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Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.
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Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.
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Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.
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INTRODUCTION: Hormonal contraceptives are among the most effective forms of reversible contraception, but many other compounds, including some antiretrovirals, have clinically meaningful drug-drug interactions (DDIs) with hormonal contraceptives. Islatravir is a novel human immunodeficiency virus nucleoside reverse transcriptase translocation inhibitor currently in clinical development for treatment and prevention of HIV infection. A phase 1 clinical trial was conducted to evaluate the DDI of islatravir and the combination of oral contraceptive levonorgestrel (LNG)/ethinyl estradiol (EE). METHODS: This was an open-label, two-period, fixed-sequence, DDI clinical trial in healthy, postmenopausal or bilaterally oophorectomized females aged 18 through 65 years in the United States between October 2016 and January 2017. A single dose of LNG 0.15 mg/EE 0.03 mg was given followed by a 7-day washout. Islatravir, 20 mg, was then dosed once weekly for 3 weeks; a single dose of LNG 0.15 mg/EE 0.03 mg was given concomitantly with the third dose of islatravir. Pharmacokinetic samples for plasma LNG and EE concentrations were collected pre-dose and up to 120 hours post-dose in each period. Safety and tolerability were assessed throughout the trial by clinical assessments, laboratory evaluations and examination of adverse events. RESULTS AND DISCUSSION: Fourteen participants were enrolled. The pharmacokinetics of LNG and EE were not meaningfully altered by co-administration with islatravir. For the comparison of (islatravir + LNG/EE)/(LNG/EE alone), the geometric mean ratios (GMRs) (90% confidence intervals [CIs]) for LNG AUC0-inf and Cmax were 1.13 (1.06, 1.20) and 0.965 (0.881, 1.06), respectively. For EE, the GMRs (90% CI) for AUC0-inf and Cmax were 1.05 (0.981, 1.11) and 1.02 (0.971, 1.08), respectively. Co-administration of all three drugs was generally well tolerated. CONCLUSIONS: The results of this trial support the use of LNG/EE contraceptives in combination with islatravir without dose adjustment.
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Etinilestradiol , Infecciones por VIH , Adulto , Anticoncepción , Anticonceptivos Orales Combinados/efectos adversos , Desoxiadenosinas , Interacciones Farmacológicas , Etinilestradiol/efectos adversos , Femenino , Humanos , Levonorgestrel/efectos adversosRESUMEN
Islatravir, an investigational nucleoside reverse transcriptase translocation inhibitor, is in clinical development for the treatment and prevention of HIV-1 infection. Because islatravir may be coadministered with other antiretroviral agents, assessment of potential drug-drug interactions are warranted. This phase 1, open-label, fixed-sequence, 2-period trial in adults without HIV (N = 12) assessed the safety and pharmacokinetic interactions of islatravir administered with dolutegravir and tenofovir disoproxil fumarate (TDF). In period 1, participants received a single oral dose of islatravir (20 mg). In period 2, participants received oral doses of dolutegravir (50 mg) and TDF (300 mg) once daily on days 1 through 11, with a single oral dose of islatravir (20 mg) coadministered on day 8. There were no clinically significant changes in islatravir, dolutegravir, or TDF pharmacokinetics following coadministration. Islatravir was generally well tolerated when administered alone or in combination with dolutegravir and TDF. Coadministration of islatravir, dolutegravir, and TDF is supported, with no clinically meaningful effect on pharmacokinetics, safety, or tolerability in participants without HIV.
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VIH-1 , Adulto , Antirretrovirales , Desoxiadenosinas , Interacciones Farmacológicas , Compuestos Heterocíclicos con 3 Anillos , Humanos , Oxazinas , Piperazinas , Piridonas , Tenofovir/farmacocinéticaRESUMEN
BACKGROUND: Islatravir (MK-8591) is a novel nucleoside analog in development for the treatment and prevention of HIV-1 infection. Islatravir has potent antiviral activity and a long intracellular half-life. SETTING: A 3-panel, randomized, double-blind, placebo-controlled, multiple-dose study in 36 adults without HIV evaluated the safety, tolerability, and pharmacokinetics of islatravir after daily administration. METHODS: Islatravir or placebo was administered orally once daily for 42 days (5 mg) or 28 days (0.25 mg; 0.75 mg). Blood samples were taken at prespecified time points for pharmacokinetic analysis of islatravir (plasma) and islatravir-triphosphate (ISL-TP; peripheral blood mononuclear cells [PBMCs]). Rectal and vaginal tissue samples were also collected in a subset of participants. Safety and tolerability were evaluated throughout. RESULTS: The pharmacokinetics of islatravir were approximately dose proportional, with concentrations approaching a steady state between days 14 and 21 in plasma and by day 28 for ISL-TP in PBMCs. Plasma exposure accumulation was 1.5-fold to 1.8-fold, and ISL-TP exposure accumulation was â¼10-fold. The apparent terminal half-life of ISL-TP was 177-209 hours. The ISL-TP pharmacokinetic trough threshold-the minimal concentration required for efficacy-of 0.05 pmol/106 cells was achieved after a single administration at all dose levels. Rectal and vaginal tissue also exhibited potentially therapeutic concentrations. Islatravir was generally well tolerated at all doses. CONCLUSIONS: ISL-TP levels in PBMCs were above the threshold projected for antiviral efficacy against wild-type HIV after a single 0.25-mg dose. Multiple once-daily dosing of islatravir in adults without HIV was generally well tolerated up to doses of 5 mg administered for up to 6 weeks.
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Desoxiadenosinas/farmacocinética , Seronegatividad para VIH , Administración Oral , Antivirales/uso terapéutico , Desoxiadenosinas/uso terapéutico , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Esquema de Medicación , Femenino , Semivida , Humanos , Leucocitos MononuclearesRESUMEN
Islatravir (MK-8591) is a nucleoside reverse transcriptase translocation inhibitor in development for the treatment and prevention of HIV-1. The potential for islatravir to interact with commonly co-prescribed medications was studied in vitro. Elimination of islatravir is expected to be balanced between adenosine deaminase-mediated metabolism and renal excretion. Islatravir did not inhibit uridine diphosphate glucuronosyltransferase 1A1 or cytochrome p450 (CYP) enzymes CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4, nor did it induce CYP1A2, 2B6, or 3A4. Islatravir did not inhibit hepatic transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 1, bile salt export pump (BSEP), multidrug resistance-associated protein (MRP) 2, MRP3, or MRP4. Islatravir was neither a substrate nor a significant inhibitor of renal transporters organic anion transporter (OAT) 1, OAT3, OCT2, multidrug and toxin extrusion protein (MATE) 1, or MATE2K. Islatravir did not significantly inhibit P-glycoprotein and breast cancer resistance protein (BCRP); however, it was a substrate of BCRP, which is not expected to be of clinical significance. These findings suggest islatravir is unlikely to be the victim or perpetrator of drug-drug interactions with commonly co-prescribed medications, including statins, diuretics, anti-diabetic drugs, proton pump inhibitors, anticoagulants, benzodiazepines, and selective serotonin reuptake inhibitors.
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Desoxiadenosinas/metabolismo , Interacciones Farmacológicas , Preparaciones Farmacéuticas/metabolismo , Inhibidores de la Transcriptasa Inversa/metabolismo , Animales , Transporte Biológico , Sistema Enzimático del Citocromo P-450/metabolismo , Desoxiadenosinas/sangre , Perros , Infecciones por VIH/tratamiento farmacológico , Humanos , Técnicas In Vitro , Células de Riñón Canino Madin Darby , Proteínas de Transporte de Membrana/metabolismo , Ratones , Transportadores de Anión Orgánico/metabolismo , ConejosRESUMEN
BACKGROUND AND OBJECTIVES: Islatravir (MK-8591) is a novel nucleoside analogue in development for the treatment and prevention of HIV-1 infection. Doravirine is a non-nucleoside reverse transcriptase inhibitor indicated for the treatment of HIV-1 infection. This study evaluated the pharmacokinetics, safety, and tolerability of islatravir and doravirine coadministration in a double-blind, placebo-controlled, randomized, fixed-sequence study. METHODS: Adult participants without HIV infection were administered oral doravirine 100 mg (n = 10) or placebo (n = 4) once daily (QD) for 5 days, immediately followed by oral islatravir 2.25 mg (n = 10) or placebo QD (n = 4) for 14 days; islatravir 2.25 mg and doravirine 100 mg QD, or placebo QD, were then coadministered for 5 days. Pharmacokinetic and safety data were collected. RESULTS: Doravirine geometric least-squares mean ratios (90% confidence intervals (CIs)) of (doravirine + islatravir)/doravirine for the area under the plasma drug concentration-time curve over 24 h (AUC0-24h), maximum plasma concentration (Cmax), and plasma concentration at 24 h post-dose (C24h) were not meaningfully impacted. Islatravir geometric least-squares mean ratios (90% CI) of (islatravir + doravirine)/islatravir for AUC0-24h and Cmax were both close to unity, 1.06 (1.01, 1.12) and 1.08 (0.91, 1.27), respectively. All study regimens were generally well tolerated. CONCLUSION: These results indicate that coadministration of islatravir and doravirine had no clinically meaningful effect on the pharmacokinetics of either drug, and support further clinical investigation of islatravir in combination with doravirine for the treatment of HIV-1 infection.
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Desoxiadenosinas/administración & dosificación , Piridonas/administración & dosificación , Triazoles/administración & dosificación , Administración Oral , Adulto , Área Bajo la Curva , Desoxiadenosinas/efectos adversos , Desoxiadenosinas/sangre , Desoxiadenosinas/farmacocinética , Método Doble Ciego , Esquema de Medicación , Interacciones Farmacológicas , Femenino , Semivida , Humanos , Análisis de los Mínimos Cuadrados , Masculino , Persona de Mediana Edad , Efecto Placebo , Piridonas/efectos adversos , Piridonas/sangre , Piridonas/farmacocinética , Curva ROC , Somnolencia , Triazoles/efectos adversos , Triazoles/sangre , Triazoles/farmacocinética , Adulto JovenRESUMEN
Doravirine, a novel nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus 1 (HIV-1), is predominantly cleared by cytochrome P450 (CYP) 3A4 and metabolized to an oxidative metabolite (M9). Coadministration with rifabutin, a moderate CYP3A4 inducer, decreased doravirine exposure. Based on nonparametric superposition modeling, a doravirine dose adjustment from 100 mg once daily to 100 mg twice daily during rifabutin coadministration was proposed. However, M9 exposure may also be impacted by induction, in addition to the dose adjustment. As M9 concentrations have not been quantified in previous clinical studies, a physiologically based pharmacokinetic model was developed to investigate the change in M9 exposure when doravirine is coadministered with CYP3A inducers. Simulations demonstrated that although CYP3A induction increases doravirine clearance by up to 4.4-fold, M9 exposure is increased by only 1.2-fold relative to exposures for doravirine 100 mg once daily in the absence of CYP3A induction. Thus, a 2.4-fold increase in M9 exposure relative to the clinical dose of doravirine is anticipated when doravirine 100 mg twice daily is coadministered with rifabutin. In a subsequent clinical trial, doravirine and M9 exposures, when doravirine 100 mg twice daily was coadministered with rifabutin, were found to be consistent with model predictions using rifampin and efavirenz as representative inducers. These findings support the dose adjustment to doravirine 100 mg twice daily when coadministered with rifabutin.
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Modelos Biológicos , Piridonas/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacocinética , Rifabutina/farmacología , Triazoles/farmacocinética , Adulto , Alquinos/farmacología , Benzoxazinas/farmacología , Simulación por Computador , Ciclopropanos/farmacología , Citocromo P-450 CYP3A/efectos de los fármacos , Citocromo P-450 CYP3A/metabolismo , Inductores del Citocromo P-450 CYP3A/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piridonas/administración & dosificación , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Rifabutina/administración & dosificación , Rifampin/farmacología , Triazoles/administración & dosificación , Adulto JovenRESUMEN
Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.
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Doravirine is a novel nonnucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1 infection. In vitro studies were conducted to assess the potential for drug interactions with doravirine via major drug-metabolizing enzymes and transporters. Kinetic studies confirmed that cytochrome P450 3A (CYP3A) plays a major role in the metabolism of doravirine, with â¼20-fold-higher catalytic efficiency for CYP3A4 versus CYP3A5. Doravirine was not a substrate of breast cancer resistance protein (BCRP) and likely not a substrate of organic anion transporting polypeptide 1B1 (OATP1B1) or OATP1B3. Doravirine was not a reversible inhibitor of major CYP enzymes (CYP1A2, -2B6, -2C8, -2C9, -2C19, -2D6, and -3A4) or of UGT1A1, nor was it a time-dependent inhibitor of CYP3A4. No induction of CYP1A2 or -2B6 was observed in cultured human hepatocytes; small increases in CYP3A4 mRNA (≤20%) were reported at doravirine concentrations of ≥10 µM but with no corresponding increase in enzyme activity. In vitro transport studies indicated a low potential for interactions with substrates of BCRP, P-glycoprotein, OATP1B1 and OATP1B3, the bile salt extrusion pump (BSEP), organic anion transporter 1 (OAT1) and OAT3, organic cation transporter 2 (OCT2), and multidrug and toxin extrusion 1 (MATE1) and MATE2K proteins. In summary, these in vitro findings indicate that CYP3A4 and CYP3A5 mediate the metabolism of doravirine, although with different catalytic efficiencies. Clinical trials reported elsewhere confirm that doravirine is subject to drug-drug interactions (DDIs) via CYP3A inhibitors and inducers, but they support the notion that DDIs (either direction) are unlikely via other major drug-metabolizing enzymes and transporters.
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Interacciones Farmacológicas/fisiología , Piridonas/metabolismo , Triazoles/metabolismo , Animales , Transporte Biológico/fisiología , Línea Celular , Citocromo P-450 CYP3A/metabolismo , Perros , Células HEK293 , Hepatocitos/metabolismo , Humanos , Cinética , Células de Riñón Canino Madin Darby , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Absorption, distribution, metabolism and elimination of doravirine (MK-1439), a novel non-nucleoside reverse transcriptase inhibitor, were investigated. Two clinical trials were conducted in healthy subjects: an oral single dose [14 C]doravirine (350 mg, â¼200 µCi) trial (n = 6) and an intravenous (IV) single-dose doravirine (100 µg) trial (n = 12). In vitro metabolism, protein binding, apparent permeability and P-glycoprotein (P-gp) transport studies were conducted to complement the clinical trials. Following oral [14 C]doravirine administration, all of the administered dose was recovered. The absorbed dose was eliminated primarily via metabolism. An oxidative metabolite (M9) was the predominant metabolite in excreta and was the primary circulating metabolite (12.9% of circulating radioactivity). Following IV administration, doravirine clearance and volume of distribution were 3.73 L/h (95% confidence intervals (CI) 3.09, 4.49) and 60.5 L (95% CI 53.7, 68.4), respectively. In vitro, doravirine is not highly bound to plasma proteins (unbound fraction 0.24) and has good passive permeability. The metabolite M9 was generated by cytochrome P450 3A (CYP3A)4/5-mediated oxidation. Doravirine was a P-gp substrate but P-gp efflux is not expected to play a significant role in limiting doravirine absorption or to be involved in the elimination of doravirine. In conclusion, doravirine is a low clearance drug, primarily eliminated by CYP3A-mediated metabolism.
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Absorción Fisiológica , Piridonas/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacocinética , Triazoles/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Administración Intravenosa , Adulto , Humanos , Masculino , Metaboloma/efectos de los fármacos , Persona de Mediana Edad , Piridonas/sangre , Piridonas/química , Piridonas/orina , Inhibidores de la Transcriptasa Inversa/sangre , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/orina , Distribución Tisular , Triazoles/sangre , Triazoles/química , Triazoles/orina , Adulto JovenRESUMEN
Regimen adherence remains a major hurdle to the success of daily oral drug regimens for the treatment and prevention of human immunodeficiency virus (HIV) infection. Long-acting drug formulations requiring less-frequent dosing offer an opportunity to improve adherence and allow for more forgiving options with regard to missed doses. The administration of long-acting formulations in a clinical setting enables health care providers to directly track adherence. MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is an investigational nucleoside reverse transcriptase translocation inhibitor (NRTTI) drug candidate under investigation as part of a regimen for HIV treatment, with potential utility as a single agent for preexposure prophylaxis (PrEP). The active triphosphate of MK-8591 (MK-8591-TP) exhibits protracted intracellular persistence and, together with the potency of MK-8591, supports its consideration for extended-duration dosing. Toward this end, drug-eluting implant devices were designed to provide prolonged MK-8591 release in vitro and in vivo Implants, administered subcutaneously, were studied in rodents and nonhuman primates to establish MK-8591 pharmacokinetics and intracellular levels of MK-8591-TP. These data were evaluated against pharmacokinetic and pharmacodynamic models, as well as data generated in phase 1a (Ph1a) and Ph1b clinical studies with once-weekly oral administration of MK-8591. After a single administration in animals, MK-8591 implants achieved clinically relevant drug exposures and sustained drug release, with plasma levels maintained for greater than 6 months that correspond to efficacious MK-8591-TP levels, resulting in a 1.6-log reduction in viral load. Additional studies of MK-8591 implants for HIV treatment and prevention are warranted.
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Desoxiadenosinas/uso terapéutico , Portadores de Fármacos/química , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/prevención & control , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Animales , Fármacos Anti-VIH , Desoxiadenosinas/química , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Macaca mulatta , Masculino , Polímeros/química , Ratas , Ratas Wistar , Inhibidores de la Transcriptasa Inversa/químicaRESUMEN
Investigation of a novel amino-aza-benzimidazolone structural class of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 2 (mGluR2) identified [2.2.2]-bicyclic amine 12 as an intriguing lead structure due to its promising physicochemical properties and lipophilic ligand efficiency (LLE). Further optimization led to chiral amide 18, which exhibited strong in vitro activity and attractive pharmacokinetic (PK) properties. Hypothesis-driven target design identified compound 21 as a potent, highly selective, orally bioavailable mGluR2 PAM, which addressed a CYP time-dependent inhibition (TDI) liability of 18, while maintaining excellent drug-like properties with robust in vivo activity in a clinically validated model of antipsychotic potential.
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Optimization of a benzimidazolone template for potency and physical properties revealed 5-aryl-1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-ones as a key template on which to develop a new series of mGlu2 positive allosteric modulators (PAMs). Systematic investigation of aryl-SAR led to the identification of compound 27 as a potent and highly selective mGlu2 PAM with sufficient pharmacokinetics to advance to preclinical models of psychosis. Gratifyingly, compound 27 showed full efficacy in the PCP- and MK-801-induced hyperlocomotion assay in rats at CSF concentrations consistent with mGlu2 PAM potency.
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Imidazoles/química , Piridinas/química , Piridonas/química , Receptores de Glutamato Metabotrópico/química , Regulación Alostérica , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Imidazoles/sangre , Imidazoles/farmacología , Imidazoles/uso terapéutico , Locomoción/efectos de los fármacos , Unión Proteica , Trastornos Psicóticos/tratamiento farmacológico , Trastornos Psicóticos/patología , Piridinas/farmacología , Piridinas/uso terapéutico , Piridonas/sangre , Piridonas/farmacología , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Relación Estructura-ActividadRESUMEN
Hit to lead optimization of (5R)-5-hexyl-3-phenyl-1,3-oxazolidin-2-one as a positive allosteric modulator of mGluR2 is described. Improvements in potency and metabolic stability were achieved through SAR on both ends of the oxazolidinone. An optimized lead compound was found to be brain penetrant and active in a rat ketamine-induced hyperlocomotion model for antipsychotic activity.
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Oxazolidinonas/química , Receptores de Glutamato Metabotrópico/metabolismo , Esquizofrenia/tratamiento farmacológico , Regulación Alostérica , Animales , Antipsicóticos , Ketamina/toxicidad , Oxazolidinonas/síntesis química , Oxazolidinonas/farmacología , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Relación Estructura-ActividadRESUMEN
Novel oxazolobenzimidazoles are described as potent and selective positive allosteric modulators of the metabotropic glutamate receptor 2. The discovery of this class and optimization of its physical and pharmacokinetic properties led to the identification of potent and orally bioavailable compounds (20 and 21) as advanced leads. Compound 20 (TBPCOB) was shown to have robust activity in a PCP-induced hyperlocomotion model in rat, an assay responsive to clinical antipsychotic treatments for schizophrenia.
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Throughput for drug metabolite identification studies has been increased significantly by the combined use of accurate mass liquid chromatography/tandem mass spectrometry (LC/MS/MS) data on a quadrupole time-of-flight (QTOF) system and targeted data analysis procedures. Employed in concert, these tools have led to the implementation of a semi-automated high-throughput metabolite identification strategy that has been incorporated successfully into lead optimization efforts in drug discovery. The availability of elemental composition data on precursor and all fragment ions in each spectrum has greatly enhanced confidence in ion structure assignments, while computer-based algorithms for defining sites of biotransformation based upon mass shifts of diagnostic fragment ions have facilitated identification of positions of metabolic transformation in drug candidates. Adoption of this technology as the 'first-line' approach for in vitro metabolite profiling has resulted in the analysis of as many as 21 new chemical entities on one day from diverse structural classes and therapeutic programs.
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Cromatografía Líquida de Alta Presión/métodos , Perfilación de la Expresión Génica/métodos , Glutatión/farmacocinética , Microsomas Hepáticos/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Perros , Humanos , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The fosfomycin resistance protein, FosX, catalyzes the hydration of the antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. Genes encoding the enzyme are found in several pathogenic microorganisms. The structure and mechanism of action of the genomically encoded FosX enzyme from Listeria monocytogenes (FosXLMATCC) obtained from the American Type Culture Collection are reported. The gene harbors 31 point mutations, and as a consequence, the protein differs in 10 amino acid residues from the previously reported FosX encoded in the genome of the EGD strain of L. monocytogenes (FosXLMEGD). The FosXLMATCC enzyme is shown to catalyze the addition of water to the C1 position of the antibiotic with inversion of configuration at C1. The reaction involves Mn(II) activation of the oxirane oxygen and E44 acting as a general base. The structure of the enzyme has been determined from six different crystal forms of the protein. The structures of the enzyme without metal bound are similar but differ in the loop regions. Perhaps the most informative structure is the one with the product bound. This structure shows that the phosphonate group of the product is bound in an orientation that is different than that of fosfomycin bound to the related enzyme, FosA. The implication is that the substrate may also be bound in a different orientation in FosX. A high-resolution structure (1.44 A resolution) of the enzyme reveals a unique conformation in which the C-terminal tail of the protein coordinates to the Mn(II) center via the carboxylate of E126. The kinetic characterization of the E126Q mutant indicates that this conformation of the protein is probably not relevant to the function of the enzyme. Kinetic analysis of mutants of active site residue E44 is consistent with its proposed roll as a general base catalyst in the addition of water to the antibiotic.
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Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Fosfomicina/farmacología , Listeria monocytogenes/genética , Secuencia de Bases , Cristalografía , Cartilla de ADN , Listeria monocytogenes/efectos de los fármacos , Modelos Moleculares , Mutación Puntual , Conformación ProteicaRESUMEN
Three similar but mechanistically distinct fosfomycin resistance proteins that catalyze the opening of the oxirane ring of the antibiotic are known. FosA is a Mn(II) and K(+)-dependent glutathione transferase. FosB is a Mg(2+)-dependent L-cysteine thiol transferase. FosX is a Mn(II)-dependent fosfomycin-specific epoxide hydrolase. The expression, purification, kinetic, and physical characteristics of six fosfomycin resistance proteins including the FosA proteins from transposon TN2921 and Pseudomonas aeruginosa, the FosB proteins from Bacillus subtilis and Staphylococcus aureus, and the FosX proteins from Mesorhizobium loti and Listeria monocytogenes are reported.