Orthosteric–allosteric dual inhibitors of PI5P4Kγ with potent antitumor activity in non-small cell lung cancer

Introduction

Phosphatidylinositol 5-phosphate 4-kinase type II gamma (PI5P4Kγ) has emerged as a promising therapeutic target in oncology due to its key role in cancer progression. However, currently available inhibitors targeting either the orthosteric or allosteric sites of PI5P4Kγ suffer from limited in vitro activity and in vivo validation.

Objectives

This study aimed to develop a highly potent and selective PI5P4Kγ inhibitor capable of simultaneously engaging both the orthosteric and allosteric sites, as well as to validate their potential in therapy for non-small cell lung cancer (NSCLC) were investigated.

Methods

Through the structural modification of the propionamide side chain of the pyridine ring and the N3 substituent of the quinazolinone, the structure–activity relationship (SAR) was elucidated, leading to the identification of the target compound n40. The binding mode of n40 to PI5P4Kγ, was elucidated by X-ray crystallography. In vitro effects were assessed through KINOMEscan Technology, cell proliferation, apoptosis, and EMT analysis. In vivo efficacy was evaluated using an HCC827 xenograft mouse model. Both single-dose (up to 400 mg/kg) and repeated-dose (20–180 mg/kg/day for 28 days) toxicity studies of n40 were conducted in C57BL/6 mice, with comprehensive monitoring of physiological parameters and organ toxicity indices.

Results

Compound n40 exhibited a strong binding affinity for PI5P4Kγ (K_d = 6.55 nM), with 3,000-fold selectivity over other two isoforms. Additionally, it displayed sub-50 nM antiproliferative activity in NSCLC cells with higher PI5P4Kγ levels. X-ray crystallography revealed n40 simultaneously binds to both orthosteric and a novel allosteric pocket. Functionally, n40 induced S-phase arrest, apoptosis, and EMT reversal and PI3K/AKT signaling inhibition in a PI5P4Kγ-dependent manner. In HCC827 xenografts, n40 achieved > 60 % tumor growth inhibition without any observed toxicity.

Conclusion

This study establishes PI5P4Kγ as a druggable target in NSCLC and demonstrates that dual orthosteric–allosteric targeting is a viable strategy to achieve potent and selective PI5P4Kγ inhibitors.