RESUMEN
PURPOSE: Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene. Migalastat, a pharmacological chaperone, binds to specific mutant forms of α-galactosidase A to restore lysosomal activity. METHODS: A pharmacogenetic assay was used to identify the α-galactosidase A mutant forms amenable to migalastat. Six hundred Fabry disease-causing mutations were expressed in HEK-293 (HEK) cells; increases in α-galactosidase A activity were measured by a good laboratory practice (GLP)-validated assay (GLP HEK/Migalastat Amenability Assay). The predictive value of the assay was assessed based on pharmacodynamic responses to migalastat in phase II and III clinical studies. RESULTS: Comparison of the GLP HEK assay results in in vivo white blood cell α-galactosidase A responses to migalastat in male patients showed high sensitivity, specificity, and positive and negative predictive values (≥0.875). GLP HEK assay results were also predictive of decreases in kidney globotriaosylceramide in males and plasma globotriaosylsphingosine in males and females. The clinical study subset of amenable mutations (n = 51) was representative of all 268 amenable mutations identified by the GLP HEK assay. CONCLUSION: The GLP HEK assay is a clinically validated method of identifying male and female Fabry patients for treatment with migalastat.Genet Med 19 4, 430-438.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Enfermedad de Fabry/genética , Mutación , alfa-Galactosidasa/genética , 1-Desoxinojirimicina/administración & dosificación , 1-Desoxinojirimicina/farmacología , Bioensayo , Línea Celular , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Enfermedad de Fabry/tratamiento farmacológico , Femenino , Células HEK293 , Humanos , Leucocitos/efectos de los fármacos , Leucocitos/enzimología , Masculino , Valor Predictivo de las Pruebas , Estudios de Validación como AsuntoRESUMEN
The preclinical characterization of WS-50030 [7-{4-[3-(1H-inden-3-yl)propyl]piperazin-1-yl}-1,3-benzoxazol-2(3H)-one] is described. In vitro binding and functional studies revealed highest affinity to the D(2) receptor (D(2L) K(i), 4.0 nM) and serotonin transporter (K(i), 7.1 nM), potent D(2) partial agonist activity (EC(50), 0.38 nM; E(max), 30%), and complete block of the serotonin transporter (IC(50), 56.4 nM). Consistent with this in vitro profile, WS-50030 (10 mg/kg/day, 21 days) significantly increased extracellular 5-HT in the rat medial prefrontal cortex, short-term WS-50030 treatment blocked apomorphine-induced climbing (ID(50), 0.51 mg/kg) in a dose range that produced minimal catalepsy in mice and induced low levels of contralateral rotation in rats with unilateral substantia nigra 6-hydroxydopamine lesions (10 mg/kg i.p.), a behavioral profile similar to that of the D(2) partial agonist aripiprazole. In a rat model predictive of antipsychotic-like activity, WS-50030 and aripiprazole reduced conditioned avoidance responding by 42 and 55% at 10 mg/kg, respectively. Despite aripiprazole's reported lack of effect on serotonin transporters, long-term treatment with aripiprazole or WS-50030 reversed olfactory bulbectomy-induced hyperactivity at doses that did not reduce activity in sham-operated rats, indicating antidepressant-like activity for both compounds. Despite possessing serotonin reuptake inhibitory activity in addition to D(2) receptor partial agonism, WS-50030 displays activity in preclinical models predictive of antipsychotic- and antidepressant efficacy similar to aripiprazole, suggesting potential efficacy of WS-50030 versus positive and negative symptoms of schizophrenia, comorbid mood symptoms, bipolar disorder, major depressive disorder, and treatment-resistant depression. Furthermore, WS-50030 provides a tool to further explore how combining these mechanisms might differentiate from other antipsychotics or antidepressants.
Asunto(s)
Antidepresivos/farmacología , Antipsicóticos/farmacología , Benzoxazoles/farmacología , Agonistas de Dopamina/farmacología , Indenos/farmacología , Receptores de Dopamina D2/agonistas , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Animales , Antidepresivos/química , Antipsicóticos/química , Reacción de Prevención/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Benzoxazoles/química , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Agonistas de Dopamina/química , Evaluación Preclínica de Medicamentos , Humanos , Indenos/química , Masculino , Ratones , Ratones Endogámicos , Microdiálisis , Actividad Motora/efectos de los fármacos , Unión Proteica , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Serotonina/metabolismo , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas del Receptor de Serotonina 5-HT2 , Inhibidores Selectivos de la Recaptación de Serotonina/química , TransfecciónRESUMEN
A 5-fluoro-tetrahydrocarbazole serotonin reuptake inhibitor (SRI) building block was combined with a variety of linkers and dopamine D2 receptor ligands in an attempt to identify potent D2 partial agonist/SRI molecules for treatment of schizophrenia. This approach has the potential to treat a broader range of symptoms compared to existing therapies. Selected compounds in this series demonstrate high affinity for both targets and D2 partial agonism in cell-based and in vivo assays.
Asunto(s)
Carbazoles/química , Agonistas de Dopamina/química , Receptores de Dopamina D2/agonistas , Esquizofrenia/tratamiento farmacológico , Inhibidores Selectivos de la Recaptación de Serotonina/química , Antagonistas del Receptor de Serotonina 5-HT1 , Animales , Carbazoles/síntesis química , Carbazoles/farmacología , Modelos Animales de Enfermedad , Agonistas de Dopamina/síntesis química , Agonistas de Dopamina/farmacología , Ratas , Receptor de Serotonina 5-HT1A/metabolismo , Receptores de Dopamina D2/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/síntesis química , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca(2+) assays (EC(50) = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 microM) and competed for binding of [(3)H]2-methyl-6-(phenylethynyl)pyridine (K(i) = 4.3 microM), but not [(3)H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Antipsicóticos/farmacología , Cognición/efectos de los fármacos , Oxadiazoles/farmacología , Piperidinas/farmacología , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Animales , Reacción de Prevención/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Hipocampo/metabolismo , Humanos , Corteza Prefrontal/metabolismo , Ratas , Receptor del Glutamato Metabotropico 5RESUMEN
The N-type voltage-gated calcium channel (Ca(v)2.2) functions in neurons to regulate neurotransmitter release. It comprises a clinically relevant target for chronic pain. We have validated a calcium mobilization approach to assessing Ca(v)2.2 pharmacology in two stable Ca(v)2.2 cell lines: alpha1(B), alpha2delta, beta(3)-HEK-293 and alpha1(B), beta(3)-HEK-293. Ca(v)2.2 channels were opened by addition of KCl and Ca(2+) mobilization was measured by Fluo-4 fluorescence on a fluorescence imaging plate reader (FLIPR(96)). Ca(v)2.2 expression and biophysics were confirmed by patch-clamp electrophysiology (EP). Both cell lines responded to KCl with adequate signal-to-background. Signals from both cell lines were inhibited by omega-conotoxin (ctx)-MVIIa and omega-conotoxin (ctx)-GVIa with IC(50) values of 1.8 and 1nM, respectively, for the three-subunit stable, and 0.9 and 0.6nM, respectively, for the two-subunit stable. Other known Ca(v)2.2 blockers were characterized including cadmium, flunarizine, fluspirilene, and mibefradil. IC(50) values correlated with literature EP-derived values. Novel Ca(v)2.2 pharmacology was identified in classes of compounds with other primary pharmacological activities, including Na(+) channel inhibitors and antidepressants. Novel Na(+) channel compounds with high potency at Ca(v)2.2 were identified in the phenoxyphenyl pyridine, phenoxyphenyl pyrazole, and other classes. The highest potency at Ca(v)2.2 tricyclic antidepressant identified was desipramine.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Transducción de Señal/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/genética , Células Cultivadas , Electrofisiología , HumanosRESUMEN
Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (Vm) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR Vm assay of rat Nav1.2 NaCh. Channels were opened by addition of veratridine, and Vm dye responses were measured. The IC50 values from various structural classes of compounds were compared to the resting state binding constant (Kr)and inactivated state binding constant (Ki)obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with Ki but not Kr. FLIPRIC50 values fell within 0.1-to 1.5-fold of EP Ki values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology.
Asunto(s)
Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Activación del Canal Iónico/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Bioensayo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Potenciales de la Membrana/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.2 , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio , Veratridina/farmacologíaRESUMEN
A fluorescent imaging plate reader (FLIPR) membrane potential (V(m)) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT(2)) in a stable rGlyT(2)-HEK cell line. Data show that glycine activation of rGlyT(2) consistently results in a concentration-dependent V(m) response on the FLIPR that is blocked by the potent and selective GlyT(2) antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [(3)H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT(2) physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT(2) inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V(m) assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT(2).