Salicylihalamide A Inhibits the Vo Sector of the V-ATPase through a Mechanism Distinct from Bafilomycin A1

Abstract

The newly identified specific V-ATPase inhibitor, salicylihalamide A, is distinct from any previously identified V-ATPase inhibitors in that it inhibits only mammalian V-ATPases, but not those from yeast or other fungi (Boyd, M. R., Farina, C., Belfiore P., Gagliardi, S., Kim, J. W., Hayakawa, Y., Beutler, J. A., McKee, T. C., Bowman, B. J., and Bowman, E. J. (2001) J. Pharmacol. Exp. Ther. 297, 114-120). In addition, salicylihalamide A does not compete with concanamycin or bafilomycin for binding to V-ATPase, indicating that it has a different binding site from those classic V-ATPase inhibitors (Huss, M., Ingenhorst, G., Konig, S., Gassel, M., Drose, S., Zeeck, A., Altendorf, K., and Wieczorek, H. (2002) J. Biol. Chem. 277, 40544-40548). By using purified bovine brain V-pump and its dissociated V1 and V0 sectors, we identified the recognition and binding site for salicylihalamide to be within the V0 domain. Salicylihalamide does not inhibit the ATP hydrolysis activity of the dissociated V1-ATPase but inhibits the ATPase activity of the holoenzyme by inhibiting the V0 domain. Salicylihalamide causes a dramatic redistribution of cytosolic V1 from soluble to membrane-associated form, a change not observed in cells treated with either bafilomycin of NH4Cl. By synthesizing and characterizing a series of salicylihalamide derivatives, we investigated the structural determinants of salicylihalamide inhibition in terms of potency and reversibility, and used this information to suggest a possible binding mechanism.