits (CK2α and CK2α'). CK2 controls several cellular processes including proliferation, inflammation, and cell death. However, CK2α and CK2α' possess different expression patterns and substrates and therefore impact each of these processes differently. Elevated CK2α participates in the development of cancer, while increased CK2α' has been associated with neurodegeneration, especially Huntington's disease (HD). HD is a fatal disease for which no effective therapies are available. Genetic deletion of CK2α' in HD mouse models has ameliorated neurodegeneration. Therefore, pharmacological inhibition of CK2α' presents a promising therapeutic strategy for treating HD. However, current CK2 inhibitors are unable to discriminate between CK2α and CK2α' due to their high structural homology, especially in the targeted ATP binding site. Using computational analyses, we found a potential Type IV ("D" pocket) allosteric site on CK2α' that contained different residues than CK2α and was distal from the ATP binding pocket featured in both kinases. With this potential allosteric site in mind, we screened a commercial library containing ~29,000 allosteric-kinase-inhibitor-like compounds using a CK2α' activity-dependent ADP-Glo™ Kinase assay. Obtained hits were counter-screened against CK2α revealing two CK2α' selective compounds. These two compounds might serve as the basis for further medicinal chemistry optimization for the potential treatment of HD.

Retinoic acid receptor alpha (RARα) antagonist ER-50891 and 15 analogs were prepared and tested in vitro for potency and selectivity at RARα, RARβ, 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.

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.

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.

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 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.

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. 1 However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. 1-2 In this paper, we describe the development and mechanism of action of new type III inhibitors that bind CDK2 with nanomolar affinity, making them the highest affinity, structurally confirmed allosteric CDK inhibitors reported. 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 efficacious contraceptive agents is seen by incubation with mouse testicular explants, where they recapitulate Cdk2 -/- and Spdya -/- phenotypes.

Despite the status of cyclin-dependent kinase 2 (CDK2) as a validated target for both anticancer and contraceptive indications, a CDK2 inhibitor with exquisite selectivity has been historically challenging, largely due to the structural similarity of the ATP-binding site where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with potential to bind a compound with desirable selectivity. Using high-throughput and virtual screening methods, we discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. We previously reported that these allosteric CDK2 inhibitors demonstrate a negative cooperative relationship with cyclin binding, are selective for CDK2 over the structurally similar kinase CDK1 and show potential as a non-hormonal contraceptive agent. In this work, we describe our screening and lead optimization efforts that led to the discovery of compounds in this series like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable with a desirably long ½ life and adequate tissue distribution in mice, demonstrating the potential of this series as a therapeutic. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for further development of this series to yield an efficacious and selective allosteric CDK2 inhibitor.

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.

Universities worldwide are establishing drug discovery centers to facilitate translation of exciting new human disease biology into therapeutic modalities. Drug hunting activities are typically focused on lead finding (high-throughput screening) coupled with some measure of chemical and pharmacokinetic optimization. Ideally, the research yields novel, selective drug-like molecules suitable for in vivo proof-of-concept studies and preclinical drug target validation. Preclinical activities are increasingly conducted in partnership with a pharmaceutical company seeking to access and supplement their drug development pipeline. Perpetually striving to gain a competitive edge and enhance efficiency, productivity, and profitability, the pharma industry is simultaneously experimenting with open- and crowd-source platforms and innovation incubators. Both enterprises, therefore, benefit from each other. This article is composed of a series of contributions (vignettes) from eight academic centers in the United States, the United Kingdom, Sweden, and Japan and one US-based pharmaceutical company (Table 2). The perspectives cover a range of topics including the rise of academic drug discovery and public–private partnerships; mission, objectives, and evolution of a particular center; resourcing; performance metrics; strategic and tactical lessons learned; attributes of successful projects; and open innovation initiatives. The accounts are punctuated with case studies to illustrate collective inventive capabilities.

WEE2 oocyte meiosis inhibiting kinase is a well-conserved oocyte specific kinase with a dual regulatory role during meiosis. Active WEE2 maintains immature, germinal vesicle stage oocytes in prophase I arrest prior to the luteinizing hormone surge and facilitates exit from metaphase II arrest at fertilization. Spontaneous mutations at the WEE2 gene locus in women have been linked to total fertilization failure indicating that selective inhibitors to this kinase could function as non-hormonal contraceptives. Employing co-crystallization with WEE1 G2 checkpoint kinase inhibitors, we revealed the structural basis of action across WEE kinases and determined type I inhibitors were not selective to WEE2 over WEE1. In response, we performed in silico screening by FTMap/FTSite and Schrodinger SiteMap analysis to identify potential allosteric sites, then used an allosterically biased activity assay to conduct high-throughput screening of a 26 000 compound library containing scaffolds of known allosteric inhibitors. Resulting hits were validated and a selective inhibitor that binds full-length WEE2 was identified, designated GPHR-00336382, along with a fragment-like inhibitor that binds the kinase domain, GPHR-00355672. Additionally, we present an in vitro testing workflow to evaluate biological activity of candidate WEE2 inhibitors including; (1) enzyme-linked immunosorbent assays measuring WEE2 phosphorylation activity of cyclin dependent kinase 1 (CDK1; also known as cell division cycle 2 kinase, CDC2), (2) in vitro fertilization of bovine ova to determine inhibition of metaphase II exit, and (3) cell-proliferation assays to look for off-target effects against WEE1 in somatic (mitotic) cells.

Multidomain bromodomain-containing proteins regulate gene expression via chromatin binding, interactions with the transcriptional machinery, and by recruiting enzymatic activity. Selective inhibition of members of the bromodomain and extra-terminal (BET) family is important to understand their role in disease and gene regulation, although due to the similar binding sites of BET bromodomains, selective inhibitor discovery has been challenging. To support the bromodomain inhibitor discovery process, here we report the first application of protein-observed fluorine (PrOF) NMR to the tandem bromodomains of BRD4 and BRDT to quantify the selectivity of their interactions with acetylated histones as well as small molecules. We further determine the selectivity profile of a new class of ligands, 1,4-acylthiazepanes, and find them to have ≥3-10-fold selectivity for the C-terminal bromodomain of both BRD4 and BRDT. Given the speed and lower protein concentration required over traditional protein-observed NMR methods, we envision that these fluorinated tandem proteins may find use in fragment screening and evaluating nucleosome and transcription factor interactions.

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR (Ki = 0.84 nM) over MOR (Ki = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, β-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED75. The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC50 = 0.17 ± 0.08 μM) and without significant contralateral activity, suggesting a lack of systemic exposure.

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.