ABSTRACT
The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms' distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.
Subject(s)
Antineoplastic Agents/pharmacology , Lymphocytes/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Allosteric Regulation , Allosteric Site , Animals , Antineoplastic Agents/chemical synthesis , Cell Line , Drug Design , Gene Expression , HEK293 Cells , Humans , Inhibitory Concentration 50 , Lymphocytes/cytology , Lymphocytes/enzymology , Mice , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sf9 Cells , Small Molecule Libraries/chemical synthesis , Spodoptera , Structure-Activity RelationshipABSTRACT
Activation of the maternal immune system (MIA) during gestation is linked to neuropsychiatric diseases like schizophrenia. While many studies address behavioural aspects, less is known about underlying cellular mechanisms. In the following study, BALB/c mice received intraperitoneal injections of polyinosinic-polycytidylic acid (Poly I:C) (20 µg/ml) or saline (0.9%) at gestation day (GD) 9.5 before hippocampal neurons were isolated and cultured from embryonic mice for further analysis. Interestingly, strongest effects were observed when the perineuronal net (PNN) wearing subpopulation of neurons was analysed. Here, a significant reduction of aggrecan staining intensity, area and soma size could be detected. Alterations of PNNs are often linked to neuropsychiatric diseases, changes in synaptic plasticity and in electrophysiology. Utilizing multielectrode array analysis (MEA), we observed a remarkable increase of the spontaneous network activity in neuronal networks after 21 days in vitro (DIV) when mother mice suffered a prenatal immune challenge. As PNNs are associated with GABAergic interneurons, our data indicate that this neuronal subtype might be stronger affected by a prenatal MIA. Degradation or damage of this subtype might cause the hyperexcitability observed in the whole network. In addition, embryonic neurons of the Poly I:C condition developed significantly shorter axons after five days in culture, while dendritic parameters and apoptosis rate remained unchanged. Structural analysis of synapse numbers revealed an increase of postsynaptic density 95 (PSD-95) puncta after 14 DIV and an increase of presynaptic vesicular glutamate transporter (vGlut) puncta after 21 DIV, while inhibitory synaptic proteins were not altered.
