Boron complexation with NAD+ inhibits ADP-ribosyl cyclase non-competitively

The purpose of this research was to identify a biochemical pathway elucidating the effects of boric acid previously studied in inhibiting the growth of prostate cancer cell lines. Recently, boric acid was shown to inhibit the growth of transplanted human prostate tumors in nude mice and prostate cancer cell lines, DU-145 and LNCaP. However, determination of biochemical pathways underlying these effects has been hampered by the lack of traceable boron isotope to reveal the site of interaction. The following results were accomplished. (1) For the first time ESI-MS and 11B NMR were used to observe the complexation of boric acid with NAD+ and NADH. The limit of detection for ESI-MS to observe the complexation was 10 muM at pH 10.3 and 50 muM at pH 7.0 with NAD+. (2) MS/MS and dGDP were utilized to determine that the binding site of boric acid was cis-2,3-ribose diol, not the hydroxyl groups on the phosphate backbone of NAD+. (3) The complexation of borate with NAD + and NADH was pH dependent with maximum formation occurring under alkaline conditions. (4) The association constants (KA) of boron complexation with various nucleotides at pH 10.3 were calculated using ESI-MS and listed from highest to lowest: NAD+>NADH ≅ AMP ≅ GMP ≅ CMP ≅ UMP>ADP ≅ GDP ≅ CDP ≅ UDP>GTP ≅ NADP+>ATP ≅ CTP ≅ UTP ≅ NADPH. The KA for boric acid complexation with NAD+ was 1770 +/- 180 L/mol at pH 10.3. (5) The complexation of boric acid with nicotinamide containing nucleotides was increased in the presence of a cationic charge on the nicotinamide group. (6) The complexation of boric acid to nucleotides increased as the number of phosphate groups decreased. (7) Only the boric acid-NAD+ complex was observed at pH 7.4, which indicated that NAD+ was the most physiologically relevant biomolecule to interact with boron in cells. (8) For the first time capillary electrophoresis (CE) was used to separate NAD+, cADPR, and ADP-ribose involved in the ADP-ribosyl cyclase reaction. The apparent Km and V max for ADP-ribosyl cyclase by CE was determined to be 1.24 +/- 0.05 mM and 131.8 +/- 2.0 muM/min (R2 0.999), respectively, through non-linear regression of the data fitted to the Michaelis-Menten equation. (9) Boric acid inhibited ADP-ribosyl cyclase non-competitively. The Ki of the inhibition was 40.5 +/- 0.5 mM. (10) Boric acid inhibited ADP-ribosyl cyclase directly. It was determined that boric acid did not interfere with hydrolase activity of the enzyme nor the inhibitory effects of nicotinamide. (Abstract shortened by UMI.)...