In Silico Discovery and Investigation of Novel Small Molecules Targeting PSMA7 for the Treatment of GBM

Abstract

The five-year survival rate for glioblastoma (GBM; WHO grade 4) remains only 5%, even with the current standard treatment of maximal safe surgical tumor resection followed by radiation treatment (RT) and concomitant and adjuvant temozolomide (TMZ). Therefore, alternative treatment therapies, such as blood-brain barrier (BBB)-permeable small molecule drugs, are needed to significantly improve patient outcomes by addressing other critical pathways in GBM progression and overcome GBM resistance to the standard treatment. Previously, we identified and validated proteasome subunit alpha type-7 (PSMA7), a subunit of the 20S proteasome, as a druggable, therapeutically vulnerable target in GBM by in vitro and in vivo methods. In this study, we performed in silico molecular docking screenings using a small molecule library to identify BBB-permeable small molecules predicted to bind to PSMA7 and investigated their potential as novel therapeutics for the treatment of GBM. In silico molecular docking screenings were performed using the National Cancer Institute Diversity Set VI small molecule library and the crystal structure of PSMA7 obtained from the native human 20S proteasome (RCSB PDB: 5LE5). Molecules were scored based on their predicted likelihood to bind to PSMA7. Top ranked molecules were screened using the SwissADME webtool, to select molecules predicted to be BBB-permeable and not a substrate for the permeability glycoprotein (P-gp), for testing with in vitro studies. Then, in vitro cell viability assays were used to determine IC50 values for molecules in human GBM PDX and normal human cell lines. The potential for molecules to sensitize GBM cells towards TMZ and/or RT was evaluated in vitro. In vitro proteasome activity assays were used to investigate whether molecules altered the three major protease activities performed by the proteasome. Computational virtual screenings using two docking algorithms (i.e., AutoDock Vina and Glide XP) identified and ranked the top small molecules most likely to bind to PSMA7. In silico screening using the SwissADME webtool predicted 99 of these molecules to be BBB-permeable and not a substrate for the P-gp. Subsequent in vitro cell viability studies determined eight of these molecules have lower IC50 values in human GBM PDX cells compared to normal human cells. Furthermore, we examined the potential of these eight molecules to sensitize GBM cells to TMZ and/or RT in vitro. Moreover, we found that three of these molecules inhibited all three major protease activities performed by the proteasome in GBM cells. Our study identified BBB-permeable small molecules predicted to target PSMA7 that demonstrated greater toxicity to GBM cells than normal cells and inhibited proteasome activities, suggesting a potential mechanism of action. Taken together, these small molecules are potential candidates for preclinical studies and may serve as novel targeted therapies for the treatment of GBM.