RESUMEN
Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.
Asunto(s)
Islas de CpG/genética , Enzimas de Restricción del ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T Reguladores/inmunología , Células Cultivadas , Metilación de ADN , Desmetilación , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Especificidad de Órganos , Cultivo Primario de CélulasRESUMEN
In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Compuestos de Anilina/farmacología , Pirimidinas/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Humanos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pirimidinas/química , Pirimidinas/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
Multiple small-molecule inhibitors of the ß-secretase enzyme (BACE1) are under preclinical or clinical investigation for Alzheimer's disease (AD). Prior work has illustrated robust lowering of central amyloid ß (Aß) after acute administration of BACE1 inhibitors. However, very few studies have assessed the overall impact of chronically administered BACE1 inhibitors on brain amyloid burden, neuropathology, and behavioral function in aged preclinical models. We investigated the effects of a potent nonbrain-penetrant BACE1 inhibitor, delivered directly to the brain using intracerebroventricular infusion in an aged transgenic mouse model. Intracerebroventricular infusion of the BACE1 inhibitor (0.3-23.5 µg/d) for 8 weeks, initiated in 17-month-old Tg2576 mice, produced dose-dependent increases in brain inhibitor concentrations (0.2-13 µm). BACE1 inhibition significantly reversed the behavioral deficit in contextual fear conditioning, and reduced brain Aß levels, plaque burden, and associated pathology (e.g., dystrophic neurites), with maximal effects attained with â¼1 µg/d dose. Strikingly, the BACE1 inhibitor also reversed amyloid pathology below baseline levels (amyloid burden at the start of treatment), without adversely affecting cerebral amyloid angiopathy, microhemorrhages, myelination, or neuromuscular function. Inhibitor-mediated decline in brain amyloid pathology was associated with an increase in microglial ramification. This is the first demonstration of chronically administered BACE1 inhibitor to activate microglia, reverse brain amyloid pathology, and elicit functional improvement in an aged transgenic mouse model. Thus, engagement of novel glial-mediated clearance mechanisms may drive disease-modifying therapeutic benefit with BACE1 inhibition in AD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/patología , Trastornos del Conocimiento/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Microglía/efectos de los fármacos , Factores de Edad , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/patología , Modelos Animales de Enfermedad , Miedo/efectos de los fármacos , Humanos , Infusiones Intraventriculares , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Transgénicos , Microglía/patología , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/patologíaRESUMEN
This Letter describes the synthesis and structure-activity relationships of a series of furo[2,3-d][1,3]thiazinamine BACE1 inhibitors. The co-crystal structure of a representative thiazinamine 2e bound with the BACE1 active site displayed a binding mode driven by interactions with the catalytic aspartate dyad and engagement of the biaryl amide toward the S1 and S3 pockets. This work indicates that furo[2,3-d]thiazine can serve as a viable bioisostere of the known furo[3,4-d]thiazine.
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Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Furanos/química , Furanos/farmacología , Tiazinas/química , Tiazinas/farmacología , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Dominio Catalítico , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Guanidinas/farmacología , Compuestos Macrocíclicos/farmacología , Prolina/análogos & derivados , Prolina/farmacología , Inhibidores de Proteasas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Péptidos beta-Amiloides/biosíntesis , Animales , Células CACO-2 , Catepsina D/antagonistas & inhibidores , Catepsina E/antagonistas & inhibidores , Perros , Guanidinas/síntesis química , Humanos , Compuestos Macrocíclicos/síntesis química , Células de Riñón Canino Madin Darby , Masculino , Ratones , Simulación del Acoplamiento Molecular , Pepsina A/antagonistas & inhibidores , Prolina/síntesis química , Inhibidores de Proteasas/síntesis químicaRESUMEN
Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.
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Trastornos del Conocimiento/tratamiento farmacológico , Epotilonas/uso terapéutico , Microtúbulos/patología , Degeneración Nerviosa/tratamiento farmacológico , Tauopatías/tratamiento farmacológico , Moduladores de Tubulina/uso terapéutico , Proteínas tau/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Trastornos del Conocimiento/complicaciones , Trastornos del Conocimiento/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Doxiciclina/farmacología , Evaluación Preclínica de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/psicología , Epotilonas/farmacología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/patología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microtúbulos/efectos de los fármacos , Tauopatías/complicaciones , Tauopatías/genética , Tauopatías/patología , Tauopatías/psicología , Moduladores de Tubulina/farmacología , Proteínas tau/antagonistas & inhibidores , Proteínas tau/biosíntesis , Proteínas tau/genéticaRESUMEN
A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aß and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aß levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aß40 and Aß42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending-dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Oxadiazoles/farmacología , Sulfonamidas/farmacología , Adolescente , Adulto , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Perros , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto JovenRESUMEN
AIM: To evaluate the single dose pharmacokinetics, pharmacodynamics, and preliminary tolerability of the γ-secretase inhibitor BMS-708163 (avagacestat) in young and elderly men and women. METHODS: All subjects received double-blinded administration of a single 50 mg dose of avagacestat in capsule form or matching placebo. Main evaluations included pharmacokinetics, safety, plasma amyloid-ß (Aß)(1-40) concentratios and exploration of Notch biomarkers. RESULTS: Avagacestat 50 mg capsule was well tolerated and rapidly absorbed among young and elderly subjects, with a median t(max) between 1 and 2 h post dose and an average half-life between 41 and 71 h. In general, subjects aged 75 years or more had higher AUC(0,∞) values than those aged less than 75 years. An exploratory analysis of Aß(1-40) serum concentrations showed a pattern of decreasing concentrations over the first 4-6 h followed by a rise above baseline that was maintained until the end of the assessment period. Adverse events were generally mild, occurring more frequently in elderly subjects, with no observed difference between subjects receiving avagacestat and placebo. No dose limiting gastrointestinal effects of avagacestat were observed and exploratory biomarkers of Notch inhibition did not change significantly. CONCLUSIONS: The favourable safety profile and pharmacokinetic effects of avagacestat in this study support its continued development, especially in the target population of elderly subjects with mild cognitive impairment or Alzheimer's disease.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Oxadiazoles/efectos adversos , Sulfonamidas/efectos adversos , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/tratamiento farmacológico , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxadiazoles/farmacocinética , Oxadiazoles/farmacología , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.
RESUMEN
Recent mouse knockout studies identified adapter protein-2-associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. BMS-986176/LX-9211 (4), as a highly selective, CNS-penetrable, and potent AAK1 inhibitor, has advanced into phase II human trials. On exploring the structure-activity relationship (SAR) around this biaryl alkyl ether chemotype, several additional compounds were found to be highly selective and potent AAK1 inhibitors with good druglike properties. Among these, compounds 43 and 58 showed very good efficacy in two neuropathic pain rat models and had excellent CNS penetration and spinal cord target engagement. Both compounds also exhibited favorable physicochemical and oral pharmacokinetic (PK) properties. Compound 58, a central pyridine isomer of BMS-986176/LX-9211 (4), was 4-fold more potent than 4 in vitro and showed lower plasma exposure needed to achieve similar efficacy compared to 4 in the CCI rat model. However, both 43 and 58 showed an inferior preclinical toxicity profile compared to 4.
Asunto(s)
Anestésicos Generales , Neuralgia , Animales , Éteres/uso terapéutico , Ratones , Neuralgia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ratas , Médula Espinal , Relación Estructura-ActividadRESUMEN
The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Lactamas/química , Lactamas/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Animales , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Lactamas/síntesis química , Lactamas/farmacocinética , Ratones , Modelos Moleculares , Ratas , Relación Estructura-ActividadRESUMEN
Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aß levels, but did not lower rat brain Aß due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.
Asunto(s)
Aminas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Indoles/síntesis química , Inhibidores de Proteasas/química , Piridinas/síntesis química , Aminas/síntesis química , Aminas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/sangre , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Indoles/química , Indoles/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Diaminas/química , Diaminas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Cristalografía por Rayos X , Diaminas/síntesis química , Diaminas/farmacocinética , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: Accumulation of amyloid-ß (Aß) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aß, but not all are equally tractable for drug discovery. RESULTS: To search for novel targets that affect brain Aß without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aß ELISA assays. Although robust Aß lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aß, including a GPR3 KO strain, which had previously been proposed as an Aß target. However, significantly increased Aß was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT). CONCLUSIONS: Thus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aß levels in the brain are rare.
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Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/genética , Técnicas de Inactivación de Genes/métodos , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Pruebas Genéticas/métodos , Manosiltransferasas/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Pentosiltransferasa/genética , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/metabolismoRESUMEN
To selectively target doxorubicin (Dox) to tumor tissue and thereby improve the therapeutic index and/or efficacy of Dox, matrix metalloproteinases (MMP) activated peptide-Dox prodrugs were designed and synthesized by coupling MMP-cleavable peptides to Dox. Preferred conjugates were good substrates for MMPs, poor substrates for neprilysin, an off-target proteinase, and stable in blood ex vivo. When administered to mice with HT1080 xenografts, conjugates, such as 19, preferentially released Dox in tumor relative to heart tissue and prevented tumor growth with less marrow toxicity than Dox.
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Antineoplásicos/química , Doxorrubicina/análogos & derivados , Descubrimiento de Drogas , Metaloproteinasas de la Matriz/química , Profármacos/química , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Doxorrubicina/farmacología , Descubrimiento de Drogas/métodos , Humanos , Metaloproteinasas de la Matriz/farmacología , Ratones , Profármacos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
A series of N-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-methoxybenzylamino)-butan-2-yl)benzamides has been synthesized as BACE inhibitors. A variety of P2 and P3 substituents has been explored, and these efforts have culminated in the identification of several 1,3,5-trisubstituted phenylcarboxyamides with potent BACE inhibitory activity.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Benzamidas/síntesis química , Inhibidores de Proteasas/síntesis química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Benzamidas/química , Benzamidas/farmacología , Línea Celular , Humanos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-Actividad , TransfecciónRESUMEN
A triazine hit identified from a screen of the BMS compound collection was optimized for potency, in vivo activity, and off-target profile to produce the bicyclic pyrimidine γ-secretase modulator BMS-932481. The compound showed robust reductions of Aß1-42 and Aß1-40 in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aß1-37 and Aß1-38 were observed, with no change in the total amount of Aß1-x produced. No Notch-based toxicity was observed, and the overall preclinical profile of BMS-932481 supported its further evaluation in human clinical trials.
RESUMEN
Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.
Asunto(s)
Edición Génica , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/fisiopatología , Animales , Línea Celular , Técnicas de Sustitución del Gen , Humanos , Ratones , Primates , Reproducibilidad de los Resultados , Visión OcularRESUMEN
Therapeutic approaches for Alzheimer's disease (AD) are guided by four disease characteristics: amyloid plaques, neurofibrillar tangles (NFT), neurodegeneration, and dementia. Amyloid plaques are composed largely of 4 kDa beta-amyloid (Abeta) peptides, with the more amyloidogenic, 42 amino acid form (Abeta42) as the primary species. Because multiple, rare mutations that cause early-onset, familial AD lead to increased production or aggregation of Abeta42, amyloid therapeutics aim to reduce the amount of toxic Abeta42 aggregates. Amyloid-based therapies include gamma-secretase inhibitors and modulators, BACE inhibitors, aggregation blockers, catabolism inducers, and anti-Abeta biologics. Tangles are composed of paired helical filaments of hyperphosphorylated tau protein. Tau-based therapeutics include kinase inhibitors, microtubule stabilizers, and catabolism inducers. Therapeutic strategies for neurodegeneration target multiple mechanisms, including excitotoxicity, mitochondrial dysfunction, oxidative damage, and inflammation or stimulation of neuronal viability. Although not disease modifying, cognition enhancers are important to treat the symptom of dementia. Strategies for cognition enhancement include cholinesterase inhibitors, and other approaches to enhance the signaling of cholinergic and glutamatergic neurons. In summary, plaques, tangles, neurodegeneration and dementia guide the development of multiple therapeutic approaches for AD and are the subject of this review.