Optimisation and mechanistic characterization of a selective benzoxazine-based MAP4K4 inhibitor in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with limited targeted therapeutic options. Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has been implicated in PDAC progression, but the development of effective MAP4K4 inhibitors requires further improvement in potency, selectivity, and mechanistic characterisation. We previously identified F389-0746 as a selective benzoxazine-based MAP4K4 inhibitor with anti-cancer activity in pancreatic cancer. Here, we used the F389-0746 scaffold to guide the selection and evaluation of benzoxazine-based analogs, followed by biochemical, structural, selectivity, and cellular characterisation of the most potent analogue. Among the identified analogs, F389-0736 showed the strongest MAP4K4 inhibitory activity, with an IC50 value of 44.0 nM, representing an approximately threefold improvement over F389-0746. Docking and structure-activity relationship analyses identified key binding interactions that support the enhanced potency of F389-0736 within the MAP4K4 binding pocket. Kinase profiling further demonstrated the selectivity of F389-0736 for MAP4K4 across the tested kinase panel. CETSA further demonstrated F389-0736-induced thermal stabilisation of MAP4K4, supporting intracellular target engagement. In pancreatic cancer cells, F389-0736 suppressed TAK1, JNK, and p38 phosphorylation and significantly reduced cell viability and proliferation, with IC50 and GI50 values of 4.41-4.65 μM and 4.03-5.12 μM, respectively. F389-0736 also inhibited clonogenic growth and induced sub-G1 accumulation with activation of apoptotic signalling. RNA sequencing identified 398 differentially expressed genes and revealed enrichment of inflammatory and stress response pathways, programmed cell death programs, oncogenic signalling perturbation, and metabolic reprogramming. These findings identify F389-0736 as a potent and selective benzoxazine-based MAP4K4 inhibitor and support its use as a chemical probe and lead scaffold for investigating MAP4K4-dependent signalling and therapeutic vulnerability in pancreatic cancer.