Integrating single-cell transcriptomics, molecular docking, and dynamics simulations to characterize protein kinase X-linked mechanisms in osteosarcoma.

BACKGROUND

Osteosarcoma (OS) progression is linked to kinase allostery dysregulation, but PRKX's allosteric role remains unknown. We aimed to decode PRKX's activation mechanism and assess its clinical potential as a biomarker and therapeutic target.

METHODS

Differential expression analysis confirmed PRKX's oncogenic role. Using single-cell RNA-seq (GSE152048) with spatiotemporal trajectory analysis, we delineated PRKX-CDC37 interactions. PRKX-knockdown via siRNA suppressed proliferation, invasion, and colony formation in vitro. Based on the AlphaFold-predicted PRKX-CDC37 complex, we performed molecular docking followed by explicit-solvent 100-ns MD simulations to evaluate inhibitor binding.

RESULTS

Multi-omics revealed PRKX-CDC37 spatiotemporal co-expression driving early TME differentiation. Ergotamine (ΔG = -11.6 kcal/mol) competitively bound PRKX's catalytic pocket via a tripartite H-bond network centered on HIS161. MD simulations demonstrated ≥95 % occupancy H-bonds and 28.6 % SASA reduction, confirming ergotamine as the lead PRKX-CDC37 inhibitor with nanomolar-scale affinity.

CONCLUSION

PRKX drives OS via a CDC37-mediated allosteric pathway, with Pocket-3 (HIS161) as a therapeutic target. Ergotamine validates PRKX-CDC37 inhibition, providing a framework for anti-OS drug design.