No effective way to slow or halt the progressive paralysis due to ALS now exists. A handle on etiology has been provided by the discovery that mutation in SOD1 causes a subset of cases. Decreased stability and decreased binding of zinc or copper, gain of a deleterious oxidative activity, binding of heat shock proteins, and aggregation, have all been proposed as causative. Yet the mystery remains. Oxidative stress seems a likely cause. We have developed manganese porphyrin derivatives that efficiently scavenge O2-, ONOO-, and CO3-. When tested in a murine model of FALS, by Dr. John Crow, one of our manganese porphyrins dramatically delayed paralysis and death (See preliminary data). We synthesized new cationic manganese porphyrins with graded hydrophobicities and would like to prepare amounts large enough for similar testing, in the hope that they may be more efficacious. We will explore the role of carbonate radical in imposing the oxidative stress that can contribute in death of motor neurons. Toward this end we will start by seeing whether CO2 exacerbates oxidative stress in suspensions of SOD-deficient strains as compared to parental strains of E. coli. Mn porphyrins should oppose these toxic effects of CO2. We will test the degree to which our Mn porphyrins suppress NOS activity in N2a and NSC-34 cells, perhaps by way of down-regulating the biosynthesis of Hsp90. In relation to the latter we will examine the effect of NO on the binding of Hsps to mutant SOD1, by immunoprecipitation. We will also examine the degree to which the wild-type and mutant SOD1s are covalently cross-linked in these cells and the effects of NO on this crosslinking. Finally, since we have shown that our manganese porphyrins can catalytically oxidize the NO cofactor, tetrahydrobiopterin, we will test this alternative, or parallel, pathway of NOS inhibition. The goal of this work is to increase our understanding of the development of ALS and at the same time to develop compounds that will be efficacious in ameliorating the symptoms of this disease. The compounds we find most effective in our models will be tested first in Dr. Crow's G93 A murine model of FALS and then forwarded to Incara Pharmaceuticals for clinical development.
