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Selective and direct targeting of the AdipoR2-CaM-CaMKII-NOS3 axis as a rapid-acting therapy for advanced acute liver failure

ABSTRACT: Drug-induced liver injuries often progress to acute liver failure (ALF) with a high mortality rate. Once ALF has commenced, therapy is minimal. Here, we report that the small bioactive compound SCM-198 (4-guanidino-n-butyl syringate) enables 100% survival of ALF mice induced by paracetamol (APAP) or thioacetamide (TAA) overdoes, even given therapeutically after the establishment of ALF. SCM-198 rapidly recovers liver functions within 6 hours. Using affinity purification and LC-MS, we identify that adiponectin receptor 2 (AdipoR2), but not AdipoR1, is a selective target of SCM-198 mediating the hepatoprotection function of SCM-198. AdipoR2 interacts with SCM-198 through the R335 residues, forming putative guanidine-arginine pairing, with a dissociation constant of 2.3 µM determined by MST and SPR. SCM-198-AdipoR2 does not signal through the AMPK or mTOR pathways, but by increasing the activity of a newly identified AdipoR2-CaM-CaMKII-NOS3 complex, which is pre-exited in liver cells. SCM-198-AdipoR2 binding causes Ca2+ influx and restores the phosphorylation level of CaMKII and NOS3, with the Y274 residue of AdipoR2 serving as a molecular switch for Ca2+ influx. This enables the rapid action of SCM-198 in regulating the levels of nitric oxide (NO) for liver protection in wild type, but not in Adipor2-/- or NOS3-/- mice. Moderately elevated NO attenuates inflammation, regulates lipid metabolism, and stimulates cell proliferation. SCM-198-activation of AdipoR2 also protects human ESC-derived liver organoids from oxidative stress, APAP, or TAA injuries. Together, these results demonstrate that selective targeting of the AdipoR2-CaM-CaMKII-NOS3 axis by SCM-198 is a rapid-acting therapeutic strategy for ALF, both in mouse models and human organoid models, and provides an avenue for selective regulation of AdipoR2 signal transduction in organ regeneration and repair.

ORGANISM(S): Homo Sapiens Mus Musculus

SUBMITTER: Gufa Lin

PROVIDER: PXD053870 | iProX | Thu Jul 11 00:00:00 BST 2024

REPOSITORIES: iProX

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Project description:In acute liver failure (ALF), liver regeneration deficiency is frequently observed, which is the leading cause of ALF and has a high mortality rate. If not treated promptly, ALF may lead to the need for a liver transplant due to limited treatment options. Here, we report the discovery of a 4-guanidino-n-butyl syringate (Leonurine), which has a hepatoprotective effect and can promote regeneration in mouse liver and human embryonic stem cell-induced liver organoids after acute liver injury (ALI). Leonurine demonstrated excellent safety and pharmacokinetics in vivo and was able to significantly reduce mortality in mice after administration of a lethal 24-hour dose of thioacetamide (TAA) or acetaminophen (APAP), which is fully protective compared to treatment with N-acetyl cysteine (NAC) loss of protection. We have synthesized a Leonurine biotinylation probe and have identified adiponectin receptor 2 (AdipoR2), a progesterone and adiponectin receptor family member, as a selective binding partner of Leonurine with a dissociation constant at micromolar levels. Depleting nitric oxide (NO) levels in hepatocytes is the leading cause of liver regeneration deficiency following ALI. The reduction of AdipoR2-Calmodulin (CaM)-endothelial nitric oxide synthase (NOS3) complex activity decreased NO production in hepatocytes, a significant contributor to the ALF phenotype induced by liver regeneration deficiency. Leonurine can form a guanidine-arginine pairing with the AdipoR2 arginine 335 (R335) site to enhance the activity level of AdipoR2 and increase the NO content in hepatocytes, promoting liver regeneration, and quickly antagonize ALF. The oxygen atom of AdipoR2 tyrosine 274 (Y274) interacts with the oxygen atom of Leonurine through hydrogen bonds, significantly enhancing the hydrophobicity of AdipoR2 arginine 275 (R275) residue, thereby activating its surface activity, reducing the molecular switch stability of R275 and aspartate 117 (D117) configurations, which increased the hydrophilic effect, thus regulating Ca2+ inflow but not significantly affecting the AMP level of hepatocytes and finally increasing CaM activity to upregulate the activity of the AdipoR2-CaM-eNOS complex, resulting in increased NO production. In vitro, the knockdown of AdipoR2 or the introduction of a mutant that does not bind to Leonurine blunts the effect of Leonurine's up-regulation of AdipoR2-CaM-eNOS complex activity. In vivo, the knock-out of AdipoR2 or NOS3 eliminated liver protection and regeneration, which Leonurine mediates. The evidence suggests that Leonurine possesses therapeutic benefits for patients without significant adverse effects. Our findings indicate that Leonurine possesses the potential to provide a rapid-acting treatment for the exercise of therapeutic benefits in ALF.

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Selective and direct targeting of the AdipoR2-CaM-CaMKII-NOS3 axis as a rapid-acting therapy for advanced acute liver failure

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