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

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.