From Sodium Channels Blockers to Dual ALKBH2/ALKBH5 Inhibitors: a Novel Therapeutic Strategy for Glioblastoma Treatment.

Glioblastoma (GBM) represents the most aggressive primary brain tumor with a median survival of approximately 15 months despite maximal treatment approaches. Standard therapy relies on surgical resection followed by temozolomide (TMZ)-based chemotherapy, yet resistance mechanisms and tumor recurrence remain major clinical challenges. The limited therapeutic options highlight the urgent need for innovative molecular targets and pharmacological strategies. Our research program began with the hypothesis that voltage-gated sodium channels could be a potential target for reducing GBM cell invasiveness. MV1035, a known sodium channel blocker, was able to inhibit migration and invasion in U87-MG glioblastoma cells but its effect was not due to sodium channel blockade. Through structure-based virtual screening SPILLO-PBSS software [1] we identified the RNA demethylase ALKBH5 as putative target to explain MV1035 effect. Cell-free assays confirmed MV1035's ability to inhibit ALKBH5 activity, resulting in increased N6-methyladenosine (m6A) RNA levels and consequent reduction of CD73 protein expression in U87-MG glioblastoma cells [2]. Moreover, MV1035 could also inhibit the DNA repair protein ALKBH2. This dual mechanism could be particularly advantageous for overcoming TMZ resistance, as ALKBH2 mediates repair of alkylating agent-induced DNA damage. In patient-derived glioblastoma stem cells (GSCs) in fact, MV1035 demonstrated synergistic effects with TMZ, significantly reducing cell viability and neurospheres formation capacity [3]. We systematically explored structure-activity relationships around the MV1035 scaffold. Opening the tricyclic imidazobenzoxazine system while maintaining a phenolic substituent yielded compound MV3009 [4], which demonstrated superior activity against both target enzymes. Further rational design led to the development of MV3030, incorporating a fumarate hydrazide moiety inspired by successful FTO (Fat mass and obesity-associated protein) inhibitors. This compound exhibited enhanced ALKBH2 inhibition (75% at 50 μM), while showing improved cytotoxicity against both U87-MG cells and patient-derived GSCs. Computational CNS-MPO analysis confirmed favorable blood-brain barrier penetration properties for the new compound. The dual ALKBH2/ALKBH5 inhibition strategy addresses two critical aspects of GBM biology: stem cell maintenance (ALKBH5) and chemotherapy resistance (ALKBH2). The ability to target resistant GSCs to TMZ while simultaneously reducing their tumorigenic potential represents a significant therapeutic advance. Conclusions: we have successfully developed a novel class of dual ALKBH2/ALKBH5 inhibitors with demonstrated efficacy against patient-derived glioblastoma stem cells. MV3030 has been shown to be particularly promising as a TMZ adjuvant, exhibiting additive effects in combination therapy and favorable CNS penetration properties. This work establishes epigenetic enzyme inhibition as a viable strategy for overcoming GBM therapy resistance and provides a strong foundation for preclinical development. References 1) SPILLO Project - https://www.spillo.ch/ 2) Malacrida, A.; Rivara, M.; et al. Bioorg Med Chem. 2020;28:115300 3) Malacrida, A.; Di Domizio, A; et al. Biology. 2022;11(1):70 4) Rivara, M; Nicolini, G.; et al. Results in Chem. 2024;9:101645