Evaluation of hepatic activity of immunomodulatory compounds and GSK3 inhibitors

Abstract

Nitric-oxide synthase (NOS), the enzyme responsible for mammalian nitric oxide (NO) generation, and cytochrome P450 (CYP450), the main enzymes involved in drug metabolism, share striking similarities. First and foremost, both are heme-thiolate proteins, which employ the same prosthetic group to perform similar chemistry. Moreover, they share the same diflavoprotein reductase as redox partner. Therefore, it makes sense that cytochrome P450 drug-mediated biotransformations might play an important role in the pharmacological modulation of NOS. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and NO modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes, in order assess the involvement of CYP450 activities in the production of NO. From the experience acquired during this aim, we developed a validated high-throughput screening (HTS) approach in 96-well plate format for the assessment and discovery of molecules with anti-inflammatory/immunomodulatory activity. The in vitro models were based on the quantitation of nitrite levels in lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophages and interleukin-8 (IL-8) in Caco-2 cells stimulated with interleukin 1β (IL-1β)