Multi-Targeting Approach in Glioblastoma Using Computer-Assisted Drug Discovery Tools to Overcome the Blood–Brain Barrier and Target EGFR/PI3Kp110β Signaling

SIMPLE SUMMARY: Treatment of glioblastoma is hampered by the activation of compensatory survival mechanisms by malignant cells that lead to drug resistance. Moreover, the blood–brain barrier (BBB) precludes the brain entrance of most drugs. We hypothesized that computer-assisted drug discovery tools would reveal novel multi-targeting drug candidates with BBB-permeant and favorable ADMET properties. We aimed to discover molecules with predicted ability to inhibit the EGFR/PI3Kp110β pathway and to validate their efficacy and safety in biological assays. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Moreover, we tested their anti-tumor efficacy and BBB safety and permeation in cell models. We found two EGFR, two PI3Kp110β, and, mostly, two dual inhibitors with anti-tumor effects. Among them, one EGFR and two PI3Kp110β inhibitors were able to cross the BBB endothelium without compromising it. These studies revealed novel drug candidates for glioblastoma treatment. ABSTRACT: The epidermal growth factor receptor (EGFR) is upregulated in glioblastoma, becoming an attractive therapeutic target. However, activation of compensatory pathways generates inputs to downstream PI3Kp110β signaling, leading to anti-EGFR therapeutic resistance. Moreover, the blood–brain barrier (BBB) limits drugs’ brain penetration. We aimed to discover EGFR/PI3Kp110β pathway inhibitors for a multi-targeting approach, with favorable ADMET and BBB-permeant properties. We used quantitative structure–activity relationship models and structure-based virtual screening, and assessed ADMET properties, to identify BBB-permeant drug candidates. Predictions were validated in in vitro models of the human BBB and BBB-glioma co-cultures. The results disclosed 27 molecules (18 EGFR, 6 PI3Kp110β, and 3 dual inhibitors) for biological validation, performed in two glioblastoma cell lines (U87MG and U87MG overexpressing EGFR). Six molecules (two EGFR, two PI3Kp110β, and two dual inhibitors) decreased cell viability by 40–99%, with the greatest effect observed for the dual inhibitors. The glioma cytotoxicity was confirmed by analysis of targets’ downregulation and increased apoptosis (15–85%). Safety to BBB endothelial cells was confirmed for three of those molecules (one EGFR and two PI3Kp110β inhibitors). These molecules crossed the endothelial monolayer in the BBB in vitro model and in the BBB-glioblastoma co-culture system. These results revealed novel drug candidates for glioblastoma treatment.