Inhibition of choline acetylase from the house fly (musca domestica l.) and mouse

Life Sciences Vol . 10, Part II, pp . 337-347, 1971 . Printed' in Great Britain

Pergamon Press

INHIBITION OF CHOLINE ACETYLASE FROM THE HOUSE FLY (MUSCA DOMESTICA L .) AND MOUSE Ching-chteh Yu and Gary M . Booth Department of Entomology 320 Morrill Hall University of Illinois at Urbane-Champaign Urbane, Illinois 61801

(Received 19 October 1970; in final form 12 February 1971) S urtma ry Thirty-one compounds have been evaluated agalnst house fly and mouse choline acetylase (ChA) . Styrylpyridlne analogs were inactive against fly ChA but active against mouse ChA . 3-Bromoacetonyltrimethylartmonlum bromide (BAT) irreversibly Inhibited fly and mouse ChA . Replacing the trimethylammonium bromide group by triethylammonium bromide completely destroyed the inhibitory activity . a-Bromoacetophenone, a,p-dYbromoacetophenone and abromo-2'-acetonaphthone possessed greater inhibitory activity agalnst mouse ChA . Alkyl bromothiolacetates which resemble part of the acetyl CoA structure Inhibited ChA, while ethyl thtolacetate which also resembles part of acetyl CoA did not Inhibit ChA . Other halogen-containing canpounds such as lodoacetamide or . ethyl bromoacetate were Inactive . Tetraalkylartmontum Ions and substituted pyridinium Ions were also inactive . N,N,-Dialkyl-2-phenylazirldinium ions possessed same inhibitory activity agalnst fly ChA while being Inactive agalnst mouse ChA : Other miscellaneous compounds such as dlisopropyl phosphorofluortdate (DFP), 2,2-bis-E chlorophenyl I,i,l-trichloroethane (DOT), 3,3-dimethyl butylacetate and hemichollnium-3 were inactive . P Hydroxyl mercuribenzoate at 10 4 M inhibited fly ChA activity 50~, whereas mouse ChA was 50~ inhibited at 6 x 10- 5 M . Styrylpyridine analogs and 3-bramoacetonyl trtmethyl artmonlum bromide (BAT) have recently been reported as inhibitors of choline acetylase from rat brain tissue (1,2,3,4,5) . The properties of choline acetylase (acetyl CoA :

choline 0-acetyl

transferase, abbreviated as ChA ; E .C . 2 .3 .1 .6) from the house fly, Musca domestics L ., have been studied by Boccaccl et al . (6), Mehrotra (7), and Mehrotra and Dauterman (8) .

These authors showed that fly ChA was different

from ChA of other animal tissues .

For example, fly ChA was found to be

insensitive to lodoacettc acid and to have Km values that were higher than

337

338

Inhibition ad Choline Acetylaae

Vol. 10, No. 6

those reported for the corresponding vertebrate enyeme . Because of the importance of acetylcholine In the transmission of nerve Impulses, its enrymic synthesis has evoked much interest .

Specifically, since

there appears to be significant differences between invertebrate and vertebrate ChA's, this enzyme may represent a potential target for a biochemical

lesion .

Study of specific insect ChA inhibitors may lead to the design of practical selective Insecticides for Insect control .

The following communication reports

the inhibition of ChA from house fly and mouse by styrylpyridlne analogs, BAT, alkylbromothlolacetates, and other miscellaneous compounds and their toxicity to flies by topical application . Materials and Methods Inhibitors :

The styrylpyridine analogs (compounds,

I,

II, IV-X on Table

were prepared according to the method of Cavallito et al . (2,3) . points agreed with the reported figures .

The melting

Compound III had a m .p . of

166-170 ° C

(f ram 2-propanol) . Compounds XI, 3-bromoacetonyl trimethyl bromide, and XII, triethyl ammonium bromide were prepared by the method of Person et al . (5) . latter chemical had a yield of 40~ and a m .p . of

The

170 ° C (decomposed) .

Butyl bromothiolacetate (XVI) was prepared according to Arndt and Bekir (10) .

The infrared spectrum has shown that the characteristic thlolester

peaks at about 1680

an I .

Compound XVII was prepared according to the method of Shaw (II) .

All

other compounds were obtained commercially . Enzyme preparation : (7) .

House fly head ChA was prepared according to Mehrotra

After second ammonium sulfate precipitation the protein concentration

was determined to be IB mg/ml

(12) .

This preparation was stable for several

weeks when stored in the deep freeze (-27° C) .

The specific activity was

I ymole of acetylcholine synthesized per mg per hour . Mouse brain ChA was prepared fran acetone powder according to Schrter and Shuster (13) .

The supernatant was used as ChA source without further

Vol. 10, No. 8 purification .

9S9

Inhibition oI Choline Acet~laee

The protein concentration was determined to be II mg/ml

(12) .

The specific activity of the ChA was 19 rmoles of acetylcholine synthesized per mg per hour . Enryme Inhibition measurements :

The method of Schrier and Shuster (13)

was used to assay the enryme activity .

The final volume of the assay medium

was 0 .2 ml with the following components :

potassium phosphate buffer pH 7 .0,

5 mM ; potassium chloride, 20 mM ; magnesium chloride, 5 mM ; choline chloride, 10 mM ; esertne, 0 .2 mM ; acetyl CoA, 0 .4 mM . 4 x

10 2

uCi of acetyl

Each assay tube also contained

I-C 14 CoA (specific activity 2 .43 mCl/mmole, .obtained

from New England Nuclear, Boston, Mass .), and enzyme preparation of 36 ug protein from fly head or 220 pg protein from mouse brain, and the desired concentration of inhibitor .

The Inhibitors were freshly prepared in distilled

water or acetone depending on solubility . final assay solution dial not exceed 5~ . activity did not change appreciably .

The acetone concentration In the At this concentration the enryme

The reaction mixture was Incubated for

I hour at 25 ° C with continuous shaking .

Styrylpyrtdlne analogs are photasensl-

ttve (14), hence these compounds were prepared in foil-wrapped test tubes in a regular fluorescent-lamp Illuminated room and the Incubation was conducted in the dark . Bioassay :

The technique of bioassay was essentially that described by

March and Metcalf (15) .

Only tamale house flies were used in the test .

Plperonyl butoxide was used to determine If the inhibitors could be synergized . Results and Discussion Table I summarizes the results of fly and mouse ChA inhibition after treatment of the enryme preparation with 31 different canpounds .

The

styrylpyridine analogs (I-X) did not inhibit fly head ChA even at very high concentrations (I x 10 3 M) .

These materials were also non-toxic when

topically applied to the house fly thorax using dosages as high as 500 u9/9 body weight .

CavaLllto et al . (2,3) reported that these compounds were good

inhibitors for rat brain ChA, presumably through a charge transfer canplex

940

Inhibition ad Choline Acetylase

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Vol . 10, No. 8

Inhibition oaf Choline Acetylase

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Vol, 10, No. 6

The present study showed that these

compounds were also good Inhibitors for mouse ChA, which is to agreement with Cavallito et al . (2,3) .

Thus, house fly ChA is different from the rat

and mouse brain ChA in regard to these Inhibitors . Person et al . (5) reported that BAT (XI) was an Inhibitor for rat brain ChA .

-5 The I~ of BAT for house fly and mouse ChA were 1 .3 x 10 M and

5 x 10-5 M respectively (Table I) .

It progressively and irreversibly

Inhibits fly ChA . The compound bromoacetonyltriethylammonium bromide (XII) was completely Inactive even at I x 10 3 M for both fly and mouse ChA .

Presumably the ethyl

groups reduce the electrostatic interaction by~increasing the distance of the quaternary nitrogen of the inhibitor and the anionic group of the enryme .

In

addition the bulky ethyl moieties could sterically hinder the "fitting" of the substrate to the active site of the enryme . a-Bromoacetophenone,a,p-dlbromoacetophenone and a-bromo-2'-acetonaphthone (X111-XV) were more inhibitory toward mouse ChA than fly ChA .

It is conceiv-

ably that a complex formation of the enryme and Inhibitor Is essential prior to alkylatlon .

In this connection, the mouse enryme Is somewhat better than

fly ChA In fofming an enryme-inhibitor canplex .

lodoacetamide (XVIII) was

devoid of any Inhibitory activity toward fly and mouse enryme which could be explained on the bests that it could not form an enryme-Inhibitor complex . The same reàson could explain the lack of activity for fodoacetic acid . Boccaccl et al . (6) reported that fly ChA was not inhibited by lodoacettc acid either to vivo or in vitro . We also prepared ethylthiolacetate and alkylbromothlolacetates which resemble part of the structure of acetyl CoA .

The results showed that n-butyl

bromothlolacetate (XVI) inhibited fly and mouse ChA at 10-5 M (Table I) . also studied methyl, ethyl or n-propyl bromothtolacetate toward fly ChA, results showed that ethyl and methyl bromothiolacetates were only slightly better inhibitors than the n-propyl or n-butyl analogs Indicating that the

The

Vol . 10, No. 6

Inhibition c[ Choline Acetylase

945

alkyl group derived from the mercaptan does not contribute or hinder the binding of the Inhibitor to the enryme .

Choline acetylase Inhibition by n-

butyl bromothiolacetate was observed to be progressive and Irreversible . Increasing the concentration of acetyl CoA resulted In decreasing the percentage of Inhibition indicating that the Inhibition was partially Additional work is needed to clarify whether or not it competes

competitive .

with the active site of the enzyme .

The compound n-butyl bromothlolacetate

has no toxicity to house files when It is applied topically at 500 ug/g However, a LD P of 220 yg/g body weight was obtained when It

body weight .

was synerglzed with piperonyl butoxide to a ratio of 1 :5 for toxicant and synergist respectively .

Piperonyl butoxide applied alone was non-toxic .

Ethylthlolacetate was used against fly ChA .

It is interesting to note

that ethylthlolacetate dId not inhibit ChA even at a concentration of 5 x 10

-3

of I x 10

M. -3

Ethylbramoacetate (XYII) also did not Inhibit ChA at concentration M.

It appears that the bromo and thiol groups are needed for

Inhibition of the enryme .

It Is probable that the sulfur atom in the thiol-

ester linkage contributes to the binding of the enzyme to form an enrymelnhibitor complex and then a nucleophilic group of the enzyme replaces the bromo atom so it is alkylated . The substituted pyridinium Ions (XIX-XXI) which are Intermediates for preparation of same styrylpyridtne analogs, were not Inhibitory against both fly and mouse ChA .

Tetraalkyl ammonium tons (XXII-XXIV) are known to compete

with the anionic site of acetylcholinesterase .

However, these compounds did

3 not Inhibit ChA at I x 10 M Indicating these ions do not interact with the anionic site of ChA . N,N-dialkyl-2-pherryl-aziridinlum Ions (XXVII, XXVIII) showed some 4 inhibitory activity against fly ChA at concentrations In the region of IÔ M . 3 However, these compounds did not Inhibit mouse ChA appreciably at I x IÔ M . These materials have been reported to alkylate acetylcholinesterase (16,17) . Other misçellaneous compounds such as DFP, DDT, 3,3-dimethyl butylacetate, and

948

Inhibition ad Choline Acetylase

Vol . 10, No. 8

hemtcholinium-3 (XXV, XXVI, XXX, XXXI) were totally inactive .

p-Hydroxyl

mercuribenzoate (XXIX) was somewhat a better inhibitor against mouse ChA than fly ChA . This study shows that fly ChA is substantially different from vertebrate ChA to regard to styrylpyridine analogs .

There are also some minor differences

in response to bromoacetophenone analogs and azirtdintum Ions .

Further

exploration of the differences (e .g . variations in the binding and catalytic sites of Insect and vertebrate ChA) may ultimately of selective inhibitors which may be useful

lead to the development

in insect control .

The newly

discovered alkylbromothlolacetates as ChA Inhibitors need additional study . Phenylchlorothtolacetate and closely related analogs are currently being Investigated .

Preliminary results Indicate these compounds are better

Inhibitors of the fly ChA than alkylbromothtolacetates .

This evidence seems

to Justify additional investigation of Insect ChA . Acknowledgements This Investigation was supported by Biomedical Sciences Research grants PH FR 07030 (46-26-18-390, 45-26-18-391) and a Research Grant from the Rockefeller Foundation (44-32-28-372) for the Development of Novel and Non-persistent Pesticide Chemicals . References I .

J .C . Smith, C .J . Cavallito and F .F . Foldes, Biochem . Pharm .

I6, 2438

(1967) . 2.

C .J . Cavallito, H .S . Yun, J .C . Smith and F .F . Foldes, J . Med . Chem .

12,

134 (1969) . 3.

C .J . Cavallito, H .S . Yun, T . Kaplan, J .C . Smith and F .F . Foldes, J . Med . Chem .

13, 221 (1970) .

4.

R .C . Allen, G .L . Carlson and C .J . Cavallito, J . Med . Chem .

5.

B .O . Persson, L . Larsson, J . Schuberth and B . Sorbo, Acta Chem . Scand . 2283 (1967) .

13, 909 (1970) .

Vol. 10, No. 6

Inhibition a~f Choline Acetylase

347

6.

M . Boccacci, G . Natallzl and S . Bettini, J . Insect Physiol . 4, 20 (1960) .

7.

K .N . Mehrotra, J . Insect Physiol . 6, 215 (1961) .

8.

K .N . Mehrotra and W .C . Dauterman, J .

9.

R .B . Baker and E .E . Reid, J . Amer . Chem . Soc . 51,

10 .

F . Arndt and N . Bekir, Ber . 638, 2390 (1930) .

II .

B .D . Shaw, J . Chem . Soc .

12 .

O .H . Lowry, N .J . Rosebrough, A .L . Farr and R .J . Randall, J . Blol . Chem .

Insect Physiol . 9, 293 (1963) . 1567 (1929) .

123, 2233 (1923) .

193, 265 (1951) . 13 .

B .K . Schrier and L . Shuster, J . Neurochem .

14 .

H .L . White and C .J . Cavallito, Biochim . Blophys . Acta 206, 242 (1970) .

15 .

R .B . March and R .L . Metcalf, Callf . Dept . Agr . Bull . 38,

16 .

B . Belleau and H . Tanl, Mol . Pharm . 2, 411

17 .

R .D . 0'B rien, Blochem . J .

113, 713 (1969) .

14, 977 (1967) .

(1966) .

I (1949) .