In an attempt to rationally design new drugs for trypanosomiasis, we have been exploring ways to increase in trypanosomes the amount of oxidant stress or the sensitivity to oxidants. Our studies have included ways to generate free radicals in the trypanosome and the nature of the enzymes that trypanosomes have to combat oxidant stress. During the previous grant period, we have investigated the following: 1) A large series of quinone derivatives were synthesized and found to be trypanocidal in vitro but without any activity in vivo. Attempts to modify the structure to prevent metabolism were without success. 2) T. brucei, T. cruzi, C. fasciculata, L. tropica were found to have an iron-containing superoxide dismutase (SOD) in contrast to the Cu, Zn-SOD and Mn-SOD found in mammalian cells. A difference in sensitivity to various inhibitors points to the potential of being able to selectively interfere with this crucial enzyme. 3) In contrast to the GSH reductase from all other organisms previously studied, the trypanosomal enzyme had an obligate requirement for a novel thiol-containing co-factor. The structure of this co-factor, trypanothione, has been recently elucidated and found to be bis-(glutathionyl) spermidine. Since trypanothione is present in all the parasitic trypanosomes, e.g. T. brucei, T. cruzi, L. mexicana and not in other organisms, it offers a unique point of attack for chemotherapeutic intervention. Accordingly, in the next grant period, we will concentrate on this new finding. 1) Further chemical and biochemical studies of trypanothione are planned in order to gain new insight in the conformation of the molecule. These are being pursued so that new agents can be designed which will react to inactivate the co-factor. 2) Trypanothione reductase from C. fasciculata and T. brucei will be isolated and studied so that new agents can be designed and synthesized to inhibit this key enzyme. 3) The biosynthesis of trypanothione also offers a point of attack. Spermidine analogues will be evaluated as potential agents. Hopefully, these studies will give new insight into drug design for trypanosomiasis.
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