INTERACTION OF USTILOXIN-A WITH BOVINE BRAIN TUBULIN

Ustiloxin A is a modified peptide derived from false smut balls on ric e panicles, caused by the fungus Ustilaginoidea virens; structurally, it resembles phomopsin A. Ustiloxin G is cytotoxic and is an inhibitor of microtubule assembly in vitro. Because of its resemblance to phomo psin A, we examined its interaction with tubulin and compared the resu lts with those obtained with phomopsin A and dolastatin 10, both of wh ich were found previously to have very similar effects. We determined that ustiloxin A inhibited the formation of a particular intra-chain c ross-link in beta-tubulin, as do vinblastine, maytansine, rhizoxin, ph omopsin A, dolastatin 10, halichondrin B and homohalichondrin B; this is in contrast to colchicine and podophyllotoxin which do not inhibit formation of this cross-link. Ustiloxin A also inhibited the alkylatio n of tubulin by iodo[C-14]acetamide, as do phomopsin A and dolastatin 10; vinblastine was almost as potent an inhibitor of alkylation as ust iloxin A, whereas maytansine, halichondrin B and homohalichondrin B ha ve little or no effect. In addition, ustiloxin A inhibited exposure of hydrophobic areas on the surface of the tubulin molecule. In this res pect, ustiloxin A was indistinguishable from phomopsin A but slightly more effective than dolastatin 10 and considerably more effective than vinblastine; this provides a strong contrast to maytansine, rhizoxin, and homohalichondrin B which have no effect on exposure of hydrophobi c areas and to halichondrin B which enhances exposure. Lastly, ustilox in A strongly stabilized the binding of [H-3]colchicine to tubulin. Th e combination of ustiloxin A with colchicine stabilized tubulin with a half-life of over 8 days, comparable with results obtained with phomo psin A and colchicine. A comparison of the structures of ustiloxin A, phomopsin A and dolastatin 10 raised the possibility that the strong s tabilization of the tubulin structure may require a short segment of h ydrophobic amino acids such as the modified valine-isoleucine sequence present in all three compounds. The rest of the structure, specifical ly the large ring of ustiloxin A and phomopsin A, may serve to place t his sequence in an appropriate conformation to interact with tubulin.