234 - Investigation into the Oxidation of CuATSM as a Mechanism for Copper Release

Abstract

Copper delivery to superoxide dismutase (SOD) by copper transport agent CuATSM (diacetyl-bis(N4-methylthiosemi-carbazone) copper(II)) is protective in mouse models of amyotrophic lateral sclerosis (ALS). CuATSM is a neutral, hydrophobic compound, able to cross into the CNS without facilitated transport. Reduction of CuII has frequently been proposed as the mechanism of copper release from the ATSM ligand. However, with a reduction potential of -0.46V, CuII reduction will be difficult to accomplish in a cellular environment. In contrast to reduction, oxidation of the ATSM ligand could serve as a mechanism of copper release in a biological system. We investigated the oxidation of CuATSM ligand in an assay utilizing 10-9M horseradish peroxidase (HRP), 2-10 µM hydrogen peroxide, and 10 to 100 µM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a competitive substrate to test for inhibition by CuATSM. Surprisingly, low micromolar concentrations of CuATSM accelerated the oxidation of ABTS by HRP. Our kinetic results suggest that CuATSM was efficiently oxidized by HRP and the CuATSM radical cation in turn oxidized ABTS, We conclude that oxidative reactions involving CuATSM are likely to occur in vivo and need to be further investigated as clinical trials are now underway in treating human ALS patients. This work is supported by funding from the DoD Congressionally Directed Medical Research Program (AL140108) and by Ice Bucket donations made to the ALSA (Research Award ALSA 320).