Pharmacological inhibition of CaMKK2 with the selective antagonist STO-609 regresses NAFLD

Brian York,Nagireddy Putluri,Naomi Gonzales,Fumin Lin,Kathrina L Marcelo,David Gooden,Jianqiang Mao,Feng Li,Anthony R Means,Suman Maity,Adam Dean,Cristian Coarfa

doi:10.1038/s41598-017-12139-3

Brian York, Nagireddy Putluri + Show 10 more

Open Access

https://doi.org/10.1038/s41598-017-12139-3

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Abstract

Binding of calcium to its intracellular receptor calmodulin (CaM) activates a family of Ca2+/CaM-dependent protein kinases. CaMKK2 (Ca2+/CaM-dependent protein kinase kinase 2) is a central member of this kinase family as it controls the actions of a CaMK cascade involving CaMKI, CaMKIV or AMPK. CaMKK2 controls insulin signaling, metabolic homeostasis, inflammation and cancer cell growth highlighting its potential as a therapeutic target for a variety of diseases. STO-609 is a selective, small molecule inhibitor of CaMKK2. Although STO-609 has been used extensively in vitro and in cells to characterize and define new mechanistic functions of CaMKK2, only a few studies have reported the in vivo use of STO-609. We synthesized functional STO-609 and assessed its pharmacological properties through in vitro (kinase assay), ex vivo (human liver microsomes) and in vivo (mouse) model systems. We describe the metabolic processing of STO-609, its toxicity, pharmacokinetics and bioavailability in a variety of mouse tissues. Utilizing these data, we show STO-609 treatment to inhibit CaMKK2 function confers protection against non-alcoholic fatty liver disease. These data provide a valuable resource by establishing criteria for use of STO-609 to inhibit the in vivo functions of CaMKK2 and demonstrate its utility for treating metabolically-related hepatic disease.

Highlights

At present, the most effective inhibitor of CaMKK2 activity is the ATP competitive inhibitor, STO-609 (7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) (Fig. 1A, red box)
Leveraging the information from our in vivo pharmacokinetic analysis of STO-609, we demonstrate that pharmacological inhibition of CaMKK2 reverses the hallmarks of hepatic steatosis in two mouse models of NAFLD
Work from our laboratory has established the importance of CaMKK2 in hypothalamic control of satiety through regulation of AMPK13

Summary

Introduction

The most effective inhibitor of CaMKK2 activity is the ATP competitive inhibitor, STO-609 (7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate) (Fig. 1A, red box). A limited number of studies have reported in vivo use of STO-609 for inhibition of CaMKK2 in the control of satiety as well as to confer protection against prostate and liver cancers[9,13,14] While these findings highlight the potential utility of STO-609 for attenuating downstream functions of CaMKK2 action in vivo, there have been no reports that evaluate the pharmacokinetics, tissue distribution, toxicity, dosing and metabolism of STO-609. Using recombinant human CYP450s and human liver microsomes, we identified CYP1A2 as the predominant P450 enzyme responsible for the metabolic conversion of STO-609 to three distinct mono-hydroxylated byproducts Translating these observations to an in vivo setting using C57BL/6 J wild type mice, we characterized the toxicity, pharmacokinetics, tissue distribution and efficacy of STO-609 for inhibiting CaMKK2 function. Taken together these data provide the research community with empirical information for the safe and effective dosing of mice with STO-609 and highlight the utility of pharmacological inhibition of CaMKK2 signaling to attenuate NAFLD

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