RESUMO
Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.
Assuntos
Desenho de Fármacos , Glucoquinase/química , Hipoglicemiantes/síntese química , Indazóis/química , Pirazinas/síntese química , Pirazóis/química , Piridinas/química , Administração Oral , Animais , Linhagem Celular Tumoral , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucoquinase/metabolismo , Teste de Tolerância a Glucose , Meia-Vida , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Indazóis/síntese química , Indazóis/farmacocinética , Indazóis/uso terapêutico , Insulina/metabolismo , Cinética , Ligação Proteica , Pirazinas/farmacocinética , Pirazinas/uso terapêutico , Pirazóis/farmacocinética , Pirazóis/uso terapêutico , Piridinas/farmacocinética , Piridinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. Several PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate centrosome- and chromatin- based microtubule spindle assembly pathways. In interphase cells PROTAC-mediated clearance of non-centrosomal AURKA modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential sensitivity of subcellular pools of substrate, governed by substrate conformation or localization-dependent accessibility to PROTAC action, a phenomenon not previously described for this new class of degrader compounds.
Assuntos
Aurora Quinase A/metabolismo , Azepinas/farmacologia , Pirimidinas/farmacologia , Animais , Aurora Quinase A/antagonistas & inibidores , Azepinas/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas/métodos , Células HeLa , Humanos , Cinética , Ligantes , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Pirimidinas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.