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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33446504

RESUMEN

Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.


Asunto(s)
Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Glicoproteínas de Membrana/genética , Microglía/metabolismo , Receptores Inmunológicos/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proliferación Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microglía/clasificación , Microglía/efectos de los fármacos , Microglía/patología , Mutación , Unión Proteica , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Factores Sexuales
2.
J Med Chem ; 63(5): 2263-2281, 2020 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-31589043

RESUMEN

ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease that plays a key role in the production of amyloid ß (Aß) in the brain and has been extensively pursued as a target for the treatment of Alzheimer's disease (AD). BACE2, an aspartyl protease that is structurally related to BACE1, has been recently reported to be involved in melanosome maturation and pigmentation. Herein, we describe the development of a series of cyclopropylthiazines as potent and orally efficacious BACE1 inhibitors. Lead optimization led to the identification of 20, a molecule with biochemical IC50 BACE2/BACE1 ratio of 47. Administration of 20 resulted in no skin/fur color change in a 13-day mouse hypopigmentation study and demonstrated robust and sustained reduction of CSF and brain Aß40 levels in rat and monkey pharmacodynamic models. On the basis of a compelling data package, 20 (AM-6494) was advanced to preclinical development.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ciclopropanos/farmacología , Inhibidores Enzimáticos/farmacología , Tiazinas/farmacología , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Masculino , Ratones , Modelos Moleculares , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/metabolismo , Ratas Sprague-Dawley , Tiazinas/química , Tiazinas/farmacocinética , Tiazinas/uso terapéutico
3.
J Biol Chem ; 293(32): 12634-12646, 2018 08 10.
Artículo en Inglés | MEDLINE | ID: mdl-29794134

RESUMEN

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.


Asunto(s)
Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Inmunológicos/química , Enfermedad de Alzheimer/patología , Cristalografía por Rayos X , Células Dendríticas/química , Células Dendríticas/patología , Variación Genética , Humanos , Ligandos , Macrófagos/química , Macrófagos/patología , Glicoproteínas de Membrana/genética , Microglía/química , Microglía/patología , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Osteoclastos/química , Osteoclastos/patología , Conformación Proteica , Dominios Proteicos/genética , Receptores Inmunológicos/genética
4.
Bioorg Med Chem Lett ; 23(23): 6447-54, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24139583

RESUMEN

γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Amidas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Picolinas/farmacología , Enfermedad de Alzheimer/enzimología , Amidas/química , Animales , Células HEK293 , Humanos , Picolinas/química , Ratas , Ratas Sprague-Dawley
5.
Cancer Biol Ther ; 5(6): 657-64, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16627989

RESUMEN

Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.


Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Neoplasias de la Mama/genética , Proteínas de la Membrana/metabolismo , Receptor ErbB-2/metabolismo , Proteína ADAM10 , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , Trastuzumab
6.
Platelets ; 14(3): 179-87, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12850842

RESUMEN

The hypothesis that glycoprotein (GP) IIb/IIIa antagonists stimulate platelets is controversial. Here, we report the results of flow cytometric measurements of platelet activation markers in a phase I dose optimization study of Roxifiban, an orally active GP IIb/IIIa antagonist. Whole blood was collected at pre-dose and during the dosing interval directly into citrate fixative so that circulating levels of platelet activation could be assessed. P-selectin expression and fibrinogen binding of single platelets were unchanged at any of the dosing intervals compared to the pre-dose values, whereas microaggregate formation was reduced. Blood was also collected in hirudin to maintain physiological calcium concentrations and stimulated with platelet agonists to test whether GP IIb/IIIa antagonists lower the threshold for platelet activation. After stimulation with a concentration range of ADP and TRAP, P-selectin expression was not altered by Roxifiban administration compared to pre-dose levels. Fibrinogen binding and microaggregate formation were reduced by Roxifiban dosing in a dose-dependent manner. Inhibition of both parameters was retained at trough and no increase above pre-dose values was observed at any time. This study provides evidence for a dose-dependent inhibition of platelet functions by an orally active GP IIb/IIIa antagonist and does not detect paradoxical activation of platelets by a GP IIb/IIIa antagonist in humans.


Asunto(s)
Amidinas/farmacología , Isoxazoles/farmacología , Activación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Adenosina Difosfato , Adulto , Amidinas/administración & dosificación , Biomarcadores/análisis , Relación Dosis-Respuesta a Droga , Femenino , Fibrinógeno/metabolismo , Humanos , Isoxazoles/administración & dosificación , Masculino , Selectina-P/biosíntesis , Profármacos , Receptores de Trombina
7.
Biochem Biophys Res Commun ; 299(4): 569-73, 2002 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-12459176

RESUMEN

Gamma-secretase is a unique protease which cleaves within the transmembrane domain of several substrate proteins. Among gamma-secretase substrates are members of the Notch family of receptors and the amyloid precursor protein. In this study we used a cell-free Notch-cleavage assay and specific gamma-secretase inhibitors to study the cleavage of Notch by gamma-secretase. Using this assay, we found that, in contrast to previous reports, the presence of valine at the P1(') position of Notch1 is not required for gamma-secretase cleavage. Our results suggest that the presence of valine at the N-terminus of the Notch intracellular domain cleavage product is important for its stability. Thus it appears that Notch cleavage is very similar to APP cleavage with respect to the lack of sequence specificity.


Asunto(s)
Acetilcisteína/análogos & derivados , Endopeptidasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Receptores de Superficie Celular , Factores de Transcripción , Acetilcisteína/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Línea Celular , Sistema Libre de Células , Células Cultivadas , Inhibidores de Cisteína Proteinasa/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Estructura Terciaria de Proteína , Receptor Notch1 , Valina/metabolismo
8.
Blood ; 99(10): 3540-6, 2002 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-11986205

RESUMEN

Glycoprotein (GP) IIb/IIIa antagonists are effective therapeutic agents, but elicit thrombocytopenia with a frequency that approaches 2%. Here, we provide evidence that thrombocytopenia in humans treated with the GP IIb/IIIa antagonist roxifiban is immune mediated. Two patients underwent conversion to a highly positive drug-dependent antibody (DDAB) status temporally associated with thrombocytopenia. Despite the continued presence of DDABs, the fall in platelet count was reversed by discontinuation of drug treatment, pointing to the exquisite drug dependency of the immune response. DDABs appear to bind to neoepitopes in GP IIb/IIIa elicited on antagonist binding. This information was used to develop an enzyme-linked immunosorbent assay (ELISA) for DDAB using solid-phase GP IIb/IIIa. A high level of specificity is indicated by the observation that DDAB binding is dependent on the chemical structure of the GP IIb/IIIa antagonist and that only 2% to 5% of human blood donors and 5% of chimpanzees present with pre-existing DDABs. Furthermore, none of 108 nonthrombocytopenic patients from the phase II roxifiban study showed an increase in antibody titer. Absorption of thrombocytopenia plasma with platelets reduced the DDAB ELISA signal, indicating that the test detects physiologically relevant antibodies. Screening patients for pre-existing or increasing DDAB titer during treatment with GP IIb/IIIa antagonists may reduce the incidence of drug-induced thrombocytopenia.


Asunto(s)
Amidinas/efectos adversos , Ensayo de Inmunoadsorción Enzimática/métodos , Isoxazoles/efectos adversos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Trombocitopenia/inducido químicamente , Administración Oral , Amidinas/administración & dosificación , Amidinas/farmacocinética , Animales , Anticuerpos/análisis , Anticuerpos/sangre , Anticuerpos/inmunología , Disponibilidad Biológica , Ensayos Clínicos Fase II como Asunto , Epítopos/química , Epítopos/inmunología , Humanos , Isoxazoles/administración & dosificación , Isoxazoles/farmacocinética , Cinética , Pan troglodytes , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Conformación Proteica , Sensibilidad y Especificidad , Trombocitopenia/inmunología
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