Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.

Abstract 4038

Poster Board III-974

Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K⁺ and Cl^- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O₂ in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs.

Acknowledgments.

This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant.