2881 - Biochemical and Biophysical Studies of Novel Small Molecules Targeting Proteasome Subunit Alpha Type-7 (PSMA7) for the Treatment of Glioblastoma

C. A. Showalter¹, P. Rajasekera², A. Kumar², K. Singh¹, S. Biehn³, B. Pérez-Soto³, S. Lindert³, H. Manring¹, S. J. Haque², and A. Chakravarti²; ¹The Ohio State University Comprehensive Cancer Center, Columbus, OH, ²Department of Radiation Oncology, Arthur G. James Hospital/Ohio State Comprehensive Cancer Center, Columbus, OH, ³Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH

Purpose/Objective(s): Glioblastoma (GBM) is the most common and aggressive primary brain tumor with less than 10% of patients surviving beyond 5 years after diagnosis. Even when treated early with the current standard of care, GBM recurs in nearly all patients. Therefore, there is an urgent need to develop novel therapies to overcome treatment resistance in GBM, including those that could be combined with the current standard of care to improve patient outcomes. In previous studies, we identified proteasome subunit alpha type-7 (PSMA7) as a potentially druggable target in GBM. Moreover, we discovered small molecules (SMs), predicted in silico to be blood-brain barrier (BBB)-permeable and target PSMA7, with greater toxicity in GBM cells compared to normal cells. In this study, we further investigated the potential of these SMs to be used as novel therapeutics for the treatment of GBM by examining their ability to inhibit PSMA7 expression, directly bind to PSMA7, block the interaction(s) between PSMA7 with its reported binding partners, and/or disrupt phosphorylation of tyrosine residues on PSMA7. Materials/

Methods: In silico molecular docking screenings were performed using the National Cancer Institute Diversity Set VI SM library and regions of interaction on PSMA7 with its reported binding partners (i.e., HIF-1alpha, Parkin, and PAC4) or regions around two tyrosine residues of PSMA7 (i.e., Y106 and Y153) that are potentially phosphorylated by cABL. Top-scoring SMs were screened using SwissADME to select SMs predicted to be BBB-permeable. Western blots (WB) were performed to examine protein expression. Surface Plasmon Resonance (SPR) was performed using a Biacore S200 to determine real-time quantitative binding affinities and kinetics for interactions between PSMA7 with select SMs. Co-immunoprecipitation (Co-IP) was used to evaluate protein-protein interactions and IP/WB for examining changes to protein phosphorylation.
Results: Preliminary in vitro screening of primary human GBM cells with eight SMs predicted to target PSMA7 was performed to identify whether they inhibit PSMA7 protein expression. Western blot analysis revealed that two SMs significantly inhibited PSMA7 in both 08-387 and 3359 cells. SPR data analysis found that three SMs had equilibrium dissociation constant (K_D) values < 10^-6 M, indicating their direct binding with PSMA7. Co-IP/WB and IP/WB studies were conducted to examine the ability of these three SMs to block interactions with their targeted PSMA7 binding partners and/or inhibit phosphorylation on PSMA7Y106 and PSMA7Y153.
Conclusion: Our study identified BBB-permeable SMs that inhibit the biological functions of PSMA7, a potential therapeutic target in GBM. These SMs are potential candidates for in vivo preclinical studies and combining these novel targeted molecules with the standard of care may aid in overcoming treatment resistance in GBM. Our group is actively investigating the binding specificity of these SMs for PSMA7.