阿米登太的药效学

The effects of amidantel (BAY d 8815) and its deacylated derivative (BAY d 9216) on wild-type and resistant mutants of Caenorhabditis elegans ROBIN A. WOODS A N D KATHLEEN M. B. MALONE Department of Biology, University of Winnipeg, Winnipeg, Man., Canada R3B 2E9 AND CHERYL A. ALBUQUERQUE A N D GEORGE TOMLINSON' Department of Chemistry, University of Winnipeg, Winnipeg, Man., Canada R3B 2E9 Received August 28, 1985 WOODS, R. A., K. M. B. MALONE, C. A. ALBUQUERQUE, and G. TOMLINSON. 1986. The effects of amidantel (BAY d 88 15) and its deacylated derivative (BAY d 9216) on wild-type and resistant mutants of Caenorhabditis elegans. Can. J . Zool. 64: 1310-1316. The anthelmintic drugs amidantel (BAY d 88 15) and its deacylated derivative (BAY d 9216) inhibited the growth of wild-type (N2) Caenorhabditis elegans but had little effect on development or reproductive capacity. Inhibition of growth correlated well with drug-induced paralysis, both becoming maximal at around 1 .O mM concentration of either drug. Egg laying was delayed by about 24 h and the rate of laying was only about 60-70% of the controls. However, the period during which eggs were laid was extended by a similar amount and the total number of eggs laid was the same for controls and drug-treated worms. Five drug-resistant mutants (TI 14, T22, T26, T21 6 , and T226) were isolated following ethylmethanesulphonate mutagenesis. All were shorter than N2 at 96 h on drug-free medium; their growth was not further impaired by either of the anthelmintic drugs. All except T I 14 exhibited a normal pattern of sexual maturation. Cultures of T I 14 at 96 h contained many immature worms. This mutant also exhibited the most impaired motility, being severely uncoordinated in liquid suspension. The other mutants could swim normally but were noticeably slower than N2. Genetic analysis indicated that each mutant was the result of a single genetic lesion, that the mutants were recessive, and that there were two genes for amidantel resistance (adtl and adt2). In vitro studies on representatives of each class (T114 and T22) indicated a defect in the acetylcholine receptor. T22 mutants showed a moderate decrease in sensitivity towards typical cholinergic agonists as well as the anthelmintic drugs, while T I 14 mutants were apparently devoid of functional pharmacological acetylcholine receptors. WOODS, R. A., K . M. B. MALONE, C. A. ALBUQUERQUE et G. TOMI~INSON. 1986. The effects of amidantel (BAY d 88 15) and its deacylated derivative (BAY d 9216) on wild-type and resistant mutants of Caenorhabditis elegans. Can. J . Zool. 64: 1310-1316. L'amidantel (BAY d 88 15), une substance anti-helminthes, ou son derive desacetyle (BAY d 9216) inhibent la croissance des Caenorhabditis elegans de type sauvage (N2), mais n'ont que peu d'effet sur le d ~ v e l o ~ ~ e m e n t ou sur la capacite reproductrice des vers. I1 y a une forte correlation entre l'inhibitation de la croissance et la paralysie qu'entralnent les substances et ces deux facteurs atteignent leur sommet lorsque la concentration de la substance atteint 1 ,O mM. La ponte est retardee d'environ 24 h et le taux de ponte n'atteint que 60-70% de la valeur enregistree chez les tCmoins. Cependant, la ponte dure plus longtemps chez les vers traites et le nombre total d'oeufs pondus est semblable a celui qui prevaut chez les temoins. Cinq mutants resistants aux produits (TI 14, T22, T26, T216 et T226) ont kt6 isolks apres une mutagenese causee par de l'ethyl-methane-sulfonate. Tous les mutants sont plus courts que N2 apres 96 h en milieu neutre; leur croissance n'est pas inhibee plus avant par les substances anti-helminthes. La maturation sexuelle est normale chez tous les mutants sauf le T I 14. Apres 96 h, les cultures de T I 14 contiennent plusieurs vers immatures. Ce mutant est aussi celui chez qui la motilite est le plus inhibee et sa coordination est fortement diminuee en suspension liquide. Les autres mutants peuvent nager normalement, mais ils sont beaucoup plus lents que les N2. L'analyse gknktique a demontrk que chaque mutant rksulte d'une lesion genetique unique, qu'il s'agit de mutants recessifs et que la resistance a I'amidantel est due a deux genes (adtl et adt2). Des etudes in vitro ont demontrk que les recepteurs de I'act5tylcholine des mutants T I 14 et T22 sont defectueux. Les mutants T22 ont rnoins de sensibilite a l'egard des antagonistes cholinergiques typiques ou a l'kgard des substances anti-helminthes, alors que les mutants T I 14 semblent depourvus de recepteurs de l'acetylcholine pharmacologique fonctionnelle. [Traduit par la revue] Introduction Amidantel, N-(4((1 -(dimethylamino)-ethy1idene)-amino))- 2-methoxyacetamide hydrochloride (ADT or BAY d 88 15), is one of a new class of p-aminophenylamidine-based anthelmin- tic drugs. Wollweber et al. (1979) showed that amidantel was effective against filariae, hookworms, ascarids, and pinworms in rats and dogs. These studies were extended by Thomas ( 1979) who showed that the drug was particularly effective against ascarids and hookworms in dogs. In both of these studies a single dose of the drug was sufficient to eliminate and kill intestinal parasites within 3 days. Rim et ul. (1980) found the drug effective in clinical trials against Ancylostomu duodenule and Ascaris lumbricoides; again a single dose was sufficient to ' ~ u t h o r to whom correspondence should be addressed. result in total elimination of the parasites. However, they noted that although amidantel was effective against Necator ameri- canus it was not superior to other drugs in common use. ADT is rapidly deacylated in vivo to the corresponding free amine (dADT or BAY d 9216) with retention of anthelmintic activity (P. Andrews, personal communication). Platzer et al. ( 1977) have advocated the use of the free-living nematode Caenorhubditis elegans as a model system for the study of nematicides. In addition to the facility with which the organism can be grown and handled, it also has a well- developed genetic system (Brenner 1974). Thus mutants resis- tant to anthelmintic drugs can be isolated and characterized, providing information on their mode of action. We have recently shown that both ADT and dADT paralyse C. elegans primarily by acting as cholinergic agonists at the WOODS ET AL. 131 1 level of the nematode acetylcholine receptor (Tomlinson et a l . 1985). The two drugs were markedly less potent than levami- sole, another anthelmintic known to function as a cholinergic agonist (Lewis et a l . 1980) in causing paralysis of intact worms. However, in an assay system using worms that had been cut laterally in two to facilitate uptake of the drugs this differential was not apparent and all three drugs were effective at less than 0.5 pM. In vitro experiments showed that both ADT and dADT were only moderately potent inhibitors of acetylcholinesterase from Electrophorus electricus and C. elegans. The effects of both drugs on cut worms were blocked by d-tubocurarine and gallamine, suggesting that their primary action is as agonists at the level of the acetylcholine receptor. As a complement to these physiological studies we report here the effects of ADT and dADT on the growth and development of C . elegans. We have also isolated and characterized mutants resistant to dADT. Genetic analysis indicates that these can be allocated to two genes and that both affect the acetylcholine receptor. Materials and methods Organisms Caenorhabditis elegans, strain N2, was kindly provided by Dr. Martin Samoiloff, Department of Zoology, University of Manitoba. Stock cultures were maintained on NG (nematode growth) agar according to the methods of Brenner (1974) using Escherichia coli OP50 or HfrC as the food source. Media and growth conditions The media and growth conditions for plate cultures were as described previously (Spence et al. 1982). Stock solutions of ADT and dADT for addition to agar media were prepared at 5 m g . m ~ - ' in sterile glass-distilled water and stored at 4°C. The drugs were added to the medium at the concentrations given in the text. Synchronized cultures of F1 larvae were prepared by the method of Hirsh et al. (1976). Large-scale cultures for enzymatic analysis were grown in 3000-mL Pyrex low-form conical culture flasks containing 200 mL of liquid NG medium seeded with E. coli OP50 or HfrC. These bacterial cultures were grown to stationary phase before the flasks were inoculated with a heavy suspension of C . elegans washed from at least four stock plates. The flasks were incubated at room temperature with gentle shaking for 5 days, when clearing of the medium was observed. At that time the worms were harvested by an initial low speed centrifugation followed by flotation on 30% w/v sucrose (Sulston and Brenner 1974). The collected worms were washed, pelleted, and flash frozen in liquid nitrogen. Growth measurements Synchronized Ll larvae were inoculated onto 3.5-cm plates of NG agar, with or without drug supplementation, and allowed to grow for 96 h. They were then killed and straightened by flooding the plates with 0.5-1.0 mL of water at 90-95OC (Byerly et al. 1976). Worms were transferred to microscope slides in a drop of water and measured using a Leitz projection microscope at a total magnification of 1 OOX (Spence et al. 1982). Determination of egg-laying rates Synchronized larvae were allowed to grow for 59 h on NG medium or NG medium supplemented with ADT (100 p g . m L 1 ) or dADT (50 p g . m ~ - ' ) . The worms were transferred to fresh plates of the same medium at intervals until 155 h after synchronization. Egg counts were made at 0 .5 and 1.5 h after transfer on three replicate plates of 10 worms. In some experiments a single count was made at 90 h after synchronization. Isolation of dADT-resistant mutants Mutants were selected on the basis of drug-induced paralysis of the wild-type strain. Adult N2 hermaphrodites were incubated in 0.05 M ethylmethanesulphonate for 4 h and washed, and 50 were transferred individually to 50 plates of NG medium and incubated until their progeny, the F1, had laid eggs. Adults and larvae were then washed from the plates and the eggs were allowed to hatch for 2 h. The resulting F2 larvae were washed onto fifty 35-mm plates supplemented with dADT (100 p g - r n ~ - ' ) . The plates were examined after 10 min and those that contained larvae resistant to the paralysing effects of dADT were recorded. Single resistant larvae were removed from these plates to initiate cultures of resistant mutants. Genetic analysis Crosses were performed by mating hermaphrodites to males induced by exposure to a temperature of 28°C. Cultures containing males were maintained by inoculating stock plates with six males and two hermaphrodites. The progeny of crosses were scored on plates containing 50 pg dADT-mL- . Drug tests Drug tests were carried out in deep-well microscope slides on the stage of a Nikon dissecting microscope as described by Tomlinson et al. (1985). Drug solutions were prepared immediately before use in the artificial perienteric solution of Del Castillo and Morales (1967), kept on ice, and diluted as required in the same buffer. Six to 10 trials, each involving 10 worms, were carried out for each drug concentration. Tests were performed on whole and cut worms. Cutting the worms removes the permeability barrier presented by the cuticle, reducing both the response time and effective drug dose. In both types of test a positive effect was recorded if one or more of the treated worms exhibited visible hypercontraction of the head region within 1 h (Lewis et al. 1980). All tests were carried out at room temperature (20°C). Acetylcholinesterase assay Worms in liquid nitrogen were freeze powdered in an equal volume of 0.1 M phosphate buffer, pH 8.0. Several freeze-thaw cycles were required to ensure adequate disintegration of the tough cuticle, as established by microscopic examination of the sample between cycles. The preparation was allowed to thaw and was then homogenized in a glass homogenizer. Samples of both the crude homogenate and the supernatant after centrifugation at 12 000 rpm for 4 min in a Beckman model 152 microfuge were assayed for activity of acetylcholinesterase (AChE) by the method of Ellman et al. (1961). The assays were performed at room temperature ( 2 2 2 1°C) in a Beckman model 25 spectrophotometer. Protein was determined by the method of Bradford (1 976). Chemicals Amidantel and deacylated amidantel were generous gifts from Dr. P. Andrews, Bayer AG, Wuppertal, West Germany. All other chemicals were purchased from the Sigma Chemical Co. Results The effects of ADT and dADT o n the growth of C. elegans The growth of wild-type C. elegans was inhibited by both ADT and dADT as shown in Fig. 1A. The decrease in length correlates well with the progressive drug-induced paralysis of whole worms (Fig. 1 B). Both parameters reached a maximum at about 1.0 mM for ADT and dADT. At this concentration length was reduced to 65% of the controls. Paralysis by both drugs is characterized by an intial hypercontraction of the head followed by a progressive thickening of the body and greatly reduced motility. At high concentration (1 .O mM and above) the worms are almost immobile and pharyngeal pumping is sporadic. Paralysis is reversible; transfer of treated worms to normal medium in sufficient buffer to dilute out the drug allows recovery of normal motility within 30 min for most worms. Effects of ADT and dADT on development and reproductive capacity Although both drugs caused a marked reduction in the size of adult worms they did not appear to impair the course of CAN. J . ZOOL. VOL. 64, 1986 Drug concn (mM) FIG. 1. Effects of drug concentration on the growth and motility of wild-type C. elegans. (A) Effects on length of ADT (0) and dADT (m). Worms were harvested and measured at 96 h. Each point is the average of at least 20 measurements. (B) Paralysing ability of ADT (0) and dADT (m). Points represent the average percentage of worms that exhibit contraction within 60 min in a solution of either drug at the indicated concentrations. Each point is the average of 6- 10 trials, 10 worms per trial. TABLE 1. Characteristics of drug-resistant mutant of C. elegans Eggs laid per worm per Mean length at 96 h (km) houf controlb ADT dADT Control ADT dADT N2 1248 985 993 7.4 5.2 5.8 T22 1047 1020 1034 - - - T26 1039 1020 1036 8.4 9.0 9.2 T216 766 806 841 - - - T226 1081 1073 1036 - - - T114 ND" - - 6.2 6.4 5.5 0 "Established with worms at 9 0 h posthatch. bControl worms were grown on NG agar in the absence of drugs. When drugs were 60 70 80 loo 140 present in the growth medium their concentrations were ADT. 100 p g . r n L 1 and dADT. 50 - . Age of egg-laying nematodes (h after hatch) pg.mL 'T,14 ' contained . a large proportion of immature worms at 9 6 h. N D , not determined. FIG. 2. Egg-laying rates of wild-type C. elegans in the absence and presence of ADT or dADT. 0 , controls, no drug present in NG medium; H, ADT (100 kg . rn~- ' ) ; A, dADT (50 k g - m ~ - ' ) . were shorter than N2 at 96 h on drug-free medium but showed no reduction of length in the presence of ADT or dADT. All development. Each of the larval moults occurred within the time range prevously reported by our laboratory (Spence et al. 1982). Reproductive development was similarly unaffected; vulvae and eggs could be discerned in treated worms at the same time as in the controls. Both ADT and dADT did have a noticeable effect on the timing and rate of egg laying; the onset of egg laying was delayed by about 24 h and the rate of laying was only 60-70% of that of the controls (Fig. 2). However, eggs continued to be laid up to 150 h with the result that the total number of eggs laid was the same for controls and drug-treated worms. Continued growth in the presence of ADT for four generations did not alter the size or egg production of treated worms. except T I 14 matured sexually and began to reproduce at the same time as the wild type. Cultures of T I 14 contained a large proportion of immature worms and egg production was severly reduced although the progeny were fully viable. This mutant also exhibited severly impaired motility. Worms moved reason- ably well on agar medium but in suspension in liquid they appeared severely uncoordinated. The other mutants swam well with the normal sinusoidal motion of C . elegans but were noticeably slower than the wild type, N2. All of the mutants were completely resistant to 1 .O mM ADT and dADT; at that concentration N2 was paralysed within minutes. Neither drug affected the growth of the mutants, nor, in the two cases tested (T26 and T I 14), the rate of egg production at 90 h. Characterization of dADT resistant mutants Genetic analysis of the resistant mutants Five dADT-resistant mutants, T I 14, T22, T26, T216, and All five mutants were crossed to N2 males and the segrega- T226, were isolated in the two hunts for mutants. The tion of the resistant phenotypes was analysed by the following characteristics of the mutants are listed in Table I . All of them protocol. Hermaphrodites from the FI were transferred individ- WOODS ET AL. 13 13 TABLE 2. Genetic analysis of dADT-resistant mutants with strain T22. However, both the minimum effective concen- No. of individuals Sensitive Resistant Ratio p for 3: 1 NOTE: F3' progeny (obtained as outlined in the text) were tested for sensitivity towards dADT (50 p g . m L 1 ) . TABLE 3. Tests for allelism among C. elegans mutants Phenotypes in F1 Cross Males Hermaphrodites T22 X TI 14 Sensitive Sensitive and resistant T22 X T26 Resistant Resistant T22 x T216 Resistant Resistant T22 x T226 Resistant Resistant NOTE: F1 progeny of the cross between T22 males and hermaphro- dites of each of the other mutants were tested for sensitivity towards dADT (50 pg .rnL1) . ually to dADT plates and allowed to produce an F2; in all cases the F2 contained resistant and sensitive worms indicating that the F 1 had been heterozygotes. Resistant F2 hermaphrodites were backcrossed to N2 males and allowed to produce an F1' and F2'. The Fl ' worms were heterozygotes (adt/+), and segregated resistant (adtladt) and sensitive (adt/+ and +/ +) genotypes in the F2'. For each mutant, 5 resistant and 15 sensitive F2' hermaphrodites were transferred to individual plates and allowed to produce an F3'. The resistant F2' individuals gave only resistant progeny. Sensitive F2 individu- als gave progeny that were either all sensitive or a combination of resistant and sensitive worms. The latter resulted from F2' heterozygotes and were scored for the ratio of sensitive to resistant worms. The results, listed in Table 2, indicate that each mutant is the result of a single genetic lesion. The sensitivity of the F l , Fl ', and F2' heterozygotes shows that the mutants are recessive. The mutants were tested for allelism by crossing them with males induced in T22. The results, given in Table 3, showed that T22 was allelic to T26, T216, and T226; all of the progeny were drug resistant. The cross with TI 14 yielded sensitive males and hermaphrodites with some resistant hermaphrodites, presuma- bly from self-fertilization of the T I 14 parent. Thus the mutants represent two genes, to which we have assigned the provisional designations adtl (T22, T26, T216, and T226) and adt2 (T1 14). Pharmacological characterization of amidantel resistance Attempts were made to define the pharmacological character- istics of amidantel resistance using one representative