RESUMEN
Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure-activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano. Potent anti-prion compounds were advanced to pharmacokinetic studies, and the resulting brain-penetrant leads from each series, together with a related N-aryl piperazine lead, were escalated to long-term dosing and efficacy studies. Compounds from each of the four series doubled the survival of mice infected with a mouse-passaged prion strain. Treatment with aryl amides altered prion strain properties, as evidenced by the distinct patterns of neuropathological deposition of prion protein and associated astrocytic gliosis in the brain; however, none of the aryl amide compounds resulted in drug-resistant prion strains, in contrast to previous studies on compounds with the 2-aminothiazole (2-AMT) scaffold. As seen with 2-AMTs and other effective anti-prion compounds reported to date, the novel aryl amides reported here were ineffective in prolonging the survival of transgenic mice infected with human prions. Most encouraging is our discovery that aryl amides show that the development of drug resistance is not an inevitable consequence of efficacious anti-prion therapeutics.
Asunto(s)
Amidas/química , Amidas/farmacología , Descubrimiento de Drogas , Enfermedades por Prión/tratamiento farmacológico , Amidas/metabolismo , Amidas/uso terapéutico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/tratamiento farmacológico , Femenino , Ratones , Enfermedades por Prión/metabolismo , Relación Estructura-Actividad , Análisis de SupervivenciaRESUMEN
Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from â¼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, ß-sheet-rich "scrapie" isoform (PrP(Sc)) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP(Sc) and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP(Sc) accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP(Sc) that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/patología , Proteínas PrPSc/metabolismo , Tiazoles/química , Tiazoles/farmacología , Animales , Encéfalo/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Proteínas PrPSc/genética , Scrapie/patología , Especificidad de la Especie , Análisis de Supervivencia , Tasa de Supervivencia , Tiazoles/farmacocinética , Tiazoles/uso terapéutico , Transgenes/genética , Resultado del TratamientoRESUMEN
PURPOSE: Previous studies showed that lowering PrP(C) concomitantly reduced PrP(Sc) in the brains of mice inoculated with prions. We aimed to develop assays that measure PrP(C) on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrP(C) levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice. METHODS: We utilized HTS ELISA assays to identify small molecules that lower PrP(C) levels by ≥30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells. RESULTS: From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose-response (EC50) in both cell lines, only studies in T98G cells identified compounds that reduced PrP(C) without killing the cells. EC50 values from 32 hits ranged from 65 nM to 4.1 µM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 µM, but only after intraperitoneal dosing. CONCLUSIONS: Our studies identified leads for future studies to determine which compounds might lower PrP(C) levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions.
Asunto(s)
Priones/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Encéfalo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Microscopía Confocal , Estructura Molecular , Isoformas de Proteínas , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Propiedades de Superficie , Distribución Tisular , Células Tumorales CultivadasRESUMEN
The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed "Compd B" (4-pyridinecarboxaldehyde,2-[4-(5-oxazolyl)phenyl]hydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.
Asunto(s)
Amidas/química , Amidas/uso terapéutico , Hidrazonas/química , Hidrazonas/uso terapéutico , Proteínas PrPSc/patogenicidad , Enfermedades por Prión/tratamiento farmacológico , Amidas/síntesis química , Amidas/farmacocinética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Hidrazonas/síntesis química , Hidrazonas/farmacocinética , Periodo de Incubación de Enfermedades Infecciosas , Estimación de Kaplan-Meier , Ratones , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Proteínas PrPSc/genética , Relación Estructura-ActividadRESUMEN
PURPOSE: To discover drugs lowering PrP(Sc) in prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases. METHODS: We tested 2-AMT analogs for EC50 and PK after a 40 mg/kg single dose and 40-210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50 after dosing. We reasoned that compounds with high AUC/EC50 ratios should be good candidates going forward. RESULTS: We evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27-40%. AUC/EC50 ratios after 3 days were >100 (total) and 48-113 (unbound). Stability in liver microsomes ranged from 30->60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter. CONCLUSIONS: IND24 and IND81 are active in vitro and show high AUC/EC50 ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease.
Asunto(s)
Proteínas PrPSc/antagonistas & inhibidores , Enfermedades por Prión/tratamiento farmacológico , Tiazoles/metabolismo , Tiazoles/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Área Bajo la Curva , Disponibilidad Biológica , Encéfalo/metabolismo , Línea Celular , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Proteínas PrPSc/metabolismo , Isoformas de Proteínas/metabolismo , Solubilidad , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Tau prions feature in the brains of patients suffering from Alzheimer's disease and other tauopathies. For the development of therapeutics that target the replication of tau prions, a high-content, fluorescence-based cell assay was developed. Using this high-content phenotypic screen for nascent tau prion formation, a 4-piperazine isoquinoline compound (1) was identified as a hit with an EC50 value of 390 nM and 0.04 K p,uu. Analogs were synthesized using a hypothesis-based approach to improve potency and in vivo brain penetration resulting in compound 25 (EC50 = 15 nM; K p,uu = 0.63). We investigated the mechanism of action of this series and found that a small set of active compounds were also CDK8 inhibitors.
RESUMEN
Recently, we described the aminothiazole lead (4-biphenyl-4-ylthiazol-2-yl)-(6-methylpyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of â¼40 %, and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrP(Sc) . Accordingly, we sought to identify analogues of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining similar or superior properties. Herein we report the discovery of improved lead compounds such as (6-methylpyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)thiazol-2-yl]amine and cyclopropanecarboxylic acid (4-biphenylthiazol-2-yl)amide, which exhibit brain exposure/EC50 ratios at least tenfold greater than that of compound 1.