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.

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.

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, 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.

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.

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.

Steroidal Antagonists of Progesterone- and Prostaglandin E-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 E, and the fungal natural product -sirenin, but these steroidal inhibitors failed to prevent calcium influx in response to elevated K and pH.

Quantifying the Selectivity of Protein-Protein and Small Molecule Interactions with Fluorinated Tandem Bromodomain Reader Proteins.

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.

FBNTI, a DOR-Selective Antagonist That Allosterically Activates MOR within a MOR-DOR Heteromer.

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 ( = 0.84 nM) over MOR ( = 160 nM).

Development of WEE2 kinase inhibitors as novel non-hormonal female contraceptives that target meiosis†.

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.

Cooperativity Between Orthosteric Inhibitors and Allosteric Inhibitor 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) in Cyclin-Dependent Kinase 2.

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.

Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures.

Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons.

The anti-parasitic agent suramin and several of its analogues are inhibitors of the DNA binding protein Mcm10.

Minichromosome maintenance protein 10 (Mcm10) is essential for DNA unwinding by the replisome during S phase. It is emerging as a promising anti-cancer target as MCM10 expression correlates with tumour progression and poor clinical outcomes. Here we used a competition-based fluorescence polarization (FP) high-throughput screening (HTS) strategy to identify compounds that inhibit Mcm10 from binding to DNA.

7-Methylation of Chenodeoxycholic Acid Derivatives Yields a Substantial Increase in TGR5 Receptor Potency.

TGR5 agonists are potential therapeutics for a variety of conditions including type 2 diabetes, obesity, and inflammatory bowel disease. After screening a library of chenodeoxycholic acid (CDCA) derivatives, it was determined that a range of modifications could be made to the acid moiety of CDCA which significantly increased TGR5 agonist potency. Surprisingly, methylation of the 7-hydroxyl of CDCA led to a further dramatic increase in potency, allowing the identification of 5.

Design, Synthesis, and Characterization of a Fluorescence Polarization Pan-BET Bromodomain Probe.

Several chemical probes have been developed for use in fluorescence polarization screening assays to aid in drug discovery for the bromodomain and extra-terminal domain (BET) proteins. However, few of those have been characterized in the literature. We have designed, synthesized, and thoroughly characterized a novel fluorescence polarization pan-BET chemical probe suitable for high-throughput screening, structure-activity relationships, and hit-to-lead potency and selectivity assays to identify and characterize BET bromodomain inhibitors.

Reduction of protein kinase A-mediated phosphorylation of ATXN1-S776 in Purkinje cells delays onset of Ataxia in a SCA1 mouse model.

Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation.

Design, Synthesis, and in Vitro and in Vivo Evaluation of Ouabain Analogues as Potent and Selective Na,K-ATPase α4 Isoform Inhibitors for Male Contraception.

Na,K-ATPase α4 is a testis-specific plasma membrane Na and K transporter expressed in sperm flagellum. Deletion of Na,K-ATPase α4 in male mice results in complete infertility, making it an attractive target for male contraception. Na,K-ATPase α4 is characterized by a high affinity for the cardiac glycoside ouabain.

Potent Pyrimidine and Pyrrolopyrimidine Inhibitors of Testis-Specific Serine/Threonine Kinase 2 (TSSK2).

Testis-specific serine/threonine kinase 2 (TSSK2) is an important target for reversible male contraception. A high-throughput screen of ≈17 000 compounds using a mobility shift assay identified two potent series of inhibitors having a pyrrolopyrimidine or pyrimidine core. The pyrrolopyrimidine 10 (IC 22 nm; GSK2163632A) and the pyrimidine 17 (IC 31 nm; ALK inhibitor 1) are the most potent TSSK2 inhibitors in these series, which contain the first sub-100 nanomolar inhibitors of any TSSK isoform reported, except for the broad kinase inhibitor staurosporine.

Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2.

The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined.

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer.

The Fungal Sexual Pheromone Sirenin Activates the Human CatSper Channel Complex.

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes.

Probing the S2' Subsite of the Anthrax Toxin Lethal Factor Using Novel N-Alkylated Hydroxamates.

The lethal factor (LF) enzyme secreted by Bacillus anthracis is a zinc hydrolase that is chiefly responsible for anthrax-related cell death. Although many studies of the design of small molecule LF inhibitors have been conducted, no LF inhibitor is yet available as a therapeutic agent. Inhibitors with considerable chemical diversity have been developed and investigated; however, the LF S2' subsite has not yet been systematically explored as a potential target for lead optimization.

From HTS to Phase I: the Institute for Therapeutics Discovery and Development at the University of Minnesota.

The high-throughput screening core at the University of Minnesota is part of the Institute for Therapeutics Discovery and Development (ITDD), a comprehensive drug discovery and development center. The Institute provides scientific services to both academic and business communities and supports translational medicine via collaborations and contractual work. The ITDD is well-known for its broad range of screening capabilities and offers extensive medicinal chemistry expertise along with GMP scale-up and pre-clinical pharmacology support.

Quantification of adverse events associated with functional MRI scanning and with real-time fMRI-based training.

Background: Although functional magnetic resonance imaging (fMRI) is in widespread research use, the safety of this approach has not been extensively quantitatively evaluated. Real-time fMRI (rtfMRI)-based training paradigms use fMRI neurofeedback and cognitive strategies to alter regional brain activation, and are currently being evaluated as a novel approach to treat neurological and psychiatric conditions.

Purpose: The purpose of this study is to determine the incidence and severity of any adverse events that might be caused by changes in brain activation brought about through fMRI or through rtfMRI-based training paradigms.

Pharmacological, pharmacokinetic, and primate analgesic efficacy profile of the novel bradykinin B1 Receptor antagonist ELN441958.

The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described.