Trypanothione reductase, an essential component of the anti-oxidant defences of parasitic trypanosomes and Leishmania, differs markedly from the equivalent host enzyme, glutathione reductase, in the binding site for the disulphide substrate. Molecular modelling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by testing 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent, with a K(i) of 6 microM, competitive with respect to trypanothione. Many of these compounds have been noted previously to have anti-trypanosomal and anti-leishmanial activity and thus they can serve as lead structures for rational drug design.

ACKNOWLEDGEMENTSThis work was supported by the Deutsche Forschungsgemeinschaft (HE 2993/5-2 to Stefan H. Heinemann), FONACIT CNPq (PI 20040000385 to Adolfo Borges), and NIH (GM48677 to Baldomero M. Olivera). We thank Dr. J. Trimmer for providing the clone for rNa V 1.4 and the MzB SW seminar group for helpful discussions. ABSTRACTThe µO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na V 1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which µO-conotoxins affect Na V channels. Rat Na V 1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily µ-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na V channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion b-toxins. In fact, MrVIA functionally competed with the scorpion b-toxin Ts1 from Tityus serrulatus, while it did not show competition with µ-GIIIA. Ts1 and µ-GIIIA did not compete either. Thus, similar to scorpion b-toxins, µO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na V channels. They "block" Na + flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.

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