RESUMEN
The radiosynthesis and GMP validation of [11 C]AMT for human use are described. Three consecutive batches were produced giving 940-3790 MBq (4%-17% RCY, decay corrected, based on [11 C]CO2 ) of the tracer. The molar activity at the end of synthesis was 19 to 35 GBq/µmol, the radiochemical purity was ≥98%, and the enantiomeric purity was >99%. While the synthesis method was automated using a new generation of synthesis equipment, tracer production system developed in house, the method should be readily applicable to other synthesis platforms with minor modifications.
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Radioisótopos de Carbono/química , Radioquímica/métodos , Triptófano/análogos & derivados , Automatización , Técnicas de Química Sintética , Control de Calidad , Trazadores Radiactivos , Triptófano/síntesis química , Triptófano/químicaRESUMEN
In this study, we explored the effect of bioisostere replacement in a series of glycogen synthase kinase 3 (GSK3) inhibitors based on the imidazopyridine core. The synthesis and biological evaluation of a number of novel sulfonamide, 1,2,4-oxadiazole, and thiazole derivates as amide bioisosteres, as well as a computational rationalization of the obtained results are reported.
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Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacología , Diseño de Fármacos , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Simulación de Dinámica Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Oxadiazoles/farmacología , Piridinas/síntesis química , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , Tiazoles/síntesis química , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.
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Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Indazoles/química , Indazoles/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dihidroorotato Deshidrogenasa , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indazoles/farmacología , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismoRESUMEN
The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Indazoles/farmacología , Neoplasias/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dihidroorotato Deshidrogenasa , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteolisis/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The original PDF version of this Article listed the authors as "Marcus J.G.W. Ladds," where it should have read "Marcus J. G. W. Ladds, Ingeborg M. M. van Leeuwen, Catherine J. Drummond et al.#".Also in the PDF version, it was incorrectly stated that "Correspondence and requests for materials should be addressed to S. Lín.", instead of the correct "Correspondence and requests for materials should be addressed to S. Laín."This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.
RESUMEN
Alzheimer's disease (AD) is characterized by aggregation of amyloid beta (Aß) into insoluble plaques. Intermediates, Aß oligomers (Aßo), appear to be the mechanistic cause of disease. The de facto PET AD ligand, [11C]PIB, binds and visualizes Aß plaque load, which does not correlate well with disease severity. Therefore, finding a dynamic target that changes with pathology progression in AD is of great interest. Aßo alter synaptic plasticity, inhibit long-term potentiation, and facilitate long-term depression; key mechanisms involved in memory and learning. In order to convey these neurotoxic effects, Aßo requires interaction with the metabotropic glutamate 5 receptor (mGluR5). The aim was to investigate in vivo mGluR5 changes in an Aß pathology model using PET. Wild type C57/BL6 (wt) and AßPP transgenic mice (tg-ArcSwe), 4, 8, and 16 months old, were PET scanned with [11C]ABP688, which is highly specific to mGluR5, to investigate changes in mGluR5. Mouse brains were extracted postscan and mGluR5 and Aß protofibril levels were assessed with immunoblotting and ELISA respectively. Receptor-dense brain regions (hippocampus, thalamus, and striatum) displayed higher [11C]ABP688 concentrations corresponding to mGluR5 expression pattern. Mice had similar uptake levels of [11C]ABP688 regardless of genotype or age. Immunoblotting revealed general decline in mGluR5 expression and elevated levels of mGluR5 in 16 months old tg-ArcSwe compared with wt mice. [11C]ABP688 could visualize mGluR5 in the mouse brain. In conclusion, mGluR5 levels were found to decrease with age and tended to be higher in tg-ArcSwe compared with wt mice, however these changes could not be quantified with PET.
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Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono/metabolismo , Oximas/metabolismo , Tomografía de Emisión de Positrones , Piridinas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Immunoblotting/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tomografía de Emisión de Positrones/métodosRESUMEN
Methods have been developed to label oligonucleotides (ODNs) in the 5'-position with (76)Br via a prosthetic group on a hexylamino-linker. The purpose of the study was to explore whether the labelling procedure would prevent specific hybridisation by using reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing of the PCR product. Antisense ODNs (30 mer, specific for rat Chromogranin A [CgA] mRNA) with phosphodiester (O-ODN) or phosphothioate (S-ODN) backbone, either unlabelled or labelled with (76)Br, served as one of the primers in individual PCR reactions. Using O-ODN as a primer, irrespective of being labelled or not, a selected 225-bp PCR fragment was successfully amplified. However, no amplification was obtained using S-ODN as a primer. The proper PCR products were only detected in the sample prepared from the adrenal gland, but not in that from the heart, liver or kidney. Autoradiographic recording of the gel, after gel electrophoresis, revealed radioactive signals corresponding to the amplified PCR products. The sequence of the PCR product matched the rat CgA mRNA sequence obtained from the EMBL database. RT-PCR is an attractive method to identify the selective binding of modified ODNs to target mRNA. This method confirmed that the labelling with (76)Br did not change the hybridisation ability of antisense O-ODN.
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Radioisótopos de Bromo/farmacocinética , Cromograninas/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos/farmacocinética , ARN Mensajero/genética , Radiofármacos/farmacocinética , Animales , Radioisótopos de Bromo/uso terapéutico , Cromogranina A , Silenciador del Gen , Marcaje Isotópico/métodos , Masculino , Hibridación de Ácido Nucleico/genética , Hibridación de Ácido Nucleico/métodos , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Especificidad de Órganos , Radiofármacos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Distribución TisularRESUMEN
The voltage-gated sodium channel Na(V)1.7 is believed to be a critical mediator of pain sensation based on clinical genetic studies and pharmacological results. Clinical utility of nonselective sodium channel blockers is limited due to serious adverse drug effects. Here, we present the optimization, structure-activity relationships, and in vitro and in vivo characterization of a novel series of Na(V)1.7 inhibitors based on the oxoisoindoline core. Extensive studies with focus on optimization of Na(V)1.7 potency, selectivity over Na(V)1.5, and metabolic stability properties produced several interesting oxoisoindoline carboxamides (16A, 26B, 28, 51, 60, and 62) that were further characterized. The oxoisoindoline carboxamides interacted with the local anesthetics binding site. In spite of this, several compounds showed functional selectivity versus Na(V)1.5 of more than 100-fold. This appeared to be a combination of subtype and state-dependent selectivity. Compound 28 showed concentration-dependent inhibition of nerve injury-induced ectopic in an ex vivo DRG preparation from SNL rats. Compounds 16A and 26B demonstrated concentration-dependent efficacy in preclinical behavioral pain models. The oxoisoindoline carboxamides series described here may be valuable for further investigations for pain therapeutics.