A novel pharmacological inhibitor of MAP4K4 activity attenuates metabolic dysfunction-associated steatohepatitis.

BACKGROUND & AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive condition and a leading driver of global liver-related morbidity. Its advanced form, metabolic dysfunction-associated steatohepatitis (MASH), is characterized by hepatic steatosis, inflammation, hepatocellular ballooning, and fibrosis. We aim to explore the importance of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) as a therapeutic target and to evaluate a novel small-molecule inhibitor, glucosyl pyrrolo-pyrimidinone (GPPD), for the mitigation of MASH.

METHODS

MAP4K4 expression was immunohistochemically quantified in liver biopsies from healthy individuals (n = 5) and patients with MASLD (n = 20). We assessed the in vitro efficacy of GPPD and genetic MAP4K4 silencing in human primary hepatocytes, mouse hepatic organoids, HepG2, and AML12 cells challenged with oleic acid. For in vivo validation, mice were fed a high-fat/high-fructose/high-cholesterol diet and treated with GPPD for 30 weeks (n = 8 per group). We analyzed the livers using single-nuclei RNA sequencing to identify the molecular mechanisms of GPPD-mediated hepatoprotection. Data were analyzed using ANOVA models and expressed as mean ± SD, with differences considered significant at p <0.05.

RESULTS

Hepatic MAP4K4 expression was significantly elevated in patients with MASLD compared with healthy controls, correlating with disease severity. In multiple in vitro models, both genetic silencing and GPPD treatment significantly attenuated steatosis. In vivo, prophylactic GPPD administration reduced body mass gain and adiposity while protecting against hepatic steatosis, inflammation, injury, and fibrosis. Mechanistically, although GPPD directly inhibited MAP4K4 activity, its hepatoprotective effects were associated with suppression of calcium/calmodulin-dependent protein kinase II (CaMK2).

CONCLUSIONS

We demonstrate that GPPD treatment mitigates steatohepatitis with fibrosis by regulating MAP4K4-CaMK2 axis. Further investigation of GPPD will advance its development as an effective future therapeutic strategy against MASH.

IMPACT AND IMPLICATIONS: In this study, we identify the MAP4K4-CaMK2 signaling axis as a critical pathway in the pathogenesis of MASH. By demonstrating that GPPD-mediated inhibition of this pathway attenuates key hallmarks of MASH, including steatosis, inflammation, and fibrosis, our findings provide a mechanistic framework for future therapeutic intervention. Moreover, targeting MAP4K4 may address unmet needs of patients who are ineligible for currently approved treatments. Collectively, this research establishes a translational foundation for the development of next-generation small molecules designed at restoring metabolic homeostasis across the disease continuum.