Rational identification of enoxacin as a novel V-ATPase-directed osteoclast inhibitor

Abstract

Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actinbinding activity has been demonstrated in the B-subunit of yeast. A conserved "profilin-like" domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the "profilin-like" domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the "housekeeping" functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.

Fig. (1)

Microfilament interacting with V-ATPase through the B-subunit. Note that because of the location of the F-actin binding site, V-ATPases in a membrane can bind microfilaments. Arbitrarily, one of the EG stator arms has been removed to allow access of microfilaments to the actin binding site on the B-subunit. In contrast, to B-subunit, the actin binding sites in the C-subunit are unlikely to be accessible in the intact enzyme and instead the interaction between C-subunit and actin may be involved in reversible assembly [128].

Fig. (2)

Structure of V-ATPase. Structure is based on the recent electron microscopy study by Harrison and colleagues (53). List of subunit isoforms was adapted from reference 33. Curved thin arrows indicate site of ATP hydrolysis and the pathway of protons through the membrane. The fat curved arrow shows the direction of turning of the rotor during enzymatic activity. Large straight arrows identify the three non-identical EG stators. ATP6AP2 (the Pro-renin receptor) is included in the schematic although its location is not known, nor whether it is associated with all V-ATPases or only with V-ATPases in specific locations. Adapted from (Neubert, J. K., R. M. Caudle, C. Dolce, E. J. Toro, Y. Bokrand-Donatelli, and L. S. Holliday. 2011. Neural modulation of orthodontic tooth movement. In Orthodontics (Mandic, ed), INTECH, Open Access Publisher, Rijeka, Croatia In press) with permission.

Fig. (3)

Organization of resorbing osteoclast. The schematic shows a side view of a resorbing osteoclast and depicts important features required for resorption. V-ATPases in vesicles are targeted to the the ruffled plasma membrane, a subdomain of the plasma membrane. The ruffled membrane is bounded by an actin ring which is composed of podosomes. The actin ring forms concurrent with the insertion of V-ATPases into the plasma membrane. The actin ring is associated with a sealing zone, a region of very tight adhesion that segregates an extracellular resorption compartment. V-ATPases pump protons into the resorption compartment lowering its pH which solubilizes bone mineral, and allows the acid cysteine proteinase, cathepsin K to degrade the organic matrix of bone. Adapted from (Neubert, J. K., R. M. Caudle, C. Dolce, E. J. Toro, Y. Bokrand-Donatelli, and L. S. Holliday. 2011. Neural modulation of orthodontic tooth movement. In Orthodontics (Mandic, ed), INTECH, Open Access Publisher, Rijeka, Croatia In press) with permission.

Fig. (4)

Small molecule inhibitors of V-ATPase binding to microfilaments. We hypothesized that small molecules (depicted as stars) that bind the actin binding surface on B-subunit would competitively inhibit binding to F-actin and inhibit osteoclast bone resorption.