Abstract Sun1005: Akt activation in leukocytes as a surrogate test for TAT-PHLPP9c efficacy

Abstract

Introduction: We previously reported that TAT-PHLPP9c enhanced neurological intact survival in both murine and swine models of cardiac arrest. TAT-PHLPP9c is a 20-amino acid peptide designed to increase Akt activity by inhibiting the binding of endogenous PHLPP1 to its adaptor NHERF1. We previously demonstrated that TAT-PHLPP9c enhanced Akt S473 phosphorylation in the heart and brain following 20-min resuscitation in mice. As a heart and brain biopsy is invasive for future clinical trials, we have developed a surrogate assay to reflect the drug activity in these critical organs. Hypothesis: We hypothesize that Akt activation in blood leukocytes could be used as a surrogate test for the efficacy of TAT-PHLPP9c in the heart or brain. Methods: Human blood neutrophils and peripheral blood mononuclear cells (PBMC) were isolated by Percoll density gradient. Mouse blood was collected from the left ventricle of the heart. Akt phosphorylation in the leukocyte subpopulation was measured by Western blot and intracellular flow cytometry. Human leukocyte experiments were performed in vitro. Mouse leukocyte studies were conducted in both in vitro and ex vivo. Results: TAT-PHLPP9c enhanced phosphor-Akt in both neutrophils and PBMCs in a dose-dependent manner, peaked at 30-100 µM, while the scramble control peptide had no effect. TAT-PHLPP9c-treated neutrophils and PBMCs increased phospho-Akt at 15- and 30-min. As western blot requires the purification step of leukocytes that interferes with the detection of transient phospho-Akt in vivo, optimized intracellular flow cytometry in fixed whole blood was established. TAT-PHLPP9c administration caused a right-shift of fluorescence of phospho-Akt in mouse granulocytes, monocytes and lymphocyte at 20-min after i.v. injection as compared to cells collected from saline-treated control mice. Conclusion: Akt activation in leukocytes can be used as a surrogate test for the efficacy of TAT-PHLPP9c in the heart and brain in vivo and potentially used as a guide for TAT-PHLPP9c. treatment.