Mohammad A. Kamal

Kinetics of human acetylcholinesterase inhibition by the novel experimental alzheimer therapeutic agent, tolserine

Characterization of the kinetic parameters of tolserine, a novel acetylcholinesterase (AChE) inhibitor of potential in the therapy of Alzheimers disease, to inhibit purified human erythrocyte AChE was undertaken for the first time. An IC(50) value was estimated by three methods. Its mean value was found to be 8.13 nM, whereas the IC(100) was observed to be 25.5 nM as calculated by single graphical method. The Michaelis-Menten constant (K(m)) for the hydrolysis of the substrate acetylthiocholine iodide was found to be 0.08 mM. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the partial non-competitive type. The value of K(i) was estimated as 4.69 nM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. Four new kinetic constants were also investigated by polynomial regression analysis of the relationship between the apparent K(i) (K(Iapp)) and substrate concentration, which may open new avenues for the kinetic study of the inhibition of several enzymes by a wide variety of inhibitors in vitro. Tolserine proved to be a highly potent inhibitor of human AChE compared to its structural analogues physostigmine and phenserine.

Kinetic analysis of the toxicological effect of tacrine (Cognex) on human retinal acetylcholinesterase activity.

For the first time, kinetic parameters of the effect of tacrine, an anti-cholinesterase inhibitor of therapeutic potential in Alzheimers disease has been studied on human retinal acetyl-cholinesterase (AChE). Tacrine inhibited the AChE activity in a concentration dependent manner, the IC(50) being about 45 nM. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.120 mM and this value was increased by 4-52.8% in the presence of tacrine. V(max) was observed to be 2.23 micromol/h per mg protein for the control system, while it was decreased by 14.73-56.25% in the tacrine treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the mixed type, i. e. a combination of competitive and noncompetitive inhibition. The values of K(i) and K(I) were estimated to be as 37.76 and 64.36 nM, respectively.

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: Kinetic characterization

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (AChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration‐dependent manner; IC50 was found to be 8.07 nM. The Michaelis‐Menten constant (Ka) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54–67% in the tacrine‐treated systems. The Vmax was 0.701 μ mole/min per milligram protein for the control system, but it was decreased by 26–69% in the tacrine‐treated systems. The Lineweaver–Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of Ki and Kt were estimated to be as 4.475 and 8.517 nM, respectively.

The inhibitory effect of cyclophosphamide on camel retina acetylcholinesterase activity

Kinetic parameters for the effect of cyclophosphamide (CP) on the camel retina acetylcholinesterase (AChE) activity were investigated for the first time in the present study. It was found that 18 micrograms of retina protein and an incubation time of 4.0 min were suitable conditions for linear of AChE activity. The CP effect was independent of time of incubation with AChE before the addition of substrate, which shows it reversible action. Moreover, dilution data prove that CP is a reversible inhibitor of camel retina AChE. Cyclophosphamide (0.2-2.4 mM) inhibited activity of camel retina in a concentration-dependent fashion, the IC50 being about 1.17 mM. The Michaelis constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.106 mM and the Vmax was 0.765 mumol/min/mg protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure noncompetitive type. The value of Ki was estimated as 0.763 mM by the primary Dixon and secondary replots of the Lineweaver-Burk plot.

The Mode of Inhibition of Human Erythrocyte Membrane-Bound Acetylcholinesterase by Cisplatin in Vitro

The effect of the well known anticancer drug cisplatin on the human erythrocyte membrane-bound acetylcholinesterase (AChE) has been investigated. It was found that cisplatin has inhibitory activity against AChE. Cisplatin (0.5-7.0 mM) inhibited AChE activity (27-82%) in a concentration dependent manner. The nature of the inhibition of AChE by cisplatin was complex and involved a partial reversible stage followed by a slow acting irreversible step. The inhibition was independent of a preincubation period up to 90 min while becoming dependent after this period. By diluting the preincubated AChE-cisplatin mixture for different times (0-24 h), the inhibition was partially reversed but again increased progressively with incubation time, ultimately reaching complete inhibition. The effect of increasing substrate concentration had the same behaviour as the dilution effect.

Investigation of the effect of anti-neoplastic drugs, cyclophosphamide, cisplatin and methotrexate on the turnover kinetics of human erythrocyte acetylcholinesterase

The present work addresses the effects of three antineoplastic drugs [cyclophosphamide (CP), cisplatin (CDDP) and methotrexate (MTX) which are in current use for the treatment of various tumors] on turnover kinetics of human erythrocyte membrane‐bound acetylcholinesterase (AcChoEase, EC 3.1.1.7). It was found that CP and MTX significantly decreased kcat and ksp, whereas CDDP decreased only the kcat value, (ksp was non‐significantly affected). In light of these findings, the CP, CDDP and MTX needs particular attention in tumor therapy and their effects on turnover number must be considered at the time of administering these drugs to cancer patients.

Investigation of the effect of tetrahydroaminoacridine on turnover kinetics of camel (Camelus dromedarius) retina acetylcholinesterase

The effect of tetrahydroaminoacridine (THA) on turnover kinetics of camel retina acetylcholinesterase has been investigated. The turnover number (Kcat) and specificity constant (Ksp) were 62.1 min‐1 and 9.92 × 105 (M. min)‐1 in the control system while the values for both parameters were lowered by 25‐68% and 48‐87% in the THA (0.05‐0.4 µM) treated systems respectively. Therefore, THAs effect on turnover number must be considered at the time of therapy to the Alzheimers disease patients.

Evaluation of the Nature of Camel Retinal Acetylcholinesterase: Inhibition by Hexamethonium

Acetylcholinesterase (AChE, EC 3.1.1.7) has been demonstrated in retinas of several species, however, the nature of the interaction of AChE with specific inhibitors are very limited in the literature and the mode of inhibition of camel retinal AChE by hexamethonium has been studied. Hexamethonium reversibly inhibited AChE in a concentration dependent manner, the IC50 value being c. 2.52 mM. The Km for the hydrolysis of acetylthiocholine iodide was found to be 0.087 mM and the Vmax was 0.63 mumol/min/mg protein. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the hyperbolic (partial) mixed type, which is considered to be a partial competitive and non-competitive mixture. The values of Ki(slope) and KI(intercept) from a Lineweaver-Burk plot were estimated as 0.30 mM and 0.17 mM, respectively, while Ki from a Dixon plot was estimated as 0.725 mM. The Ki was greater than KI indicating that hexamethonium has a greater affinity of binding for the active site than the peripheral site of the camel retina AChE.

Investigation of the effect of lannate on kinetic parameters of acetylcholinesterase: Slightly concave mixed-type of inhibition system

The effect of lannate on kinetic parameters of camel retina acetylcholinesterase is investigated in the present study. The Ks for the hydrolysis of acetylthiocholine iodide was found to be 0.086 mM in the control system; a value that increased in the lannate treated systems. The Vmax was 0.853 μmole/min/mg protein for the control system while it decreased in the lannate treated systems. Dixon as well as Lineweaver‐Burk plots and their secondary replots indicated that the nature of the inhibition is of the slightly concave mixed type, which is considered to be a semi‐pure competitive and semi‐partial as well as semi‐pure non‐competitive mixture. The values of Ki(slope) and KI (intercept) were estimated as 0.143 μM and 0.179 μM respectively. The KI was greater than Ki indicating that lannate has a greater affinity of binding for the peripheral site than the active site of the camel retina AChE.

Human erythrocyte acetylcholinesterase inhibition by cis-diamminediaquaplatinum (II): a novel kinetic approach.

The present work addresses the analyses of some novel kinetic parameters (k(t), K(v), t50, K(ir), t(c), m(c), IC50, IC99 and Ki) of human erythrocyte membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7) inhibition by cis-diamminediaquaplatinum II (PDC). PDC is under a clinical trial for use as an antineoplastic drug. The authors recently reported that PDC and cisplatin have the ability to inhibit AChE activity in vitro. Therefore this study was designed to determine the estimation of time constant (k(t)), velocity constant (K(v)), 50% inhibition time (t50), inhibition rate constant (K(ir)), transition concentration (t(c)), meeting concentration (m(c)), 50% inhibition (IC50), 99% inhibition (IC99) and inhibition constant (Ki) by novel methods. The details are described in the text.