(A) Percent inhibition generated from pelleting assays for 19 small molecules tested at 100 μM concentration is shown. (B) Human B2 subunit (amino acids 1 – 112) at 1 μM concentration was mixed with 2 μM of microfilaments and pelleted in the presence of the following concentrations of enoxacin: (1) no actin control, (2) vehicle control, (3) 1 μM enoxacin, (4) 10 μM enoxacin, (5) 25 μM enoxacin, (6) 100 μM enoxacin. The amount of B2-MBP in supernatants and pellets is shown.

Treatment of mature osteoclasts with enoxacin reduced actin ring and ruffled membrane formation. Mouse marrow cultures on tissue culture plates were stimulated with calcitriol to for 6 days to produce osteoclasts. The cells were then scraped free and loaded atop bone slices and treated with vehicle or enoxacin as indicated. After 3 days, the cells were fixed and stained with anti-E subunit antisera to detect ruffled membranes (A,B) and with phalloidin to detect actin rings (C,D). B and D are typical fields from vehicle control cultures. A and C are fields from cultures treated with 100 μM enoxacin. Tabulation fo actin rings and ruffled membranes shows that both were reduced in a dose dependent manner by enoxacin. Asterisks indicate P < 0.05 compared with control value by Student’s t test.

Enoxacin did not block osteoblast formation or mineralization at concentrations where osteoclast numbers were sharply reduced. (A) Typical field from a bone slice with mouse marrow a top a bone slice and cultured in the presence of vehicle fixed and stained after 3 days for alkaline phosphatase activity to detect osteoblasts (blue staining). (B) Typical field from a bone slice in which the cultures were handled identically to A, but in the presence of 10 μM enoxacin. (C–H) Enoxacin had no detected effect on growth and mineralization by MC3T3-E1 subclone 4 osteoblasts at concentrations of 100 μM or less. MC3T3-E1 subclone 4 cells were obtained from ATCC (Manassas, VA) and were inoculated in 24-well plates at a density of 2 × 10⁴ cells/well and were maintained in αMEM D10. To induce mineralization, we treated cultures with with 50 μG/mL ascorbic acid and 10 mM/β-glycerophosphate for 15 days. Cells formed monolayers and mineralized in vehicle (C), 100 μM enoxacin (D), 25 μM enoxacin (E), 10 μM enoxacin (F), 5 μM enoxacin (G), and 1 μM enoxacin (H). Mineral deposits were detected as black flecks in all of the images. Some are indicated by small arrows in panel C in order to provide an example. The monolayer of MC3T3 cells appears as striations in the bright field micrographs. The scale bar = 10 μm in A and B and 15 μm in C–H. (I) Quantitation of number of mineral particles per well determined by analysis of Von Kossa stained cells using Image J.

Identification of enoxacin by molecular docking. A homology model of human V-ATPase subunit B is shown in the top panel colored based on sequence similarity to profilin-1 (graded blue, cyan, green, yellow, orange, red to represent low sequence similarity (blue) to high (red) based on a BLOSUM62 matrix. Magenta spheres depict the site selected for molecular docking. The box represents boundaries of the scoring grid used for molecular docking. The chemical structure of enoxacin is shown in the inset of the top panel. The bottom panel shows enoxacin in the orientation posed by molecular docking using DOCK6.1 (UCSF), yellow for carbon, blue for nitrogen, red for oxygen, light blue for fluorine, white for hydrogen. The molecular surface of the V-ATPase B2 is colored cyan for carbon, blue for nitrogen, red for oxygen. Enoxacin may interact with a structural pocket in V-ATPase comprised of side chains from Tyr68, Val89, Thr268, Ile269, Glu308, and Arg314. In addition to van der Waals contacts between these residues and enoxacin, a H bond interaction is predicted between the Oε1 atom of Arg314 and N4 of enoxacin.

Enoxacin inhibits the number of TRAP+ cells differentiating from mouse marrow cultures. (A–C). Comparison of typical fields from mouse marrow cultures treated with calcitriol to stimulate osteoclast formation and (A) vehicle, (B) 10 μM, and (C) 100 μM enoxacin for 7 days. (D) Numbers of TRAP+ cells per well categorized by number of nuclei differentiating in response to calcitriol in the presence of varying concentrations of enoxacin. Asterisks indicate P < 0.05 compared with control value by Student’s t test. Scale Bar = 30 μm.

Enoxacin dose dependently reduce bone resorption by mouse marrow cultures. Left panel: scanning electron micrographs of bone slices resorbed by mouse marrow cultures in the presence of vehicle (A), 10 μM enoxacin (B), 25 μM enoxacin (C), 100μM enoxacin (D). The scale bar = 25 μn. Right panel: tabulation of the percent of the control amount of pit numbers, area per pit, and percent of total area resorbed. The N was 4 for each condition.

Effects of fluoroquinolones related to enoxacin on interaction between B-subunit and F-actin and on differentiation of mouse marrow osteoclasts. (A) Reduction of B-subunit bound to F-actin in pelleting assay by 100 μM of the various fluoroquinolones. (B) Number of TRAP+ cells per well in the presence of 50 μM of the indicated compound after 6 days in culture. Statistical analysis was performed using Student’s t test. Asterisk indicates P < 0.05 compared with vehicle control, double asterisk indicates P < 0.05 compared with both the vehicle control and with enoxacin.