bk-2009-1014.ch010

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Chapter 10

Pharmacological Mapping of the Acetylcholinesterase Catalytic Gorge in Mosquitoes with Bis(n)-Tacrines Troy D. Anderson1, Sally L. Paulson1, Dawn M. Wong2, Paul R. Carlier2 , and Jeffrey R. Bloomquist1 Departments of 1Entomology and 2Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A. 24061

New insecticides are needed for control of mosquitoes, such as Anopheles gambiae, the major vector of malaria. Acetylcholinesterase is a proven insecticide target site, but conventional organophosphate and carbamate compounds are plagued by concerns about human toxicity and resistance. A pharmacological approach with novel, bivalent bis(n)-tacrines was used to map the catalytic gorge of this enzyme from human and several mosquito species (Anopheles gambiae, Culex restuans, Aedes aegypti, and Aedes albopictus). We screened bivalent bis(n)-tacrines having methylene linkers from 2-12 carbons in length, where proper spacing would allow for high potency binding via interaction with both the catalytic and peripheral sites on the enzyme. The tacrine monomer had fairly similar potency across species (somewhat less for Culex restuans), indicating a common mode of binding at the catalytic site. A greater maximal potency for a bis(n)-tacrine was observed against human AChE than any of the mosquitoes tested. With the exception of Anopheles gambiae, the mosquitoes showed a clear tether length dependence, with tether length most critical in Aedes aegypti. This finding has implications for identifying the targeted amino acid residues in or near the gorge. Despite the greater potency of bis(n)-tacrines against vertebrate than mosquito acetylcholinesterase, the information gleaned from this study should help inform the molecular design of selective anticholinesterase insecticides in other chemical series. © 2009 American Chemical Society In Advances in Human Vector Control; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Introduction Mosquito vectored diseases cause extensive mortality in humans (1). By far, the most important vector borne disease is malaria, which is estimated to cause over 1 million deaths a year, world-wide. Other important diseases spread by mosquitoes include: dengue, yellow fever, and encephalitis, and the total number of global deaths from these diseases is estimated to be about 50,000/yr (1). Acetylcholinesterase (AChE) is a serine hydrolase that hydrolyzes the neurotransmitter acetylcholine (ACh) in the nervous system of both insects and humans, and is a proven target site for organophosphate and carbamate insecticides (2). However, the widespread resistance of numerous insect species to these insecticides, and poor selectivity towards humans, limits the utility of these compounds in pest control programs. The gene encoding AChE in the marine fish Torpedo californica is homologous to the ace-1 gene found in several insect species (2,3). Sussman et al. (4) first revealed that the tertiary structure of T. californica AChE contains a deep and narrow active site gorge (Fig. 1) with important subsites for ACh binding/hydrolysis and thereby possible interaction with inhibitors. These subsites include the catalytic triad, cholinebinding site, peripheral anionic site, and the acyl-binding pocket (5), all of which appear to be conserved across many invertebrate species (6). Inhibitors of AChE may interact with more than one site on the enzyme. For example, tacrine prevents the hydrolysis of ACh by occupying the active site near the catalytic serine (7,8). Similarly, inhibitors can impede cholinergic substrate access by binding to the peripheral site of AChE (7,8), located at the entrance of the catalytic gorge (Fig. 1). Inhibitors that simultaneously bind to the active and peripheral sites of AChE can occupy much of the gorge, and inhibit enzyme activity with high affinity (7,8). For example, bifunctional tacrine molecules (Fig. 2), optimally tethered with an alkylene chain, can interact simultaneously with both sites of the enzyme, resulting in greater potency compared to that of tacrine itself (7). Determining the optimal spacing of bivalent inhibitors by altering the tether length can act as a molecular ruler, and indicates the distance between the catalytic and peripheral sites, as shown previously for compounds anchored at the catalytic site (9). We used pharmacological studies to explore the geometry of the active site gorge of AChE in several mosquito species. Specifically, the gorge was probed with a series of bis(n)-tacrines to provide some insights on gorge volume and the distance between the catalytic and peripheral sites. Results were compared to human AChE. The resulting information should be useful in the design of bivalent inhibitors of high potency and selectivity.

In Advances in Human Vector Control; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2009.

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Peripheral Site

W279 N

E327

OH

C

C

H440

N

NH

Catalytic Site


OH N

W84

S200

Figure 1. A simplified schematic representation of the AChE gorge with some key amino acids that define the peripheral aryl site and the catalytic active site. ACh enters the gorge at the peripheral site (arrow), and the hydrolysis products (acetate and choline) exit from the catalytic site.

N

H N

(CH2)n

H N

N

Figure 2. Chemical structure of the bis(n)-tacrines used in this study, with ‘n’ representing the number of carbons that form the tether linkage. The bis(n)tacrines were synthesized and purified to >99.5% using established methods (10).

AChE Assay Residual AChE activity was determined according to the method of Ellman et al. (11), with slight modifications. Malarial mosquitoes, Anopheles gambiae (G3 strain), were taken from colonies cultured in the Department of Entomology at Virginia Polytechnic Institute and State University. The mosquitoes Aedes aegypti, Aedes albopictus, and Culex resutans were collected from local wild populations. Adult mosquitoes were homogenized in 1 ml ice-cold 0.1 M sodium phosphate (pH 7.8) containing 0.3% (v/v) Triton X-100. The homogenate was centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was used for assay. The AChE preparations were pre-incubated with tacrine or each methylene-linked tacrine dimer (1-10,000 nM) for 10 min at room



146 temperature. The residual AChE activity of the supernatant was measured using an enzyme kinetic microplate reader (Dynex Technologies, Chantilly, VA, USA) at 405 nm immediately after the addition of 5’5-dithiobis-(2-nitrobenzoic acid) (DTNB) and acetylthiocholine (ATCh). The final concentrations of DTNB and ATCh were 0.3 and 0.4 mM, respectively. Recombinant human AChE (lyophilized powder; Sigma-Aldrich, St. Louis, MO), with a quoted specific activity of 2790 U/mg, was diluted to 600 U/mL with 0.1 M sodium phosphate (pH 7.8) containing 0.3% (v/v) Triton X-100, frozen, and stored at −80 °C. Immediately prior to assay, a frozen human AChE sample was thawed and diluted 1000-fold with the same buffer before use. Nonlinear regression analysis of the residual AChE activity was used to determine the negative logarithm of IC50 for each inhibitor using Prism software (GraphPad Software Inc., San Diego, CA, USA).

Inhibition of AChE Activity by Bis(N)-Tacrines Figure 3 and Table 1 reveal both similarities and differences in the responses of mosquito and human AChEs to inhibition by bis(n)-tacrines. The monomeric tacrine was among the least active compounds in each species, and with the exception of Cx. restuans, the sensitivity to this compound across species was similar (Table 1). This finding suggests that the binding of tacrine within the active site is comparable, but there is some difference in or near the catalytic site of Cx. restuans AChE that lowers affinity. Patterns of inhibition are visually apparent in Figure 3. In all cases, the IC50 decreased with increasing tether length compared to the monomer, and then increased again as tether length approached 12 methylenes, the maximum tested. There was a relatively flat response in An. gambiae, whereas the other species showed a greater tether length dependence reflected by a maximal potency at 7-8 methylenes. There is also a more steep tether length dependence in Ae. aegypti compared to any other species, since potency declines over 14-fold with a single carbon change from optimal tether length (n = 8) in Ae. aegypti, while potency declines < 3-fold with a one carbon change from optimal tether length for the others (Table 1). This difference suggests more efficient dual site binding in Ae. aegypti that rivals that of human AChE. Finally, the bis(n)-tacrines are uniformly more active against human AChE than any of the mosquito AChEs, at all tether lengths (Table 1). Thus, these compounds are negatively selective for mosquitoes. Other studies (10, 12) found that cockroach (Blattella germanica) and rat AChEs are, like Ae. aegypti, Ae. albopictus and Cx. restuans, most potently inhibited at a tether length of 7-8 carbons. Although absent in human and minimal in Ae. albopictus, “bumps” were observed in the pattern of mosquito responses to the bis(n)-tacrines (Fig. 3). The most pronounced was at n = 6 in An. gambiae, with smaller ones at n = 5 in Cx. restuans and Ae. aegypti, suggesting a steric clash that impeded binding within the gorge. By way of comparison, the cockroach and rat enzymes have a “bump” at n = 2 tether length (10, 12), unlike any of the species studied here. This finding suggests that the gorge in these species is spatially constrained nearer the catalytic site.


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10000

10000

An. gambiae

1000

1000

100

100

10

10

1

M 2

3

4

5

6

7 8

9 10 12

1

Cx. restuans

M 2

3

4

5

6

7

8

9 10 12

5 6

7

8

9 10 12


10000 Human 1000

Log IC50 (nM) 100 10 1 10000

M 2

3

4

Ae. aegypti

5 6 7 8 10000

1000

1000

100

100

10

10

1

1 M 2

3 4

5

6

7

8

9 10 12

9 10 12 Ae. albopictus

M 2

3

4

Figure 3. Comparison of IC50 values for tacrine monomer (M) and tether-length dependence of dimeric bis(n)tacrines, where numbers represent tether length in methylene units.



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Protein Modeling X-ray crystallographic studies of bis(7)-tacrine complexed with TcAChE (PDB ID 2ckm), (13) found that one tacrine unit binds to the W84 cholinebinding site (Fig. 4), sandwiched between the aromatic side chains of W84 and F330, at the bottom of the active-site gorge (13). This arrangement has also been observed in structures of monomeric tacrine (PDB ID 1ACJ) (14) and other tacrine-based bivalent inhibitors bound to TcAChE (PDB ID 1ODC, 1UT6, 1Q83, 1Q84, 1ZGB. 1ZGC. 2CEK) (13, 15-17). We conclude that a similar binding mechanism operates in mosquitoes. The X-ray crystal structure of bis(7)-tacrine complexed to TcAChE also reveals a π-complex sandwich of tacrine with W279 and Y70 at the peripheral site (13). Comparison of the TcAChE and human AChE sequences suggests an identical binding mode would be realized for hAChE. The general lower overall potency of bis(n)-tacrines against mosquito AChEs relative to vertebrates may be due, in part, to the presence of I70 instead of the Y72 found in the human peripheral site (Fig. 4). We have proposed that such a substitution would diminish π-π/cation-π interaction at the peripheral site of An. gambiae AChE with the second tacrine moiety (3). However, such an explanation does not explain the high potency of bis(8)-tacrine against Ae. aegypti, which posses the same I70 substitution as An. gambiae. We are currently investigating enzyme-ligand interactions that could account for the high potency binding of this molecule in Ae. aegypti.

Conclusions Mosquito species differ considerably in their responses to bis(n)-tacrines. Although these compounds are useful probes of the geometry of the AChE catalytic gorge, we do not view them as lead compounds, since they have no contact activity against insects (data not shown). The lack of toxicity is likely due to the presence of basic nitrogens that prevent penetration to the target site. Our studies found a greater overall potency for these compounds against human AChE than any of the mosquitoes tested, similar to a previous study comparing rat and German cockroach. The greater potency against vertebrate than insect AChE suggests structural and/or functional differences that creates possibilities for further structure-activity investigation. This speculation could be further investigated by using Ala-scanning site directed mutagenesis of residues thought to interact with tacrines, coupled with extensive in silico molecular modeling. Such studies could help inform the molecular design of selective anticholinesterase insecticides.



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Figure 4. Molecular model using Pymol® of bis(7)-tacine complexed with TcAChE (bold text labels), including relevant amino acid residues of human and An. gambiae AChE. Figure is slightly modified from Carlier et al. (3).


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