Human Platelet Acetylcholinesterase Inhibition by Cyclophosphamide: A Combined Experimental and Computational Approach

Abdulaziz      A. Al-Jafari; Shazi      Shakil; Marcella      Reale; Mohammad      A. Kamal

doi:10.2174/187152711799219280

Abstract

Acetylcholinesterase (AChE)-inhibition is an area of priority research as various roles have been attributed to AChE in neurodegenerative disorders and cancer as well. In the present study, a comparative multiple 4 dimensional (4D)-approach was applied to analyze human platelet AChE-inhibition by cyclophosphamide (CP). AChE activity was assessed by measuring the hydrolysis of acetylthiocholine iodide (ASChI). The different perspective of analysis was based on two classical (Lineweaver-Burk as well as Dixon) plots, built-in equations of GOSA and a recently introduced graphical approach. Thus, various kinetic constants such as KI, Ks, Km, ksl, Vmao, Ki, ksli, Slmax, °Ks, K1/2, kcat and ksp were estimated. Previous findings of AChE (from different sources) inhibition by CP were also compared. This study extends the elucidation of the kinetic approach of analysis and quantifying enzyme-substrate and enzyme-inhibitor interactions, which is crucial to bringing any drug from bench to bedside. The acyl pocket of human AChE was found to interact with CP through the amino acid residues Y70, Y121, W233, F288, F290, Y334, F408 and Y442, while the anionic sub-site of catalytic site (CAS) interacted with the ligand through residues W84, N85, G116, G117, Y121, S122, G123, L127, Y130, E198, Y334, H443 and G444. CP displayed variable docking poses with the peripheral anionic site (PAS) of human AChE. The findings of kinetic analysis were reinforced by the results of docking experiments. Both the applied approaches strongly indicate partial mixed type of inhibition pattern for the study enzyme (AChE) and its inhibitor (CP).

Keywords: cyclophosphamide, docking, enzyme-inhibition, human platelet acetylcholinesterase, hydtrophobic interactions, kinetics, autoimmune encephalomyelitis, ACHE, QMEAN, PM0077380, Thiotepa