RESUMO

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.

Assuntos

Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Inibidores de Proteínas Quinases/química , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Descoberta de Drogas , Humanos , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes , Relação Estrutura-Atividade

RESUMO

Baby Hamster Kidney cells (BHK-21) are commonly used in research and the biopharmaceutical industry. This work aimed to model the kinetic performance in batch operation mode of BHK-21 cells cultured in two stirred tank configurations using different dissolved oxygen concentrations and pH control strategies. Viable and dead cell concentrations, as well as glucose, glutamine, lactate and ammonium concentrations, were monitored. Statistical multiple linear regression, logistic equation and multiplicative Monod kinetic models were fitted. Statistical models for viable cells concentration as a function of nutrient and metabolite concentrations were significant (R2 >0.91). Logistic model parameters: intrinsic growth rate, cell density level in the medium and time for reaching maximum cell concentrations were within 0.061-0.083 h-1, 1.85-5.39 x 109 cell L-1 and 52-90 h ranges, respectively. A Monod-type model was satisfactorily fitted to the experimental data. Relative errors were lower than 10% for six monitored state variables in most of the assessed experimental conditions. The three models developed in this work can be used in bioprocesses involving BHK-21 with good fitting.