Identification of two new inhibitors of the hepatic glucose-6-phosphatase system

Abstract

A high-throughput screening assay aimed at the detection of inhibitors of the translocase components of the hepatic glucose-6-phosphatase (G6Pase) system was set up in a microplate format using untreated and Triton X-100®-disrupted rat liver microsomes. The assay measured the phosphate released from glucose-6-phosphate (G6P) using a standard calorimetric method. We identified two structurally unrelated compounds, 2-hydroxy-5-nitrobenzaldehyde and chlorogenic acid, which inhibited the hydrolysis of G6P in untreated microsomes, each with IC50 values of 338 μM and 226 μM, respectively, but were devoid of activity in disrupted microsomes. Thus, the two compounds exhibited a high degree of specificity for translocase components. The effects of 2-hydroxy-5-nitrobenzaldehyde bear a resemblance to the effects of pyridoxal phosphate. Studies with compounds structurally related to 2-hydroxy-5-nitrobenzaldehyde suggest that both a phenolic OH-group in ortho position to the aldehyde group and a suitable electron-withdrawing group in position 3 or 5 of the aromatic ring are indispensable for the activity of this class of inhibitors. The inhibition pattern of chlorogenic acid is distinct from that of phloretin and is dependent on a free carboxyl group. The products of chlorogenic acid hydrolysis, quinic acid and caffeic acid, are inactive. 2-Hydroxy-5-nitrobenzaldehyde type inhibitors and chlorogenic acid are potent new inhibitors for investigating the structure and function of the translocase components of the G6Pase system.