EFFECT OF OLIGOMERIZATION ON THE INHIBITION OF ACETYLCHOLINESTERASE BY CURRENT AND POTENTIAL ALZHEIMER'S THERAPEUTICS

Amelia Pousson

Mentor: Dr. Ashima Saxena

Division of Biochemistry

Walter Reed Army Institute of Research

Washington, D.C.

Richard Montgomery High School

250 Richard Montgomery Drive

Rockville, MD

August 12, 1997

ABSTRACT

In the mammalian brain, acetylcholinesterase exists in several molecular forms; the monomer, dimer, and tetramer. Anticholinesterases are currently being used to treat patients suffering from Alzheimer's disease in an attempt to balance their cholinergic system. The inhibition of monomeric and tetrameric forms of FBS AChE by tacrine hydrochloride (tacrine), donepezil (E2020), and Huperzine A (HupA) was evaluated in order to understand the manner in which different molecular forms are inhibited by these inhibitors. Monomeric FBS AChE and tetrameric FBS AChE were incubated with the various inhibitors for thirty minutes to an hour, and assayed for activity. The data was analyzed using V_app v. [I], K_m v. [I], and V_app/K_m v. [I], plots to determine the values of K_I and K_I. Differences were noted in the mechanism of inhibition of the two forms of FBS AChE by these compounds. Characterizing the dissociation constants for the inhibition of monomeric and tetrameric forms of FBS AChE is an important step in understanding the role that these drugs will play in the future treatment of Alzheimer's disease.

INTRODUCTION

Acetylcholinesterase (AChE) is an enzyme that exists in several structurally distinct forms, including the monomer and tetramer, both of which are present in mammalian brains. The monomeric form is generally less prevalent than the tetramer form, however, in patients with Alzheimer's disease, recent research seems to suggest that the concentration of monomeric AChE in the brain is elevated. Like tacrine hydrochloride (tacrine), which the FDA approved for use in treating patients with Alzheimer's disease in 1993, and donepezil (E2020) (Aricept; marketed by Eisai America, Inc, Teaneck, NJ and Roerig Division of Pfizer, Inc, New York, NY), which was just approved (JAMA. 1997;277:10), Huperzine A (HupA) is a reversible AChE inhibitor that prevents the degradation of endogenous acetylcholine. Due to their role in improving the function of the central cholinergic synapses, tacrine, E2020 and HupA are all under investigation to understand how they perform, both on their own, and in comparison to one another. The goal of this study was to categorize the mechanism of inhibition and calculate the dissociation constants for the various forms of FBS AChE with attention to possible Alzheimer's Disease treatment applications.

MATERIALS AND METHODS

Electrophoretically pure AChE from FBS was purified as described (De La Hoz et al., 1986). BSA diluent/blocking solution concentrate was from Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD. Acetylthiocholine (ATC) and 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) were procured from Sigma Chemical Company, St. Louis, MO. Tacrine, was procured from Sigma Chemical Company, St. Louis, MO, and dilutions were made at concentrations of 2.3 mg/ml in water. E2020 procured from Eisai America Inc, Teaneck, NJ, and dilutions were made at concentrations of 0.33 mg/ml in 0.0001 N HCl. Hup A and its methyl analogs, kindly supplied by Dr. Alan Kozikowski, were made at concentrations of 1-2 mg/ml in 0.01 N HCl. AChE activity was measured in 50 mM sodium phosphate, pH 8.0, using 0-1 mM ATC and 1 mM DTNB. Enzyme assays were conducted using a Beckman DU-64 Spectrophotometer equipped with a Kindata (Kinetics) Softpac module or a Molecular Devices Thermomax micro titer plate reader.

RESULTS AND DISCUSSIONSteady State Assays (Huperzine A and its methyl analogs)

In these studies, 100 �l of either mFBS AChE or tFBS AChE (1-2 U/ml) was incubated with 0-9 �l of HupA overnight at room temperature. Residual enzyme activity was measured in 1.5 ml disposable cuvettes using a Beckman DU-64 spectrophotometer. Assays used 33 �l of ATC at a concentration of 30 mM and DTNB also at a concentration of 30 mM in 1 ml of sodium phosphate buffer, pH 8.0, with 5 �l of inhibited enzyme. The interaction of HupA with the enzyme can be described by the following scheme, where k_on is the rate of the binding of enzyme to inhibitor, k_off is the rate of inhibitor releasing from the enzyme, and E is the enzyme:

k_on

E + I EI

k_off

Scheme I

Data was analyzed in Lotus 1-2-3's spreadsheet, and K_I was calculated using the following equation, where [I] is the concentration of the inhibitor (HupA):

Table 1. Dissociation Constants for inhibition of FBS AChE (Steady State Assay)

Date	(-) Huperzine A		C-10 axial monomethyl Huperzine A		C-10 equatorial monomethyl Huperzine A		10, 10' dimethyl Huperzine A
	Tetramer	Monomer	Tetramer	Monomer	Tetramer	Monomer	Tetramer	Monomer
6-7-97	6.5�1.1	9.6�1.5	1.7�0.8	2.7�0.7	21.3�2.8	30.5�4.2	---	32.3�3.9
6-8-97	6.9�1.6	7.2�2.0	6.9�1.1	4.8�1.5	34.1�8.0	37.2�7.1	24.7�5.8	24.6�4.1
6-9-97	7.2�0.5	8.2�0.9	5.3�0.6	5.9�0.7	27.5�2.1	34.6�2.5	16.9�1.9	23.0�2.5
Avg.	6.9�0.3	8.4�0.9	4.7�2.7	4.4�1.7	27.6�6.4	34.1�3.4	20.8�5.5	26.6�4.9

No significant differences were noted between the K_I of the monomer and tetramer, for (-)HupA and its methyl analogs. This method of analysis yielded the value of the dissociation constant, however, the mechanism of enzyme inhibition by HupA and its methyl analogs could not be ascertained.

Kinetic Assays using the Microtiter Plate (E2020, Tacrine and Huperzine A)

Both mFBS AChE and tFBS AChE were diluted to approximately 0.06 U/ml and 0.12 U/ml in 50 mM sodium phosphate, pH 8.0 containing 0.05% BSA. 250 �l was aliquoted into each of eight eppendorf tubes, 0.7 ml size. Added to each were varying concentrations of the inhibitor. These were incubated for at least thirty minutes, or overnight at room temperature. Ten tubes with ATC in 50 mM sodium phosphate, pH 8.0 containing 0.05% BSA were then prepared, with ATC concentrations ranging from 934.76 to 9.28 �M. 200 �l of 30 mM DTNB was added to all tubes immediately prior to the assay. The enzyme alone or enzyme/inhibitor complex was aliquoted; 10 �l per well into the microtiter plate, in duplicate. 290 �l of buffer was then added to each column of the plate, using a multi-channel pipette. The plates were read for twenty minutes on a Molecular Devices Thermomax microtiter plate reader, coupled with a MacIntosh computer running the associated software. The dilution method can be seen further in Appendix 1. A ribbon diagram of AChE with acetylcholine in the active site, as used in this, and the previous method, can be seen in Figure 1.

The interaction of an inhibitor (I) with an enzyme (E) can be described by the following scheme

k_cat

++

I I

K_I K_I

EI + S ESI

Scheme 2

where ES is the enzyme-substrate complex and P is the product. K_I and K_I are the inhibition constants reflecting the interaction of inhibitor with the free enzyme and the enzyme-substrate complex, respectively. The raw data generated on the microtiter plate reader was analyzed in Prism 2.0 using V_app v. [I], K_m v. [I], and V_app/K_m v. [I], plots to calculate the values of K_I and K_I. Figure 2 shows the plots of V_app v. [I], and Figure 3 shows V_app/K_m v. [I], with summarized data for each inhibitor, for the monomeric and tetrameric forms of FBS AChE. The data points are the mean of all the data collected, and the curves were obtained by nonlinear regression of the data using the following equations (a-1 site inhibition, b-2 site inhibition):

Table 2. Dissociation Constants for inhibition of FBS AChE (Kinetics Assay)

Inhibitor	Tetrameric FBS AChE		Monomeric FBS AChE
	KI	KI	KI	KI
Huperzine A	0.12 +/- 0.08	0.42 +/- 0.06	0.21 +/-0.02	0.30 +/- 0.036
Tacrine	107.3 +/- 17.1	118.3 +/- 11.6	41.8 +/- 6.6	144.4 +/- 28.4
E2020	8.6+/- 1.04	9.85 +/- 1.04	7.1 +/- 1.1	13.4 +/- 1.8

For tFBS AChE inhibited with tacrine, K_I and K_I are statistically the same, due to the error. However, the K_I for mFBS AChE inhibited with tacrine is two standard deviations away from the K_I for the tetrameric form. The mFBS AChE, when inhibited with tacrine also shows a difference between the K_I and the K_I. This difference in the K_I for mFBS AChE and tFBS AChE is on the order of a factor of three. The generally higher K_I that can be seen with tacrine, E2020, and HupA indicates that these inhibitors "prefer" to bind to the enzyme, rather than the enzyme-substrate complex. Also, of these three inhibitors, it is clear that Hup A is most efficient at inhibiting the enzyme. The efficiency of HupA at inhibiting the enzyme may be due to the fact that it appears to have two places to which it can bind on the enzyme, and two on the enzyme substrate complex. The large errors in finding the second values for both are due to the sharp drop in activity that HupA causes, even in very low concentrations. Also notable are the differences between corresponding K_I values for the monomeric and tetrameric forms of FBS AChE. For E2020 it is not significant, however, for both tacrine and HupA, the difference is significant, in the range of two to three standard deviations. This suggests that due to conformational changes that occur when the monomeric "sub-units" form the tetramer, it becomes more difficult for the inhibitor to bind to the active site, similar to what occurs when the substrate has bound to the active site. Also, the mixed-type inhibition that can be seen in graphs of the analyzed data for tFBS AChE is less pronounced in the monomer, with the monomer having a slightly more uncompetitive mode of inhibition, as well as a more complex mechanism.

CONCLUSIONS

Several conclusions may be drawn from this study. First, is the value of the kinetic microtiter plate assay for evaluating the efficacy of inhibitors. A large amount of data is gathered in a short amount of time, and therefore the raw data does not have to be linearized in order to obtain K_I, allowing more accurate results. This method yields valuable information about the mechanism of inhibition, which can help identify differences in inhibitors even with similar K_I values. Second, as can be clearly seen with HupA, the K_I values for FBS AChE differed radically depending on the method used. When the steady state method was used, the results were similar to those gathered by other researchers, that is to say, in the range of 6-10 nM. However, when the same samples were analyzed using the kinetic microtiter plate method, the values were much lower, in the 0.05-0.5 nM range. This has implications for the evaluation of new inhibitors. Finally, the greater complexity of the monomer-inhibitor reactions versus the tetramer-inhibitor reactions suggests that there may yet be more that is not understood about the mechanism of inhibition of tFBS AChE and mFBS AChE. Despite the unknowns that are present in this study, the increased information that it has provided about the impact of the method on the results of a K_I determining assay, as well as the characterization of the mechanism of inhibition of FBS AChE by tacrine, E2020 and HupA should provide the basis for further research on these therapeutics, perhaps one day ending with a suitable treatment for Alzheimer's Disease.

ACKNOWLEDGMENT

Many thanks are offered to my mentor, Dr. Ashima Saxena at the Walter Reed Army Institute of Research. Thank you also to Mr. German Caranto and Dr. Shawn Feaster for their help. This research was supported by the collaborative program between George Washington University's Science and Engineering Apprenticeship Program and the Walter Reed Army Institute of Research.

REFERENCES

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Enzymes of the Cholinesterase Family Ed. Quinn, D. M., Balasubramanian, A. S., Doctor, B.P., and Taylor, P. New York: Plenum Press, 1995.