Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies

Abstract

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 10⁴ M^-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

Keywords: adenylate kinase; atorvastatin; fluorescence spectroscopy; fluvastatin; inhibitors; pleiotropic effect; pravastatin; rosuvastatin; simvastatin.

Figure 1

Chemical structures of statins used in the study.

Figure 2

hAK1 structure (PDB ID: 1Z83) with the depicted tyrosine residues.

Figure 3

The fluorescence quenching of hAK1 in the presence of (A) atorvastatin, (B) fluvastatin, (C) pravastatin, (D) rosuvastatin and (E) simvastatin at 37 °C. (left) Emission spectra of hAK1 in the presence of statin, (right) dependence of fluorescence intensity on statin concentration. The concentration of hAK1 was 2 μM. The concentrations of the five statins from a to h were 0, 2, 4, 8, 16, 24, 32 and 40 μM, respectively.

Figure 4

Results of blind docking of RVS to hAK1 performed with the SwissDock server. (A) All predicted binding sites for RVS (pink sticks) in hAK1. (B) The conformation of RVS at the selected possible binding regions in close vicinity of tyrosine residues. Ap₅A is shown as spheres; possible hydrogen bonds are marked in red dashed line.

Figure 5

Effect of (A) atorvastatin, (B) fluvastatin, (C) pravastatin, (D) rosuvastatin and (E) simvastatin on the hAK1 enzymatic activity in the presence of ADP or ATP and AMP as substrates, 1 mM each. $ p < 0.0001 compared to the control with ADP, # p < 0.0001 and § p < 0.001 compared to the control with ATP and AMP as substrates.

Figure 6

(A) Double reciprocal plots of hAK1 inhibition by rosuvastatin (RVS). (B) A graphical presentation of hAK1 noncompetitive inhibition by statins.

Figure 7

(A) Superposition of hAK1 (cyan) (PDB ID: 1Z83) and AKst (light pink) (PDB ID: 4QBH) showing the main structural difference between these two AKs. (B) The LID domain in AKst in a closed (light red, PDB ID: 4QBH) and open (green, PDB ID: 1ZIN) state. (C) Comparison of amino acid sequences of AKs with marked functional domains AMPbd and LID and the NTP-binding site (P loop). Asterisk indicates a conserved amino acid residue at each alignment position.

Figure 8

Electrostatic potential mapped on the solvent-accessible surface of (A) hAK1 (PDB ID: 1Z83) in the closed state and (B) AKst in the open (PDB ID: 1ZIN) and closed (PDB ID: 4QBH) state. Cofactor Ap₅A is shown in sticks representation. Electrostatic potential was calculated using APBS plugin 2.1 to PyMOL.