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.