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 (3H-(S)-RH-7281) and subsequent analysis of radiolabeled protein revealed a highly specific covalent binding to β-tubulin. 3H-(S)-RH-7281 also bound covalently to the β-subunit of isolated bovine tubulin. Binding of 3H-(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 3H-(S)-RH-7281 in the assay correlated well with their fungitoxicity. The experimental antimicrotubule fungicide zarilamide was shown to be a competitive inhibitor of 3H-(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 β-tubulin and disruption of the microtubule cytoskeleton.