Fodor A et al. (1987) Worm Breeder's Gazette "competition of exogenous methoprene and precocenes in nematodes: the end of the story."

Timar T, Hosztafi S, Dinya Z, Fodor A, Kiss I

[Worm Breeder's Gazette, 1987]

We have accomplished a ten year long project aimed to learn whether competition between the juvenile hormone (JH) analogue methoprene (JHA) and precocenes (P's) (chromene derivatives capable to destruct the JH producing organ (CA) in sensitive insect species tissue specifically) in C. elegans (Fodor, et. al., Gen. Comp. Endcr. 46: p. 99 (1982)) can or cannot be explained by a comparable 'anti-JH' action of P's in nematodes. Neither JH or CA like organ has been discovered in nematodes so far. There are only a few indirect data showing that insect juvenile hormones may influence certain nematodes pathogenizing insects. We adopted a 'structure/activity' approach including design, synthesis and test P analogues on nematodes in the presence and absence of JHA. If (at least part of) those analogues which capable to destruct the CA of a sensitive insect (Locusta migratoria) were also effective in nematodes and their effect could be compensated by JHA exogenously, then this hormone (analogue) should play a physiological role in the P-poisoned nematodes. If those P's could be competed by JHA, which proved effective (as 'anti-JH' compounds) in insects, but those which exerted only aspecific toxicity could not be, then it would be logical to suggest, that P's are the same kind of 'suicide compounds' for nematodes as for insects. More than 200 P derivatives were synthesized (Tim r, Hosztafi) and tested on C. elegans (Fodor) and L. migratoria (Kiss). After a detailed quantitative structure/activity relation (QSAR) analysis ( Dinya, et. al., QSAR Strat. Des. Bioact. Compd. Proc. Eur. Symp. Struct.-Act. Relat. 5th (1984) Publ. 1985) several new P analogue were designed, synthesized and tested on L. migratoria and on C. remanei var. Bangaloriensis. (We choose this nematode strain because half of its population consists of males, therefore it is easy to distinguish male adultoids from other type of retarded worms.) Altogether, 121 molecules were retested C. remanei and 17 of them was found to exert some significant biological effect. These compounds were retested again several times both in the absence and in the presence of 1 mg/ml NGM dose of JHA: altogether, more that 144,000 C. remanei embryos were counted, treated and scored afterwards. The tests on nematodes were carried out as described in our attached paper. The most characteristic data concerning precocene activity in nematodes were the following: (1) LC50: the half lethal dose (in g/ml) at which half of the embryos develops to worms (calculated by probit analysis); (2) AD50: the dose ( g/ml) at which half of the embryos develops to normal adults; (3) EC50: the dose ( g/ml) at which half of the nematodes on the plates found as 'normal' fertile adults; (4) The maximum frequency of 'adultoid mini worms' during the experiments. [See Figures 1- 2] The main conclusions are the following: About structure/activity relations: (1) All the three (P1-P3) precocene is effective in nematodes and their effects can be compensated by exogenous JHA. (2) The longer the chain of the R7 substituent the less the effect of the compounds in nematodes. (3) The 7-proparglyoxy analogues are much more effective in nematodes than any other C7 substituted compound. (compare P1 to TT51; P2 to K460; P3 to TT80; TT56 to TT58 or 3,4-diCl-P1 (inactive) to FI121.) (4) The asymmetrically disubstituted analogues are much more effective than the symmetrically disubstituted ones (compare TT80 to K460). It is true, if R7 is longer than R6. (5) Me substitution at C5 position inactivates the originally potent P's (compare TT58 to TT51) but restore the activity of originally inactive (for instance, 7-sBuO-P1) analogues (compare it to TT56). 8-MeO substitution eliminate specific P activity (compare TT51 to K464). (6) Both 8-Me and 8-MeO substitution increase toxic rather than JH compatible biological activity of P's. 8-MeO analogues are more toxic than 8-Me ones, but the consequences of the action of 8-MeO compounds in nematodes can be cured more efficiently by JHA than those concerning 8-Me compounds (compare TT100 to K475). About JHA competition experiments: JHA competed the effects of all precocenes which effected both insects and nematodes. However, the data concerning K354 and FI121 show, that there are analogues which effective only in nematodes and their effects can also be cured by exogenous JHA. Although there are aspecifically toxic analogues (like K454 or 2,3,5-triMe-7 propargO-P1) which cannot be compensated by methoprene, we cannot conclude, that our data unambiguously support the idea of existence JH-like hormones in nematodes. It seems very probable, however, that JH-like compounds can interfere with the lethal metabolism of P's.

Fodor A et al. (1980) Worm Breeder's Gazette "a counteraction between insect juvenile hormone analogue methoprene and antijuvenile hormone precocene 2."

Kiss I, Deak P, Fodor A

[Worm Breeder's Gazette, 1980]

Data presented at Cold Spring Harbor this year showed, that 200 and 400 g per ml precocene-2 in NGM food dramatically decrease the final size of lon-2(e678)/lon-2(e678) hermaphrodites which were placed onto these plates at different ages. It was also demonstrated that the same doses proved to be toxic to the adults. Both effect could be partly compensated by different doses of methoprene to the food. Both the precocene and the JHA was more effective in higher concentrations, and we might suppose, that the precocene-'mini-adults' represent hypojuvenile hormone states. For to say it we need some more evidence. Following the life and growth of dauer larvae placed onto precocene and control plates we found that the molts happened almost the same time, but the size of animals on precocene plates hardly increased. By looking at them under a light microscope, these 'mini-adults' look like perfectly normal but small adults. John Sulston was so kind to observe some of our animals by Nomarski microscope and he also found them normal. Now we are looking for intermediate developing forms having both larval and adult cuticle, gonad and vulva structures. Excluding the possibility of the repellent/antifeeding effects of precocene, we found that when the worms have a choice of moving to bacteria with or without any of the drugs, they choose the latter. When they have to choose between JHA and precocene, they prefer JHA, but if they could avoid it, they don't lay eggs on it. (e678 worms were used as dauers and scored as adults; e1034 chemotactic mutant was the control.) Looking for chance for genetic differences in the reactions to precocene, we compared 22 mutant and N2 stocks on the base of adult test and found that one of the mutants resistant to levamisole proved to be highly resistant to precocene too. This mutant was isolated and generously provided by Jim Lewis: X37(tmr3). We are going to isolate mutants resistant to precocene and also looking for developmentally intermediate forms among precocene 'mini- adults'.