Inhibition of ATP synthase by chlorinated adenosine analogue

Abstract

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; F(O) intermembrane base and F1 domain, containing alpha and beta subunits. Crystal structures of both alpha and beta subunits that bind to the substrate, ADP, are known in tight binding (alpha(dp)beta(dp)) and loose binding (alpha(tp)beta(tp)) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the alpha(tp)beta(tp) state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (alpha(dp)beta(dp)) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF(3)(-). Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase.

Figure 1. 8-Cl-Ado does not affect O₂ consumption

MM.1S cells were incubated with either no drug (control), Ado (A) or 8-Cl-Ado (B), and O₂ consumption was measured using a respirometer. The experiments were performed in triplicate with similar results.

Figure 2. Inhibition of ATP synthase decreases ATP and 8-Cl-ATP accumulation but not monophosphate (MP) or diphosphate (DP) accumulation

MM.1S cells were pretreated with no drug (black bars) or 2 µg/mL oligomycin (a pharmacological inhibitor of ATP synthase, gray bars) for 30 min, then either [³H]Ado (A, D), [³H]8-Cl-Ado (B, D) or [³H]8-Cl-cAMP (C) was added to the cells for indicated times or for 3 hours (Figure 2D). Accumulation of [³H]AMP, [³H]ADP, and [³H]ATP or [³H]8-Cl-AMP, [³H]8-Cl-ADP, and [³H]8-Cl-ATP was measured as described and are reported as ±SEM. At the end of incubations, the endogenous ATP pool in untreated cells was 2600 µM; in oligomycin alone, 8-Cl-Ado alone, and oligomycin + Ado treated cells was between 2350 and 2400 µM; in oligomycin + 8-Cl-Ado-treated cells it was 1960 µM; and 2800 µM in Ado treated cells. The results obtained were statistically significant (p < 0.05)

Figure 3. Docking of 8-Cl-ADP into ATP synthase

(A) shows the docked 8-Cl-ADP by GOLD and AutoDock, and the crystal ADP structure. GOLD docked structure is colored based on atom types and AutoDock docked structure is in blue. Crystal ADP is shown in magenta, and the chlorine group is shown in green. The surfaces are in the net representation. (B) depicts the surface analysis of the catalytic binding pocket, revealing a concave region to accommodate the –Cl group (green). Mg²⁺ (shown in cyan) forms charge-charge interactions with the diphosphate group. The color coded surface based on atom types is for the receptor binding pocket. The docked 8-Cl-ADP (from GOLD) is displayed in stick model (atom type-based color) and ADP is shown in magenta. For 3C, 8-Cl-ADP is in stick and transparent sphere display. complementarity instead of clash was observed between –Cl (green sphere) and the pocket surface, leading to stable binding of 8-Cl-ADP. This is due to the concave region mentioned above and the tilting of the adenine purine ring

Figure 4. Binding of 8-Cl-ADP to ATP synthase

(A) is a stereo-view (wall-eyed) for the final docked (colored by atom types) and starting (light cyan) conformations of 8-Cl-ADP. Better geometry was obtained for the docked conformation. The lines represent key enzyme residues, and the –Cl group is shown in green. (B) shows that only one new hydrogen bond was observed between the binding pocket residues (α-Arg373) and 8-Cl-ADP β-phosphate group. ADP is shown in magenta and 8-Cl-ADP is colored by atom types. ATP synthase is displayed in secondary structure cartoon diagrams and some residues are displayed as color lines. Hydrogen bonds are represented by dashed lines (magenta for ADP and black for 8-Cl-ADP), and the cyan sphere represents Mg²⁺.

Figure 5. Inhibition of phosphorylation of ADP to ATP in cells with intracellular 8-Cl-ATP

MM.1S cells were either untreated (black lines) or treated with 10 µM 8-Cl-Ado (gray lines) for 24 h, washed and then incubated with [³H]Ado for indicated times. Accumulation of phosphorylated [³H]Ado metabolites such as AMP (A), ADP (B), and ATP (C) was measured by HPLC as described in the Materials and Methods. Data points and error bars represent average of three experiments with S.D., and the results obtained were statistically significant (p < 0.05).

Figure 6. Surface analysis and binding of the product 8-Cl-ATP to ATP synthase

(A) shows the surface analysis of the α_tpβ_tp subunit binding pocket, also revealing a concave region to accommodate the –Cl group (green). The green surface represents Mg²⁺, and the color coded surface based on atom types is for the receptor binding pocket. Atom type-based color coded stick represents docked 8-Cl-ATP. Docked ATP is shown in yellow sticks and magenta represents the crystal ANP. (B) (wall-eyed) provides a comparison of the catalytic sites in the docked 8-Cl-ATP-F₁ and crystal ADP-BeF₃ ⁻-F₁ structures. In the 8-Cl-ATP-F₁ structure, γ-phosphate is bound in a similar position to those three fluorides mimicking ATP binding in the active enzyme. The catalytically essential α-Arg373, β-Arg189 and β-Lys162 are closer to the nucleotide in the docked complex than those in the ADP-BeF₃ ⁻-F₁ structure, creating a tighter binding interface for the nucleotide. (C) Docking Results from AutoDock and GOLD. The metabolites of Ado and halogenated analogues were docked into the diphosphate (α_dpβ_dp) and triphosphate (α_tpβ_tp) binding sites of ATP synthase structure.