Optimisation and mechanistic characterization of a selective benzoxazine-based MAP4K4 inhibitor in pancreatic cancer cells.
10 affiliations
- 1Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
- 2Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
- 3Warshel Institute for Computational Biology, School of Medicine, The Chinese University of Hong Kong (Shenzhen), Shenzhen, Guangdong, China.
- 4Division of Translational Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA.
- 5Department of Digital Biology, Graphen Drugomics Inc., New York, NY, USA.
- 6CRISPR Gene Targeting Core, Taipei Medical University, Taipei, Taiwan.
- 7TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan.
- 8Department and Graduate Institute of Pharmacology, National Taiwan University College of Medicine, Taipei, Taiwan.
- 9Ph.D. Program in Innovative Medical Technology Research and Development Industry, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
- 10Cancer Center, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan.
Abstract
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.
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BibTeX
@article{wu2026optimisation,
title = {{Optimisation and mechanistic characterization of a selective benzoxazine-based MAP4K4 inhibitor in pancreatic cancer cells.}},
author = {Yi-Wen Wu and Chun-Lin Yang and Yu-Ting Fang-Chin and Yun-Hsuan Yeh and Bor-Jiun Chen and Tzu-Ying Sung and Jung-Chun Chu and Tony Eight Lin and Shih-Chung Yen and Jui-Hua Hsieh and Sing-Han Huang and Cheng-Ying Chu and Kai-Chien Yang and Kai-Cheng Hsu and Shiow-Lin Pan},
journal = {Journal of enzyme inhibition and medicinal chemistry},
year = {2026},
month = oct,
doi = {10.1080/14756366.2026.2743434},
url = {https://europepmc.org/article/MED/42885518},
note = {CC BY},
}
RIS
TY - JOUR
TI - Optimisation and mechanistic characterization of a selective benzoxazine-based MAP4K4 inhibitor in pancreatic cancer cells.
AU - Yi-Wen Wu
AU - Chun-Lin Yang
AU - Yu-Ting Fang-Chin
AU - Yun-Hsuan Yeh
AU - Bor-Jiun Chen
AU - Tzu-Ying Sung
AU - Jung-Chun Chu
AU - Tony Eight Lin
AU - Shih-Chung Yen
AU - Jui-Hua Hsieh
AU - Sing-Han Huang
AU - Cheng-Ying Chu
AU - Kai-Chien Yang
AU - Kai-Cheng Hsu
AU - Shiow-Lin Pan
PY - 2026
DA - 2026/10/16
JO - Journal of enzyme inhibition and medicinal chemistry
DO - 10.1080/14756366.2026.2743434
UR - https://europepmc.org/article/MED/42885518
AB - 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.
SN - 1475-6366
SN - 1475-6374
ER -