GO-6976

Various tyrosine kinase inhibitors (TKIs) have been developed to target human epidermal growth factor receptor (EGFR) for cancer therapy. However, many patients treated with first-line TKIs are clinically observed to eventually establish a gatekeeper T790M mutation in the ATP-binding site of the EGFR kinase domain, which is primarily responsible for acquired drug resistance to cancers. Over the past decades, a number of noncovalent, wild-type-sparing and ATP-competitive inhibitors (NWAIs) were reported to selectively target the T790M mutant over wild-type kinase, which are independent of the traditional inhibitor classification system that categorizes EGFR TKIs into four generations. Here, we systematically investigated the intermolecular interaction of wild-type EGFR (EGFRWT) and its T790M mutant (EGFRT790M) with 15 existing NWAI inhibitors, paying attention to the structural and energetic responses of inhibitor ligands to the gatekeeper mutation. It was revealed that the NWAIs can be typed into three classes I, II and III, which can form S[Formula: see text] interactions, hydrophobic (van der Waals) contacts and weak hydrogen (halogen) bonding with the side-chain thioether moiety of the mutant Met790 residue, respectively, thus conferring additional affinity and specificity to inhibitor ligands upon the T790M mutation. In addition, we further performed 2D-chemical similarity search to identify new class I NWAIs, from which two Staurosporine analogs (i.e. UCN01 and ZHD0501) were identified to have a good selectivity for EGFRT790M over EGFRWT. They can be exploited as promising leading chemical scaffolds to further develop potent, selective, wild-type-sparing NWAI inhibitors of EGFRT790M gatekeeper mutant.

Calcium signaling pathways play a crucial role in neuronal and glial function, yet the effects of inhibiting specific components of these pathways remain poorly understood. Here, we investigated how various inhibitors affect calcium dynamics in neurons and astrocytes within hippocampal co-cultures under normal conditions and during bicuculline-induced epileptiform activity. We found that phospholipase C (PLC) inhibitor U73122 and protein kinase C (PKC) inhibitors BIM IX and Go 6976 independently induced epileptiform activity in neurons, characterized by synchronized calcium oscillations similar to those caused by bicuculline. Notably, these inhibitors did not affect astrocytic calcium dynamics. In contrast, IP3 receptor inhibitor 2-APB and calmodulin inhibitor calmidazolium triggered significant calcium responses in both neurons and astrocytes. The 2-APB application led to an immediate increase in neuronal calcium levels and cessation of calcium oscillations, while also inducing varied calcium responses in astrocytes. Calmidazolium caused elevated calcium levels in both cell types, with neurons maintaining calcium oscillations at increased baseline levels. Interestingly, PI3 kinase inhibitor AS-605240 and ryanodine receptor inhibitor dantrolene showed no significant effects on calcium dynamics in either cell type. Electrophysiological recordings confirmed that both PLC and PKC inhibition induced paroxysmal depolarization shifts similar to those observed during bicuculline-induced epileptiform activity. These findings reveal previously unknown effects of commonly used signaling pathway inhibitors on neuronal excitability and calcium homeostasis, which should be considered when designing experiments and interpreting results involving these compounds. Our results also suggest a potential role for PLC and PKC in maintaining the excitation-inhibition balance in neuronal networks.