[JM12] 

Figure 3 - CuATSM restores copper to SODWT. Representative mass spectrum generated from brain homogenate of 15-day-old SODWTxCCS mice. Spectra were deconvoluted and quantitated by comparing human SODWT with the internal standard bovine SODWT. SODWTxCCS mice express about 100uM SODWT in the brain at 15 days. Half this pool of SODWT is “mature”, containing two metals and the C57-C146 disulfide. Treatment with CuATSM significantly increases the fraction of SOD containing two metals and the C57-C146 disulfide.

 

In summary, treatment with CuATSM rescued the early development crisis seen in SODWTxCCS mice (Figure 1) and restored copper to brain COX (Figure 2) and SOD (Figure 3). Next, using COX and SOD as biomarkers for copper delivery, we investigated how modulating copper delivery affected the early crisis seen in SODWTxCCS mice. Our approach was to systematically test a large number of CuATSM chemical derivatives differing in their potential to release copper.

CuATSM is a planar, neutral, slightly hydrophobic molecule consisting of a bisthiosemicarbazone ligand in complex with a CuII atom (Figure 4). It is proposed that CuII in CuATSM is reduced to CuI and released in cells possessing highly reducing environments resultant from damage to mitochondria (Donnelly et al., 2012). Because CuATSM has an extremely low CuIIàCuI reduction potential relative to biological systems, very little copper is likely to be released in vivo. To investigate how modulating copper delivery affected the early crisis seen in SODWTxCCS mice, and to explore the possibility of creating a more effective copper delivery agent, we synthesized a series of bisthiosemicarbazone derivatives with increasing CuIIàCuI reduction potentials. We then systematically tested their efficacy in rescuing the early development crisis seen in SODWTxCCS mice, as measured by copper delivery to the biomarkers COX and SOD.

We first synthesized bisthiosemicarbazones with 2, 1, or 0 methyl groups on the di-imine backbone, called CuATSM, CuPTSM, and CuGTSM, respectively (Figure 4). Despite these small structural differences, cyclic voltammetry revealed considerably different reduction potentials of -0.46V, -0.31V, and -0.24V, respectively FIG 6. These reduction potentials predict that the ligated copper in CuATSM is least likely of the three to be chemically reduced (and released) in a biological context, while CuGTSM is the most likely. These reduction potentials agree with reported literature values[JM13] .

We treated SODWTxCCS mice from days 4-15 with CuATSM, CuPTSM, or CuGTSM. At the planned age of 15 days we measured brain COX activity and SOD maturity. Fifteen days was chosen as the primary comparison date because preliminary studies indicated that clear differences are apparent between untreated and CuATSM-treated SODWTxCCS mice at this age, and because 15 days is near-terminal for SODWTxCCS mice without intervention.

CuATSM and CuPTSM treatments significantly increased COX activity and SOD maturity by day 15, compared to untreated control. However, the increase in COX activity and SOD maturity, compared to control, seen in CuGTSM-treated animals was slightly less than that seen in CuATSM- and CuPTSM-treated animals, and was not statistically significant. There were no significant differences in COX activity or SOD maturity between treated groups.

The effects of bisthiocarbazone treatment on COX and SOD were not necessarily reflected in animal weights, for which there were no significant differences. However, each treatment group exhibited a markedly different behavioral phenotype that suggests differential effects on animal health. Untreated and CuGTSM-treated SODWTxCCS pups were immobile and moribund, while CuATSM-treated pups were active and beginning to walk. CuPTSM-treated pups were intermediate in phenotype severity.