Mode of action of zoxamide (RH-7281), a new Oomycete fungicide

RH-7281 (zoxamide) rapidly arrested nuclear division in Phytophthora capsici germlings and destroyed the microtubule cytoskeleton. In studies using isolated bovine tubulin, RH-7281 inhibited the in vitro assembly of tubulin into microtubules. Treatment of P. capsici mycelium with the tritiated active enantiomer of RH-7281 (H-3-(S)-RH-7281) and subsequent analysis of radiolabeled protein revealed a highly specific covalent binding to beta -tubulin. H-3-(S)-RH-7281 also bound covalently to the beta -subunit of isolated bovine tubulin. Binding of H-3-(S)-RH-7281 to tubulin in P. capsici was found to be saturable in a whole-cell binding assay in 96-well microtiter plates. The ability of structural analogs of RH-7281 to inhibit binding of H-3-(S)-RH- 7281 in the assay correlated well with their fungitoxicity. The experimental antimicrotubule fungicide zarilamide was shown to be a competitive inhibitor of H-3-(S)-RH-7281 binding to tubulin in P. capsici, indicating a common binding site on tubulin. We conclude that the fungitoxic mechanism of RH-7281 involves inhibition of nuclear division as the result of covalent binding to beta -tubulin and disruption of the microtubule cytoskeleton. (C) 2001 Academic Press.