Differential binding of bispyridinium oxime drugs with acetylcholinesterase

Abstract

Aim: To performe a time-dependent topographical delineation of protein-drug interactions to gain molecular insight into the supremacy of Ortho-7 over HI-6 in reactivating tabun-conjugated mouse acetylcholinesterase (mAChE).

Methods: We conducted all-atom steered molecular dynamics simulations of the two protein-drug complexes. Through a host of protein-drug interaction parameters (rupture force profiles, hydrogen bonds, water bridges, hydrophobic interactions), geometrical, and orientation ordering of the drugs, we monitored the enzyme's response during the release of the drugs from its active-site.

Results: The results show the preferential binding of the drugs with the enzyme. The pyridinium ring of HI-6 shows excellent complementary binding with the peripheral anionic site, whereas one of two identical pyridinium rings of Ortho-7 has excellent binding compatibility in the enzyme active-site where it can orchestrate the reactivation process. We found that the active pyridinium ring of HI-6 undergoes a complete turn along the active site axis, directed away from the active-site region during the course of the simulation.

Conclusion: Due to excellent cooperative binding of Ortho-7, as rendered by several cation-pi interactions with the active-site gorge of the enzyme, Ortho-7 may be a more efficient reactivator than HI-6. Our work supports the growing body of evidence that the efficacy of the drugs is due to the differential bindings of the oximes with AChE and can aid to the rational design of oxime drugs.

Figure 1

Chemical structure of the two oxime drugs. (A) HI-6, [1-(2-hydroxy-iminomethylpyridinium)-1-(4-carboxyamino)-pyridinium dimethylether]. and (B) Ortho-7, [1,7-heptylene-bis-N,N'-2-pyridiniumaldoxime]. The carbon, nitrogen, and oxygen atoms are numbered for referral purpose. The two pyridinium rings, one directed towards the active triad region and one directed towards the PAS region of the AChE enzyme, are indicated.

Figure 2

Unbinding of a drug molecule from the active-site gorge of mAChE (the active serine of which is shown in blue colored space filling representation) through the application of an external time-dependent harmonic force in the Z-direction as depicted by a spring. The artificial spring, characterized by a force constant, is attached to the center of mass of the drug molecule (shown by space filling representation) and is pulled with a constant velocity until it completely dislocates from the gorge.

Figure 3

Pulling force and center of mass distance (A), water bridge (B), and hydrophobic atom pair numbers (C) between the drug and the enzyme shown as a function of time during the exit of the drug, Ortho-7 from mAChE gorge. In (B), the grey-colored bars indicate events in which a given amino acid residue of the enzyme that forms a water bridge with Ortho-7 also forms direct hydrogen bonds between themselves. The plots in parts (A) and (C) are prepared by 10 point adjacent averaging of the SMD results. The double-sided arrows in (A) represent the fall in the force profile from an intermittent peak and subsequent rise in center of mass distance between the enzyme and the drug.

Figure 4

Same as Figure 3 but for the drug, HI-6.

Figure 5

Number of direct hydrogen bonds as a function of time between amino acid residues of mAChE and Ortho-7 during the process of leaving the active-site gorge. The amino acid residues and atom numbers of the drug molecule with which it forms hydrogen bonds are mentioned. The grey-colored bars are indicative of the simultaneous formation of water bridges between the protein and the drug with an intervening molecule of water in addition to a direct hydrogen bond, which is otherwise plotted here. Note that Ser 203 is found to form only water bridges and no direct hydrogen bond.

Figure 6

Same as Figure 5 but for the drug molecule, HI-6.

Figure 7

Free energy of direct protein-drug hydrogen bonding of the two complexes (circles: mAChE.Ortho-7, squares: mAChE.HI-6). Black- and grey-colored symbols represent forward hydrogen bonding (where the drug molecule is the hydrogen donor) and backward hydrogen bonding (where the drug molecule is the hydrogen acceptor), respectively.

Figure 8

Time-dependent minimal distance between the (A) peripheral pyridinium ring of Ortho-7 and the PAS residues, Tyr 72 (grey line) and Trp 286 (black line), (B) active-site pyridinium ring of Ortho-7 and the phenyl ring of Tyr 337 (grey line) and the indole ring of Trp 86 (black line), (C) peripheral pyridinium ring (carboxylamide-pyridinium) of HI-6 and the PAS residues, Tyr 124 (grey line) and Trp 286 (black line), and (D) active-site pyridinium ring of HI-6 and Tyr 337 (grey line) and Phe 338 (black line) during the process of the drugs leaving the active-site gorge of mAChE.

Figure 9

Center of mass distance between the peripheral pyridinium moiety of the drugs during their exits from the active-site gorge, and the hydrophobic aromatic residues of the PAS of mAChE, as indicated in the plots. Grey lines are the results for the SMD simulation of the mAChE.Ortho-7, and the black lines are for mAChE.HI-6 complexes.

Figure 10

Center of mass distances between the APR of the drugs and the hydrophobic aromatic residues (other than those at the PAS) of mAChE as indicated in the plots. In all the plots, the SMD pulling results shown by grey lines are for the mAChE.Ortho-7, and the black lines are for the mAChE.HI-6 complexes.

Figure 11

Center of mass distances between Ser 203 and the active pyridinium moiety of the drugs, as marked in the plot, during their exit from the active-site gorge of mAChE.

Figure 12

Temporal spatial distance during SMD pulling between the center of mass of active-site Ser 203 and the N20 atom (grey line) and the N2 atom (black line) of the two pyridinium rings of Orhto-7 that was initially close to active triad and the peripheral site, respectively. Relative orientations and separations of the drug molecule with respect to Ser 203 (shown by space filling representation) are also shown at regular intervals. The grey circle encircling O28 atom of the drug is the oximate oxygen. See Supplementary information S-1 for the snapshots of protein-drug hydrogen bonds and water bridges at the time steps indicated in the figure by arrows.

Figure 13

Temporal spatial distance during SMD pulling between the center of mass of active-site Ser 203 and the N2 atom (grey line) and the N3 atom (black line) of the two pyridinium rings of HI-6 that was initially close to active triad and the peripheral site, respectively. Relative orientations and separations of the drug molecule with respect to Ser 203 (shown by space filling representation) are also shown at regular intervals. The grey circle encircling O1 atom of the drug is the oximate oxygen. See Supplementary information S-2 for the snapshots of protein-drug hydrogen bonds and water bridges at the time steps indicated in the figure by arrows.

Figure 14

Results of all-atom superimposed structures of the two complexes, mAChE.HI6 and mAChE.Ortho-7. Shown at different times are the active site Ser 203 and the two drugs. The Ser 203 residue of the two complexes is shown in space-filling representation, green for mAChE.Ortho-7 and gold for mAChE.HI6 complex. Ortho-7 drug is displayed in licorice representation and HI-6 is displayed in ball-and-stick representation. The oximate oxygen atoms of the two drugs (Ortho-7: O28, HI-6: O1) are also shown.

Scheme 1

AChE inhibition by OP compound, tabun. Aging of the tabun conjugated enzyme and its oxime-drug based reactivation.