Selective inhibition of schistosome acetylcholinesterase by dansylated acetylcholine analogs

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  • C,mp Bu,them Pht~ml Vo]. 68C. pp 22'4 to 2.~) 0306-4492 81.030229.0250200.'0 Pergamon Prc~s Lid 1981 Printed in Great Brllaln

    SELECTIVE INHIBITION OF SCHISTOSOME ACETYLCHOLINESTERASE BY DANSYLATED

    ACETYLCHOLINE ANALOGS

    MARY J. DOTSON, S. H. CHt: and GILBERT R. HILLMAN

    Department of Pharmacology and Toxicology. University of Texas, Medical Branch, Galveston, TX 77550, and Division of Biomedical Sciences, Brown University, Providence. RI 02912, U.S.A.

    (Received 20 June 1980}

    Abstract---I. The inhibitory effects of a series of dansylated choline compounds on schistosome and bovine erythrocyte acetylcholinesterase have been examined.

    2. Several of the drugs produced a significant preferential inhibition of the schistosome acetylcholines- terase.

    INTRODUCTION anesthesia with ether. The adult paired schistosomes were removed from the portal and mesenteric veins by hook

    There is evidence supporting the presence of acety]- dissection and placed in a tube of cold 0.1 M sodium phos- cholinesterase (ACHE) in schistosomes (Bueding, phate buffer at pH 7.0. 1952: Gear & Fripp, 1974). This enzyme is generally The schistosome acetylcholinesterase was prepared using required for the metabolic breakdown of acetyl- the method of Bueding (1952). Worms were homogenized choline (ACh) at cholinergic synapses. Since both the with a glass micro-tissue grinder. The homogenate was schistosome and vertebrate nervous system utilize transferred to a microcentrifuge tube with 0.5 ml phosphate acetylcholinesterase, it would be useful to characterize buffer and centrifuged for 3 rain at 12,000 g. The superna- the schistosome enzyme and to examine the feasibility tant was removed and more buffer was added to wash the of inhibiting the parasites enzyme selectively in the schistosome residue. The suspension was recentrifuged and

    the supernatant removed. Phosphate buffer (25 gl per schis- presence of the vertebrate host's enzyme, tosome) was added to the washed homogenate residue. The

    A moderate degree of selective inhibition of schisto- suspension was immersed in ice and sonicated with an some AChE by hycanthone has been described in a ultrasonic cell disrupter (Heat Systems-Ultrasonics, Inc.) previous report (Hillman & Senti, 1975)i Another using a 40% pulsed cycle to avoid excessive heating. report (Gear & Fripp, 1974) discusses the relative Bovine erythrocyte acetylcholinesterase (Sigma Chemical properties of AChE from four schistosome species. Co.) was diluted with 0.1% of bovine serum albumin to Metrifonate, an organophosphate anticholinesterase, an activity equal to the schistosome cholinesterase pre[, has been used as an antischistosomal agent (Jewsbury aration.

    Both the bovine and schistosome acetylcholinesterase et al., 1977). However, the problem of synthesizing an activity were determined by a colorimetric method (EIIman, effective inhibitor of schistosome AChE has not been 1961 ). The reaction mixture consisted of 0.1 M phosphate approached using studies of the structural require- buffer at pH 7.a, acetylthiocholine bromide from 0.0375 to ments necessary for inhibition of schistosome enzyme. 0.225 raM. 5,5'-dithiobisnitrobenzoic acid (DTNB) 0.01 M

    In the present study we have examined a series of test compounds at concentrations from 0 to 0.05 raM; and dansylated choline compounds. These studies are either the bovine erythrocyte or schistosome cholinester- designed to determine whether modification of this ase. The buffer, substrate, DTNB, inhibitor and enzyme group of chemical structures can produce compounds were combined and mixed in a cuvette with a total volume having selective inhibitory properties against AChE of 0.5 ml. Cuvette temperature was maintained at 30~C. A

    Gilford spectrophotometer was used to monitor and calcu- from schistosome or from vertebrate sources. late the rate of absorbance change at 412 nm. The rate ol

    This same series of compounds has also been stud- non-enzymatic hydrolysis, determined in an enzyme-free led with respect to their effects on cholinergic neuro- control reaction, was subtracted from the experimental muscular systems in schistosomes (Hillman et al., rate. 1980) some of the compounds had significant effects The K,,, K, Ithe competitive inhibition constant} and K{ on the motor activity of the schistosomes and all have (the non-competitive constant} for all compounds were cal- been examined as fluorescent labels of ACh binding culated according to the equations of Krupka & Laidler sites in schistosomes. (1961) and Wilkinson (1961). Both bovine erythrocyte and

    schistosome acetylcholinesterase were measured at various substrate and inhibitor concentrations.

    MATERIALS AND METHODS RESULTS AND DISCUSSION

    Syntheses of the experimental compounds have been de- The percentage inhibition by the dansyl compourld scribed previously (Hillman et al., 1980). Their structures under a given set of reaction conditions was deter- are shown in Fig. I.

    Drs S. K. File and J. H. Smith of the University of Texas mined using the formula: Medical Branch in Galveston maintain a mouse-snail cycle activity without inhibitor - activity with inhibitor of Schisrosoma mansoni which was used to provide a supply activity without inhibitor of infected mice. Mice were sacrificed 4.5-60 days postinfec- tion by cervical dislocation after injection with heparin and 100 = "o inhibition.

    229

  • 230 MARY J. DOTSON et al.

    HsC \ /CHs HsC CH s N \ N /

    / CH,~ /CHz CH ~ SO~NHCH z CH z N SO z NHCH z CH z N

    \CH 3 \ C H z CH 3

    HsC \ /CH.~ H3C \ / C H 3

    N N

    /CH 3 /CH 2 CH s SOzNHCH z CH 2 CH 2 N \CH5 NHCH 2 CH z CHz N\CH2CH s

    HsC \ /CH3 HsC \ / C H 3 N N

    /c,H, SOz-- N N ~ C H s SO z NHCH z CH t CHzN \C,H I

    Fig. I. Structures of Compounds I-VI.

    All compounds except !! caused a significantly Acknowledgements--The authors are grateful to Drs higher inhibition of the schistosome enzyme (P < 0.05 Sharon File and Jerome Smith for supplying schistosome by t-test) than of the vertebrate enzyme under some materials. This research was supported by USPHS Grant conditions. At 0.02 mM inhibitor and 0.15 mM sub- No. DHEW 5R22 AI 14103 and AI 15536. strate concentration, all compounds except !I and !11 were preferentially inhibitory; !11 exhibited preferential effects at 0.02/0.0375 and 0.05/0.0375 inhibitor/sub- strate concentrations and VI had significant preferen- REFERENCES

    tial action at 0.02/0.15, 0.05/0.0375, 0.005/0.0375, B u E o ~ E. (1952~ Acetylcho|inesterase activity of Sehisto- 0.02/0.15 and 0.005/0,15 mM inhibitor/substrate con- soma mansoni. Br. J. Pharmac. 7. 563-566. centrations. No compound inhibited the vertebrate ELLMAr~ G. L., COURT~EY K. D.. ANDRV, S V. & FE THERSXON enzyme more strongly than the schistosome enzyme. R.M. (1961) A new and rapid colorimetric determi-

    The values of Ki were in the range of 50--500 #M, nation of acetylcholinesterase activity. Biochem. Phar- for both schistosome and vertebrate enzymes. The mac. 7, 88-95. noncompetitive K~ was larger than the competitive K~ GEAR N. R. & FRtPP P. J. (1974) Comparison of the in all cases, indicating that little noncompetitive inhi- characteristics of acetylcholinesterase EC 3.1.1.7 present bition occurred. Only compound V! had a signifi- in four species of Schistosoma. Comp. Biochem. PhysioL

    47B, 743-752. cantly different K, for the two enzyme preparations: HtLLM^N G. R.. CHU S.-H. & DOTSON M. J. (1980) Effect of Ki = 0.045 mM for the vertebrate enzyme, dansylated acetylcholine analogs on Schistosoma man- Kl = 0.0093 mM for the schistosome enzyme soni. d. pharmac. Sci. 69, 516--520. (P < 0.05). HtLLMAN G. R. & SENFT A. W. (1975) Anticholinergic

    These findings demonstrate that selective inhibition properties of the anti-schistosomal drug hycanthone. of the schistosome enzyme can be achieved. Corn- Am. J. trop. Med. Hyg. 24, 827-834. pound VI, the N-butyl derivative had the most JEWSBURY J. M., COOKE M. J. & WEBER M. C. (1977) Field strongly selective properties of the compounds tested, trial of metrifonate in the treatment and prevention of While this compound may not yet possess enough schistosomiasis infection in man. Ann. trop. Med. Parasit.

    71, 67-83. selectivity to be useful for chemotherapeutic purposes, KRupK^ R. M. & L IDLER K. J. (1961) Molecular mechan- extension of this and other structure--activity series isms for hydrolytic enzyme action, d. Am. chem. Soc. 83, should provide further information on which to base 1445-1458. the design of schistosome-selective cholinesterase in- WILmNSOr~ G. N. (1961) Statistical estimations in enzyme hibitors, kinetics. Biochem. J. 80, 324--332.

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