A novel macrolide/fluoroketolide, solithromycin (CEM-101), reverses corticosteroid insensitivity via activation of protein phosphatase PP2A

Introduction: Activation of PI3 kinase causes oxidative stress-induced corticosteroid (CS) insensitivity via HDAC2 reduction. We have recently demonstrated that a novel macrolide/fluoroketolide, solithromycin (Soli, CEM-101) restores CS sensitivity via HDAC up-regulation due to PI3K signaling inhibition (ATS2010). However, the mechanism of this effect has not been elucidated. Aims: To investigate the role of a serine/threonine phosphatase PP2A on regulation of the PI3K pathway as the target of Soli. Methods: CS sensitivity was determined by IC 50 of dexamethasone (Dex) on TNFα-induced IL-8 production in U937 monocytic cell. Activities of HDAC2 and PP2A were measured by fluorescence-based activity assay. Phosphorylation levels of Akt as a marker of PI3K activation were determined by Western blotting. Okadaic acid (OA) was used to inhibit PP2A as needed. Results: OA enhanced H 2 O 2 -induced Akt phosphorylation and HDAC2 reduction in U937 cells, and recombinant PP2A reduced Akt phosphorylation levels. Soli restored Dex sensitivity under H 2 O 2 exposure, but pretreatment with OA abrogated Soli-mediated restoration of Dex sensitivity, inhibition of Akt phosphorylation, and HDAC2 activation. In addition, PP2A immunoprecipitates from the membrane fraction and recombinant PP2A were directly activated by Soli. Conclusions: PP2A might be a negative regulator of PI3K signalling. Soli activates PP2A directly, inhibits Akt phosphorylation, then restores HDAC2 activity, resulting in the restoration of CS sensitivity under oxidative stress. Thus, Soli may be a potential treatment for steroid insensitive diseases such as COPD and severe asthma.