8-Chloro-adenosine (8-Cl-Ado) is an adenosine analogue that is entering clinical Phase I trial. It is phosphorylated to the triphosphate form (8-Cl-ATP) to act as a cytotoxic agent. Studies using nucleoside kinase proficient or deficient cell lines demonstrated that 8-Cl-Ado was phosphorylated to its monophosphate form (8-Cl-AMP) by Ado kinase, which was further metabolized to its diphosphate form (8-Cl-ADP), then converted to 8-Cl-ATP. Previous studies in multiple myeloma cell line (MM1.S) showed that 10 μM 8-Cl-Ado resulted in accumulation of 400-500 μM 8-Cl-ATP with a parallel decline in the cellular bioenergy. The endogenous ATP concentration (3-4 mM) was reduced by more than 50% after a 12-hr incubation. This phenomenon was also observed in several solid tumor cell lines and in primary leukemia cells. A majority of cellular ATP is generated via oxidative phosphorylation in the mitochondria. Complex V of the mitochondrial respiratory chain is ATP synthase, which catalyzes the conversion of ADP into ATP. Based on this background, we hypothesized that 8-Cl-ADP is a substrate of ATP synthase, and 8-Cl-ADP/8-Cl-ATP inhibit ATP synthesis by this enzyme. Using an inhibitor of ATP synthase, oligomycin, we determined whether 8-Cl-ATP formation was through oxidative phosphorylation. Compared to only 8-Cl-Ado incubated cells, MM cells pre-treated with oligomycin accumulated only ~35% of 8-Cl-ATP. This was similar to the inhibition of ATP formation in oligomycin treated cells. However, the accumulation of mono- and diphosphates was not affected. These data indicated that similar to ADP, 8-Cl-ADP may be a substrate for ATP synthase. Using the crystal structure of this protein, ADP and 8-Cl-ADP were docked into the catalytic pocket of the enzyme by molecular docking packages GOLD and AUTODOCK. The results from both programs demonstrated that 8-Cl-ADP was bound more tightly to the protein than ADP, suggesting that 8-Cl-ADP may be a better substrate of ATP synthase than its native substrate ADP. This provides a potential mechanism for the rapid accumulation of 8-Cl-ATP, with a simultaneous decline in ATP. A negative regulator of ATP synthase is ATP, which when bound to the inhibitory site, reduces the activity of the enzyme. To assess if noncognate 8-Cl-ATP could serve as an inhibitor of ATP synthase, ATP and 8-Cl-ATP were docked into the binding pocket of the enzyme. The results revealed that 8-Cl-ATP was bound more favorably to the synthase than ATP. To validate this modeling observation experimentally, MM.1S cells were incubated with 8-Cl-Ado to buildup intracellular 8-Cl-ATP, washed, then ATP formation was monitored using [3H]Ado as a substrate. The level of newly synthesized [3H]ATP was indeed reduced in MM.1S cells that were pre-treated with 8-Cl-Ado. Taken together, our cellular pharmacology results are in agreement with the computational docking studies of ATP synthase, and provide a possible mechanism for cellular bioenergy depletion by 8-Cl-Ado.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA