Oximes: inhibitors of human recombinant acetylcholinesterase. A structure-activity relationship (SAR) study

Abstract

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring.

Figure 1

Chemical structures of assayed mono-pyridinium reactivators.

Figure 2

Chemical structures of assayed bis-quaternary ammonium reactivators.

Figure 3

Chemical structures of the bis-pyridinium reactivators with an oxygen substitution or double bond in the connecting linker.

Figure 4

Experimental versus predicted pIC₅₀ values for the 21 compounds in this study. The IC₅₀ prediction was based on the eight descriptors identified by principle component analysis. The “leave one out”—Q², correlation was 0.86. Red dots are compounds used in the training set and blue are from the test set. In general, extension of the linker length increased the inhibition potency of the bis-pyridinium oximes. Compounds 4, 6, 24 are currently approved as organophosphorus poisoning (OP) antidotes in various parts of the world.

Figure 5

Comparison of various IC₅₀ values of bis-pyridinium compounds in hrAChE and EeAChE. In general, the hrAChE enzyme is more easily inhibited by oxime reactivators than the EeAChE enzyme.

Figure 6

Superimposition of TcAChE active site (yellow) on EeAChE active site (blue) residues within R = 8 Å spherical selection calculated by super (A) root-mean square deviations (RMSD) (super) = 0.400 Å and align (B) RMSD (align) = 0.416 Å algorithm. Alignment of 13 flexible residues is rendered in the same manner (C) RMSD (super) = 0.586 Å; (D) RMSD (align) = 0.536 Å.

Figure 7

Overall superimposition of TcAChE on EeAChE colored by C_α RMSD (“super” algorithm used, 449 C_α carbon atoms matched). Dark blue is a good alignment; higher deviations are in orange/yellow/red. Residues omitted in the alignment are white. Minimum C_α distance: 0.04 Å, maximum C_α distance: 5.25 Å, average C_α distance: 0.71 Å, C_α RMSD = 0.545 Å.

Figure 8

The top-scoring pose for compound 13 (green) in in the active site of TcAChE. The original spatial conformation of amino acid residues are rendered in blue, amino acid residues in computationally determined alternate conformations are shown in yellow, and the rest of the enzyme is displayed in a cartoon representation. For the sake of clarity, original conformation of Trp279 is not depicted since no structural modification was observed.

Figure 9

The top-scoring pose for compound 13 (green) in the active site of EeAChE. The original spatial conformation of amino acid residues are rendered in blue, amino acid residues in computationally determined alternate conformations are shown in yellow, and the rest of the enzyme is displayed in a cartoon representation.

Figure 10

Compound 13 (green) docked into the hrAChE active site showing its interactions with various amino acid residues. The original spatial conformations of amino acid residues are shown in blue, amino acid residues in computationally determined alternate conformations are shown in yellow, and the rest of the enzyme is displayed in a cartoon representation. Other residues with important interactions with the oxime are shown in magenta. Significant changes are seen in several important active site residues upon oxime binding.