General discussion—Sessions I and II

General Discussion Sessions I and II PRESENT AND FUTURE PROBLEMS IN VECTOR CONTROL BRUCE-CHWATT: Mr Wright has quite properly stressed the immense achievements of the global malaria eradication programme. May I indicate what remains to be done? Today, in the fourteenth year of this unique international public health programme, some 400 million people still live in areas where malaria eradication programmes have not been undertaken and where malaria control measures have little effect on the overall transmission of this disease. There is little doubt that the part of the task that remains unfinished is going to be much more difficult than anything we have yet done. This fact alone should indicate not only the continuing need for DDT but, to an even greater extent, the need for new and improved insecticides. It has been pointed out that of approximately 1 300 candidate insecticides only 5 or 6 have shown some promise; but this is not a bad yield in comparison with the far less impressive results of our recent search for better antimalarial drugs. It should be remembered that both insecticides and drugs are only " tools " that can assist us in our endeavour to control or eradicate a disease such as malaria. The way in which they are used depends upon the socio-economic conditions of the society in which they are used. Consequently, the eradication of malaria depends not only on the technical means that are available but, to an even greater extent, on the will of the community to employ these means for its social and economic advance. Final success in the malaria eradication programme cannot be achieved without the existence of a network of basic health services that can deal with the remaining foci of the disease. It is in this sphere that the affluent societies can help the developing countries to accelerate their advance towards a better future. NARAHASHI: How serious is the effect of persistent insecticides such as DDT on the environment in developing countries? Is WHO trying to develop new insecticides for malaria control because of the effects of persistent insecticides on the environment, because of the development of resistant strains of insects, or for some other reason? WRIGHT: The search for new insecticides for malaria eradication is based entirely on the development of resistance in insects. In a statement on the place of DDT in public health programmes, the Director- General of WHO has said that indoor spraying of DDT in routine antimalaria operations does not involve a significant risk to man or wildlife. KENAGA: Cost has been mentioned as one of the three criteria that must be met if a given insecticide is to be acceptable as a substitute for DDT. What is the price requirement that must be met? WRIGHT: Companies developing new insecticides as alternatives to DDT could use as guides the price of carbaryl for carbamates and that of malathion for organophosphorus compounds. BARNES: It might be of interest to note that one organophosphorus insecticide-trichlorfon-has re- cently been used successfully as a drug to treat schistosomiasis of the urinary tract and to arrest the excretion of eggs in the urine. WRIGHT: A systematic search is being made by WHO for new and effective molluscicides and a number of these compounds are now undergoing evaluation. The subject was not included in the programme of this Conference as it falls outside the administrative scope of the Insecticide Evaluation Programme as it now exists. DESIGN OF NEW INHIBITORS OF ACETYLCHOLINESTERASE OoMs: The first remark I should like to make concerns the process of "aging" mentioned by Dr Aldridge. Investigations in my laboratory have shown that this aging process occurs only with the cholinesterases and not, or only to a slight extent, with the other B-esterases. It is an enzyme-catalysed C-O splitting of the P-O-C bond with the formation of a carbonium ion. With acetylcholinesterase, the group involved is very probably an undissociated carbonyl group, and the aging depends on the - 1132620 GENERAL DISCUSSION-SESSIONS I AND II stability of the carbonium ion. A further point is that secondary alkyl groups, for example, are much more rapidly removed than primary alkyl groups. Secondly, I should like to discuss further a subject mentioned by both Dr Fukuto and Dr Metcalf, the stereospecificity of cholinesterase for optically active organophosphorus compounds. The first thing that can be observed is that acetylcholinesterase is sub- stantially more stereospecific than " butyrylcho- linesterase ". A fairly large number of compounds was investigated with asymmetry centres in the alkyl group, in the central phosphorus atom, and in the leaving group as well as combinations of these. The stereospecificity patterns of acetylcholinesterase and of butyrylcholinesterase, observed by inhibition with asymmetrical organophosphorus compounds, are shown in Fig. 1. Fig. 1 Stereospecificity of acetylcholinesterase (X ---X) and of butyrylcholinesterase (O ---- 0) *R 0 0 / Y H H RMe S *C-/ *Cc -S- Me Me Me 4- L 3- 0 2- 1- 0 - /I \ ~~x <esteti site s i te Stereospecificity of acetylcholinesterase is low for asymmetry in the alkyl groups, high for asym- metry on the phosphorus atom, and declines with asymmetry on the leaving group the farther it is situated from the central phosphorus atom. The other thing that can be observed for acetylcholin- esterase is that there seems to be a relation between stereospecificity and specificity. For inhibitors with high rate constants, in the order of 107 litre mol-1 min-', the ratio between the rate constants of the fastest and slowest optical isomers can be as high as 3-4 orders of magnitude. In reply to a question posed by Dr Weiden, we did measure the association step and the subsequent phosphorylation step separately in some cases. As an example, we found that a ratio of 3 000 in the bimo- lecular rate constants breaks up in a ratio of 500 in the dissociation constant of the complex and a ratio of 6 in the rate of the phosphorylation reaction. One of the interesting things one can do with these optical isomers is to find out if organophos- phorus compounds with different leaving groups, but otherwise identical structure, inhibit the enzyme in the same way. Optical isomers of the compounds shown in Fig. 2 were prepared and were stereochem- Fig. 2 WHO 10307 ically correlated using reactions of known stereo- chemical course. In all cases, the same configuration reacted most rapidly with the acetylcholinesterase M\ SHC0 M Me/ SCH2CH2NMe iPrO 0 Me/ \F iPrO 0 Me 0/ \0O NO2 114 GENERAL DISCUSSION-SESSIONS I AND II with ra (=k(-)/k(+)) values of 1 200, >4 000, and >20, respectively. This result seems to indicate that all three compounds react in the same way with acetylcholinesterase and that the so-called anionic site, which is certainly very important in the forma- tion of enzyme-substrate complexes, is probably not involved in the multipoint interaction of enzyme and inhibitor, thus resulting in the high degree of stereospecificity. HEILBRONN: The morning session has clearly shown that there is a need for standardization of the enzyme tests performed with organophosphorus compounds and carbamates. If the anticholinesterase activity of these compounds is regarded as a main factor in their insecticidal activity and if, therefore, it is desired to determine their anticholinesterase activity, then the enzymes that are used to determine this must be carefully selected. Either they should give information on the reaction between enzyme and anticholinesterase only, and then the enzymes used should be as pure and as water-soluble as possible, or the test should approach as closely as possible the events occurring in the living animal, when a homogenate of an organ should be used or the anti- cholinesterase activity should be measured in vivo. The latter method would give a value for the anti- cholinesterase activity that included such factors as penetration, metabolism, and rate of excretion. A careful and standardized selection of the organs from which the cholinesterase is taken is also necessary in view of the well known species and tissue variation in cholinesterases. In order to illustrate how a cholinesterase changes its properties when in solution or when fixed to (for example) a membrane, as many cholinesterases are in vivo, let me describe a few experiments in which the kinetics of a free acetylcholinesterase were com- pared with those of the same cholinesterase coval- ently bound to agarose (Axen, R., Heilbronn, E. & Winter, A. (1969) Biochim. biophys. Acta (Amnst.), 191, 478). The Km values, the values of V, the rates of activity, and the rates of inhibition all changed, as did the pH-activity curve of the enzyme. Clearly this has to be so, as the microenvironment of an enzyme influences the behaviour of the enzyme and most probably that of its substrates and inhibitors. Possibly even some secondary reactions, such as the rate of spontaneous reactivation and the rate of aging of a phosphorylated cholinesterase, are affected. It is thus clear that tests for anticholinesterase activity should be carried out under standardized conditions and that the origin of the selected cholin- esterases and their state of purity should be specified. Only then will it be possible to compare results obtained in different laboratories and to draw conclusions about the usefulness of the compounds tested. NEAL: The in vitro metabolism of diethyl aryl phosphorothioates by the mixed-function oxidase enzymes of rabbit liver microsomes produces two principal metabolites. These are the corresponding diethyl aryl phosphate and diethylphosphorothioic acid plus the corresponding phenol. These reactions are regulated by what appear to be two separate mixed-function oxidase enzymes. We recently performed an experiment to examine the effect of structure on the rate of metabolism of a series of diethyl aryl phosphorothioates to these two metabolites. In these studies we synthesized a series of diethyl aryl phosphorothioates that had the following substituents in the aromatic group: p-NO2, m-NO2, m-CF3, p-Cl, m-CH3, p-CH3, and p-OCH3. A compound that contained phenol as the aromatic group was also synthesized. These compounds, labelled with 32p, were incubated in various concentrations with rabbit liver microsomes and the maximum rate of metabolism, Vmax, was calculated using the Michaelis-Menton equation. A plot of log Vmax against the Hammett sigma constant resulted in a biphasic curve for each meta- bolite. The inflexion point of both curves occurred at a value of sigma close to zero, which corresponds to phenol as the aromatic group. Statistical analysis of the data supported a biphasic linear relationship between the maximum rates of metabolism of the diethyl aryl phosphorothioates to these two products and the Hammett substituent constants. The smallest Vmax for the formation of diethyl phosphorothioic acid was obtained with the com- pound that had phenol as the aromatic group. Aro- matic groups containing both electron-donating and electron-withdrawing groups had an increasingly larger Vmax for the formation of this compound than did the compound containing phenol, depending on the electron-donating or electron-withdrawing power of the substituent. In contrast, the compound containing phenol had the largest Vmax for the formation of the corresponding diethyl phenyl phosphate. Compounds containing aromatic groups substituted with both electron-withdrawing and electron-donating groups displayed a decreasingly smaller Vmax for the formation of this compound than the compound containing phenol, depending 115 GENERAL DISCUSSION-SESSIONS I AND II on the electron-withdrawing or electron-donating power of the substituent. In non-enzymatic systems, a biphasic response of a kinetic parameter to changes in the electron-with- drawing or electron-donating properties of an aromatic group usually indicates a change in the rate- limiting step of the reaction. It is difficult to apply this explanation to the biphasic response obtained in these studies, primarily because it is attractive to postulate a common intermediate in the metabolism of the diethyl aryl phosphorothioates to these two products. A possible explanation may be that a change in the mechanism of the reactions leads to the loss of the sulfur in the case of diethyl aryl phosphate formation and to the loss of the aromatic group in the formation of the diethylphosphoro- thioic acid, with the change from electron-with- drawing to electron-donating groups. HEILBRONN: The many different results that are obtained from kinetic measurements ofphosphoryl- ation, carbamylation, and spontaneous reactivation of different cholinesterases call for studies of the cholinesterase molecule itself. An effort must be made to purify and crystallize these enzymes and to study their primary, secondary, and tertiary struc- ture. This will give us new information on the reaction between these enzymes and anticholin- esterase agents. Whether or not it will give us better insecticides depends, of course, on other factors also. DONNINGER: I agree with Dr Heilbronn. The path that we advocate is costly and difficult, but I believe that real progress in this field may well depend on our taking it. We need proper informa- tion on the active centre of the enzyme, the type of information that is obtained by X-ray crystallogra- phic studies and by studies of protein chemistry. Pure enzyme in quantity is needed. The group at Columbia University will, it is to be hoped, provide such information for the eel enzyme. Comparative studies with a mammalian and with an insect enzyme should be rewarding. HOLLINGWORTH: Dr Heilbronn's point is well taken and I do not wish to debate the utility of purifying cholinesterases and studying their properties; such work is essential. However, it would be well to keep in mind that what may be good biochemistry may not be good toxicology. These and other significant enzymes deserve study at all levels if we wish to understand their toxicological significance in the living organism. The properties of membrane- bound enzymes (such as many cholinesterases) may well undergo changes in the process of bringing these enzymes into solution. Therefore, the beauti- fully crystalline enzyme may have doubtful relevance to the original enzyme in vivo, even though it delights the biochemist. I think it is debatable whether studies of such purified enzymes are always more useful to the toxicologist than the study of cruder, but more realistic, preparations if we wish to obtain comparative data for a range of organisms. DONNINGER: I am not suggesting that purification and crystallization of acetylcholinesterase is a panacea that will solve all our problems. Studies at all levels of organization are of course necessary. The crystalline enzyme is not an end in itself, but will make it possible to undertake other studies- X-ray crystallographic studies, protein chemistry, and proper kinetic work, to mention a few. ALDRIDGE: We ought not to expect to deduce, from the study of substrates, much that will be useful in the study of inhibitors. Although some broad generalizations might emerge, I think any detailed comparison is not possible. This is because in the hydrolysis of substrates we are measuring the entire reaction, whereas the reaction with inhibitors such as organophosphorus compounds concerns what for substrates is the period of transient kinetics. It has been suggested that we should purify cholinesterase completely and then determine the chemistry of the interaction between the inhibitor and the enzyme. I am sure this will provide extra scientific informa- tion, but the problem facing us here is how to approach the design of better insecticides. I believe that we can make progress by using impure enzyme preparations, for thus far there is no evidence that the constants for the separate steps in the reaction differ with enzyme preparations of widely differing purity. DONNINGER: Since the inhibition of acetylcholin- esterase by anticholinesterase agents is a chemical reaction in which one of the reactants is the enzyme, and since there is good evidence that an enzyme- inhibitor complex is formed before covalent bonds are broken and formed, it seems to me that know- ledge of the active centre of the protein should assist us in the design of more effective anticholin- esterase agents. HEILBRONN: If we require new insecticides that are selective anticholinesterase agents, we must learn more about the active centre of cholinesterases. The 116 GENERAL DISCUSSION-SESSIONS I AND II ideal procedure would be to search for differences in the active centres of the cholinesterases of insects and of mammals, including man, but this would be extremely costly. However, we need information about the groups in the enzyme that take part in the different steps of the reaction between the enzyme and the inhibitor. For organophosphorus com- pounds these steps are the formation of an EI complex, phosphorylation of the enzyme, and reac- tivation or aging. Elucidation of the latter step is particularly important, since it hinders the treatment of intoxications with organophosphorus compounds. The purification of eel acetylcholinesterase has not so far given us any information, owing to the fact that its tertiary structure has not been studied. Only by such studies can we clarify the shape and nature of the active centre of the enzyme and the roles played by the polypeptide chains. Impure enzyme preparations may be used for testing the anti- cholinesterase activity of new insecticides (but not for testing their general toxicity), provided that every laboratory uses the same enzyme source, and provided also that the enzyme preparations are free from other enzymes that bind or hydrolyse the anticholinesterase. MAIN: It has been asked what can be learned from the study of purified cholinesterase that cannot equally well be learned from the study of crude enzyme preparations. Perhaps such questions are based on the tacit assumption that every possible organophosphorus and carbamate structure has already been synthesized. However, it becomes clear that this is not so when it is observed how closely most successful compounds have been modelled on the substrate acetylcholine and on our concepts of how the substrate fits the active site. This raises the question of how much is known about the fit of acetylcholine to the active site. The fact is that many important kinetic observations remain unexplained-for example, inhibition at high substrate concentrations is poorly understood. The reality of the " anionic site " remains open to ques- tion, as does that of other binding and orientation sites. Indeed, there is some question as to whether organophosphorus compounds orient and react in a manner that is truly analogous to that of substrate and carbamate reactions. In view of this lack of knowledge, it seems to me that considerable struc- tural innovation may be possible in the design of organophosphorus compounds and carbamates. It is clear, however, that the kinetic studies necessary to answer these questions cannot be conducted with impure preparations. Furthermore, the results of kinetic studies are rarely definitive: they must be confirmed by physical evidence, and this requires purified cholinesterases. Examination of the cho- linesterase structure that is emerging from all the studies that have been conducted shows the necessity of having available purified cholinesterases. This structure involves the possible presence of an allo- steric site, as suggested by the results of Aldridge and Reiner. Such a site could be of profound physio- logical significance in terms of feedback control, but it could also lead to the design of new insecticide structures. We must also consider the subunit agglomerate structure that cholinesterase appears to possess. This results in multiple molecular forms of significantly different kinetic properties, again sug- gesting possibilities for the design of novel organo- phosphorus and carbamate structures. The same could also be said of potential therapeutic agents. Certainly the development of selective insecticides will depend on an understanding of the structure of cholinesterases. It is probably not unreasonable to say that significant progress in the development of organophosphorus compounds will depend on the ready availability of highly purified cholinesterase preparations. METCALF: The complete purification of at least one species of acetylcholinesterase is clearly desirable. However, there are perhaps 10 000 species of inju- rious insect, and the purification of the enzymes from all of them would be unbelievably complex. We have recently examined in detail, using the Main kinetic method, the reaction between a series of organo- phosphorus compounds and carbamates and the enzymes from the housefly, the honey-bee, and the house cricket, all purified several hundred-fold. Although the overall '50 values were roughly com- parable for the three acetylcholinesterases, k2 and k3 values sometimes showed 10-20-fold variations for the same inhibitor. This suggests that each insect species may have a structurally distinct acetylcho- linesterase. The same degree of complexity exists for other enzymes-e.g., ribonuclease-that have been studied on a comparative basis. Studies of the kinetics of inhibition by carbamates and phosphates of crude and highly purified red-cell or fly-head acetylcholinesterase have given identical results. It seems debatable, therefore, whether much progress in the design of better insecticides could be made by the study of pure enzymes. 117 GENERAL DISCUSSION-SESSIONS I AND 1I BOWERS: Is protein chemistry, especially enzyme chemistry, sufficiently advanced to permit an under- standing of structure-activity relationships if the enzyme used were indeed pure? Are there examples of other enzymes whose purification has permitted the development of more effective chemical inhi- bitors ? DONNINGER: I think that X-ray crystallographic and certain protein chemistry studies should give us information on the structure of the active centre. KENAGA: The idea for the insecticide Zectran t was based on a spatial modification of physostigmine, a potent cholinesterase inhibitor. Shulgin synthesized a m-tert-butyl-p-dimethylaminophenyl methylcar- bamate that mimics the spatial position of the heterocyclic N in physostigmine, and it was found to be a highly active insecticide. Further modifica- tion of the m-alkyl substituents resulted in the potent insecticide Zectran. DONNINGER: It may be of interest to outline the reasoning that led us to synthesize the methomyl group of oxime carbamates. In the belief that a particular conformation of acetylcholine would be " frozen " in the enzyme-substrate complex, we set out to investigate what conformation was involved. Acetylcholine analogues in which the central carbon- carbon bond of acetylcholine was fixed in known absolute configuration were synthesized. The imine bond was used to this end. Quaternary ammonium was replaced by tert-butyl and this by alkylthio in the expectation that oxidation in vivo of the sulfur to sulfoxide and sulfone might increase the anticho- linesterase activity. 118