Development of novel tetrazole-based pyruvate kinase M2 inhibitors targeting U87MG glioblastoma cells

Abstract

Glioblastoma (GB), the most aggressive and life-threatening primary brain tumor in adults, poses significant therapeutic challenges. Tumor pyruvate kinase M2 (PKM2) has been implicated in the proliferation and survival of glioma cells. In this study, we designed and synthesized a series of 23 novel tetrazole-based derivatives. The compounds were thoroughly characterized using ¹H, ¹³C, ¹⁹F NMR, along with HRMS analysis. Among them, 1-(imidazo[1,2-a]pyrimidin-3-yl)-2-(5-(naphthalen-2-yl)-2H-tetrazol-2-yl)ethan-1-one (9b) exhibited potent and selective antiproliferative activity against U87MG glioma cells, with minimal effects on bEnd (brain endothelial cell line) non-glioma cells. It emerged as a potent PKM2 inhibitor, with an IC₅₀ of 0.307 µM. Apoptosis assays and cell cycle analysis revealed that compound 9b induced early apoptosis and caused G1 phase arrest. A significant decrease in pyruvate concentration further suggested PKM2 inhibition. In silico studies confirmed the binding affinity to the PKM2 inhibitory site, and RT-PCR data demonstrated its inhibitory activity against PKM2. Additionally, it reduced lactate levels, indicating its potential impact on cellular metabolism. Collectively, these findings suggest that the most potent compound holds promise as a therapeutic candidate against GB.

Keywords: Apoptosis assay; Cell cycle analysis; Glioblastoma; Lactate assay; Pyruvate assay; Pyruvate kinase M2; Tetrazole.