A small molecule MST1/2 inhibitor accelerates murine liver regeneration with improved survival in models of steatohepatitis

Abstract

Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.

Keywords: Hippo; MASH; TAZ; YAP; medicinal chemistry.

Fig. 1.

mCLC846 inhibits MST1/2 and protects islet cells from toxic stimuli in vitro. A) Chemical structures of neratinib and mCLC846 with respective half maximal inhibitory concentrations (IC₅₀) for MST1, MST2, and EGFR. B) Normalized cell viability and C) insulin production of INS1E exposed to TG with or without mCLC846 (n = 3). D) Immunoblot of MST1 and cleaved caspase 3 (Cl.Casp3) from INS1E cell lysates following exposure to TG and/or mCLC846. E) Normalized insulin secretion from INS1E following exposure HG in the presence of PA, mCAR432, mCLC846 (µM), or mCLC846 and PA (n = 3). F) Human islet donor cell lysate immunoblot for MST1 and cleaved caspase 3 under conditions in E, representative sample from two donors.

Fig. 2.

mCLC846 accelerates murine liver regeneration following hepatectomy. A) Murine partial hepatectomy schema. B) Liver-to-body-weight ratio (%) following murine partial hepatectomy treated with vehicle or mCLC846 (mg/kg/dose) 40 and 72 h posthepatectomy (n = 3–6). C) Immunoblot for PCNA of liver lysates 40-h posthepatectomy treated with vehicle or mCLC846 (mg/kg/dose). D) Posthepatectomy plasma analysis of ALT, ALP, BUN, and total bilirubin in vehicle and mCLC846 (50 mg/kg/dose)-treated mice (n = 5). E) Representative H&E–stained liver resection and liver remnants treated with vehicle or mCLC846 (50 mg/kg/dose). Scale bar = 200 µm.

Fig. 3.

mCLC846 transiently activates YAP in vitro in Hu1545 cells and is important for liver regeneration acceleration. A) Immunoblot detection of Mps one Binder (MOB1), Yes-associated protein (YAP), and their phosphorylated states in Hu1545 cell lysates following exposure of mCLC846 (3 µM) and/or H₂O₂. B) Immunoblot detection of YAP and its phosphorylated state following exposure to increasing concentrations of mCLC846 (µM). C) Representative immunofluorescence microscopy images of Hu1545 cells stained for YAP or TAZ following exposure to mCLC846 (3 µM). Image insets displaying DAPI overlay. Scale bar = 50 µm. D) Mean fluorescence intensity of nuclear YAP or TAZ in Hu1545 cells. Dashed line = median and dotted lines = interquartile range. E) Hu1545 transcript quantification by real-time-PCR following exposure to mCLC846 (3 µM), mCLC846 exposure 6 h, and 24 h after washout of mCLC846, normalized to vehicle expression (n = 3). F) Relative cell viability of Hu1545 cells following exposure to vehicle or mCLC846 (300 nM) for 72 h (n = 3). G) Murine resection liver specimen lysates pretreated with vehicle or mCLC846 phosphorylated MOB1 immunoblot with loading controls. Each lane represents a representative biological replicate (n = 2). H) Liver-to-body-weight ratio (%) in control YAP^fl/fl/TAZ^fl/fl and mice with YAP/TAZ genetically ablated in hepatocytes, YAP^Δhep/TAZ^Δhep treated with vehicle or mCLC846 (50 mg/kg/dose; n = 4–6).

Fig. 4.

mCLC846 induces accelerated liver regeneration through enhancement of normal regenerative pathways. A) Venn diagram of differentially expressed genes, 2 > Log₂-fold change <−2, 40 h posthepatectomy in vehicle- and mCLC846-treated mice. B and C) Heatmap and transcript expression quantification of cell cycle genes in murine livers at baseline and 40 h posthepatectomy in vehicle- and mCLC846-treated mice (n = 3).

Fig. 5.

mCLC846 prevents mortality following partial hepatectomy in diet-induced MASH models. A) Procedural schematic for MASH induction with a high FFC diet. B) Chow fed control mice liver-to-body-weight ratio (%) following hepatectomy with vehicle or mCLC846 (50 mg/kg/dose) treatment (n = 3–6). C) Representative immunofluorescence images and quantification of BrdU incorporation in murine liver sections 40 h posthepatectomy (n = 3–4). D) Murine MASH Kaplan–Meier survival curve posthepatectomy in vehicle- (n = 18) and mCLC846-treated animals (n = 16). Log-rank analysis. Vehicle n = 18 and mCLC846 n = 16. E) Representative immunofluorescence images and quantification of BrdU incorporation in MASH murine liver sections 72 h posthepatectomy (n = 3–5).