RESUMEN
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.
Asunto(s)
Anticoncepción , Masculino , Ratones , Animales , Receptor alfa de Ácido Retinoico , Relación Estructura-ActividadRESUMEN
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.
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Alprostadil/antagonistas & inhibidores , Compuestos de Azabiciclo/farmacología , Benzamidas/farmacología , Canales de Calcio/metabolismo , Progesterona/antagonistas & inhibidores , Esteroides/farmacología , Aldosterona/química , Aldosterona/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Levonorgestrel/química , Levonorgestrel/farmacología , Masculino , Semen/efectos de los fármacos , Semen/metabolismo , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Esteroides/química , Relación Estructura-ActividadRESUMEN
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, ß-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.
Asunto(s)
Analgésicos Opioides/farmacología , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/agonistas , Analgésicos Opioides/química , Animales , Calcio/metabolismo , Células HEK293 , Humanos , Inyecciones Espinales , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Estructura Molecular , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismoRESUMEN
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.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Anticonceptivos Femeninos/administración & dosificación , Meiosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Bovinos , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Humanos , Oocitos/efectos de los fármacos , Oocitos/metabolismoRESUMEN
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. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.
Asunto(s)
Ataxia/metabolismo , Ataxina-1/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células de Purkinje/metabolismo , Serina/metabolismo , Animales , Ataxia/genética , Ataxia/patología , Ataxina-1/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Fosforilación/fisiología , Células de Purkinje/patología , Serina/genéticaRESUMEN
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. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 µM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.
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Anticonceptivos Masculinos/farmacología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/biosíntesis , Baculoviridae/genética , Caseínas/metabolismo , Proteínas del Citoesqueleto , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Fosfoproteínas , Fosforilación/efectos de los fármacos , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Recombinantes/genética , Estaurosporina/farmacologíaRESUMEN
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.
Asunto(s)
Anticoncepción , Testículo , Ratones , Animales , Masculino , Receptor alfa de Ácido Retinoico/metabolismo , Benzopiranos/farmacologíaRESUMEN
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.
Asunto(s)
Anticonceptivos Masculinos , Masculino , Humanos , Animales , Ratones , Quinasa 2 Dependiente de la Ciclina , Relación Estructura-Actividad , Anticonceptivos Masculinos/farmacología , Recuento de Espermatozoides , Semen/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
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.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina , Ciclinas , Inhibidores de Proteínas Quinasas , Animales , Ratones , Anticoncepción , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Ciclinas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
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. METHOD: Quantitative adverse event self-report data were obtained from 641 functional imaging scans in 114 chronic pain patients participating in a research clinical trial examining repeated fMRI scans and rtfMRI-based training. Participants recorded potential adverse events during non-scanning baseline, fMRI scanning, or rtfMRI-based training sessions. RESULTS: There were no significant increases in the number of reported adverse events following fMRI or rtfMRI scanning sessions compared to baseline non-scanning sessions in a chronic pain trial (N = 88). There were no reported adverse events of any kind for over 90% of sessions during the course of rtfMRI-based training. When adverse events were reported, they were almost exclusively mild or moderate in severity and similar to those observed in a non-scanning baseline session. There was no increase in adverse events reported by participants receiving feedback from any of four brain regions during repeated rtfMRI-based training scans compared to non-scanning baseline sessions. For chronic pain patients completing the rtfMRI-based training paradigm including up to a total of nine scan sessions (N = 69), neither the number nor severity of reported events increased during the fMRI or rtfMRI scanning portions of the paradigm. There were no significant increases in the number of reported adverse events in participants who withdrew from the study. CONCLUSION: Repeated fMRI scanning and rtfMRI training, consisting of repeated fMRI scanning in conjunction with cognitive strategies and real-time feedback from several regions of interest in multiple brain systems to control brain region activation, were not associated with an increase in adverse event number or severity. These results demonstrate the safety of repetitive fMRI scanning paradigms similar to those in use in many laboratories worldwide, as well as the safety rtfMRI-based training paradigms.
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Neuroimagen Funcional/efectos adversos , Imagen por Resonancia Magnética/efectos adversos , Seguridad del Paciente/estadística & datos numéricos , Adulto , Encéfalo/fisiopatología , Dolor Crónico/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neurorretroalimentación , Encuestas y CuestionariosRESUMEN
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.
Asunto(s)
Canales de Calcio , Motilidad Espermática , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Humanos , Masculino , Progesterona/metabolismo , Progesterona/farmacología , Semen/metabolismo , Espermatozoides/metabolismoRESUMEN
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.
Asunto(s)
Naftalenosulfonatos de Anilina/química , Óxidos N-Cíclicos/química , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Indolizinas/química , Inhibidores de Proteínas Quinasas/química , Compuestos de Piridinio/química , Roscovitina/química , Regulación Alostérica , Naftalenosulfonatos de Anilina/metabolismo , Óxidos N-Cíclicos/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Sinergismo Farmacológico , Humanos , Indolizinas/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Compuestos de Piridinio/metabolismo , Roscovitina/metabolismoRESUMEN
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.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Descubrimiento de Drogas/métodos , Halogenación , Histonas/química , Histonas/metabolismo , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Dominios Proteicos/efectos de los fármacos , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
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 5.6 nM TGR5 agonist 17.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Receptores Acoplados a Proteínas G/agonistas , Línea Celular , AMP Cíclico/metabolismo , Descubrimiento de Drogas , Humanos , Metilación , Simulación del Acoplamiento Molecular , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 µM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 µM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 µM 2-BP or 100 µM glutamate for 24 h, while 300 µM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.
RESUMEN
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. Of the five active compounds identified, only the anti-parasitic agent suramin exhibited a dose-dependent decrease in replication products in an in vitro replication assay. Structure-activity relationship evaluation identified several suramin analogues that inhibited ssDNA binding by the human Mcm10 internal domain and full-length Xenopus Mcm10, including analogues that are selective for Mcm10 over human RPA. Binding of suramin analogues to Mcm10 was confirmed by surface plasmon resonance (SPR). SPR and FP affinity determinations were highly correlated, with a similar rank between affinity and potency for killing colon cancer cells. Suramin analogue NF157 had the highest human Mcm10 binding affinity (FP Ki 170 nM, SPR KD 460 nM) and cell activity (IC50 38 µM). Suramin and its analogues are the first identified inhibitors of Mcm10 and probably block DNA binding by mimicking the DNA sugar phosphate backbone due to their extended, polysulfated anionic structures.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas de Mantenimiento de Minicromosoma/antagonistas & inhibidores , Suramina/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cinética , Proteínas de Mantenimiento de Minicromosoma/genética , Estructura Molecular , Unión Proteica , Suramina/análogos & derivados , Suramina/química , XenopusRESUMEN
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. With the goal of discovering selective inhibitors of the Na,K-ATPase α4 and of sperm function, ouabain derivatives were modified at the glycone (C3) and the lactone (C17) domains. Ouabagenin analogue 25, carrying a benzyltriazole moiety at C17, is a picomolar inhibitor of Na,K-ATPase α4, with an outstanding α4 isoform selectivity profile. Moreover, compound 25 decreased sperm motility in vitro and in vivo and affected sperm membrane potential, intracellular Ca2+, pH, and hypermotility. These results proved that the new ouabagenin triazole analogue is an effective and selective inhibitor of Na,K-ATPase α4 and sperm function.
Asunto(s)
Anticoncepción/métodos , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Masculino , Ratones , Ouabaína/análogos & derivados , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Relación Estructura-Actividad , Testículo/enzimologíaRESUMEN
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.
RESUMEN
Testis-specific serine/threonine kinaseâ 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 (IC50 22â nm; GSK2163632A) and the pyrimidine 17 (IC50 31â nm; ALK inhibitorâ 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. The novel, potent pyrimidine TSSK2 inhibitor compound 19 (IC50 66â nm; 2-[[5-chloro-2-[2-methoxy-4-(1-methylpiperidin-4-yl)anilino]pyrimidin-4-yl]amino]-N-methylbenzenesulfonamide) lacks the potential for metabolic activation. Compound 19 had a potency rank order of TSSK1>TSSK2>TSSK3>TSSK6, indicating that potent dual inhibitors of TSSK1/2 can be identified, which may be required for a complete contraceptive effect. The future availability of a TSSK2 crystal structure will facilitate structure-based discovery of selective TSSK inhibitors from these pyrrolopyrimidine and pyrimidine scaffolds.