Chapter 23
Sites of Action for Neurotoxic Pesticides Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 09/14/18. For personal use only.
Panel Discussion Role of Biochemical, Physiological, and Neurochemical Research in the Insecticide Discovery Process 1
Chairman: A. E. Lund Panel members: D. W. Gammon , S. M. Sieburth , M. A. Brown , M. E. Schroeder , K. R. Jennings 2
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E. I. du Pont de Nemours & Co., Wilmington, DE 19898 FMC Corporation, Princeton, NJ 08543 University of California, Berkeley, CA 94720 Shell Agricultural Chemical Company, Modesto, CA 95352 American Cyanamid, Princeton, NJ 08540 2
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A. E. Lund: I want to provide a context for our discussion on the role of biochemical, physiological, and neurochemical research in this process. Figure 1 shows a schematic view of insecticide discovery that highlights some key steps taken by most agricultural chemical companies. Probably no company fully integrates all the functions shown here, and many undoubtedly have chosen not to develop one or more steps in the process as I have drawn it. Traditionally, the discovery process begins when a chemist synthesizes a new compound. The chemist may choose to make compounds based on an understanding of vertebrate pharmacology, biologically active natural products, or proven insecticidal chemistry; or he may choose to make completely novel structures for which no biological information is available. In any case the compounds are evaluated for toxic effects in insect species which usually represent desirable insecticide markets. The objective of this initial testing is to eliminate inactive compounds from further tests, and to roughly define the species spectrum of activity. Active compounds continue to higher level testing where attributes deemed important to successful product marketing are measured and compared with standards. The most attractive compounds are tested in initial field trials from which product development candidates are selected — again based on a knowledge of the relevant markets. This sequence between chemical synthesis and product development is, and will surely continue to be, the heart of the insecticide discovery process. The compelling reason for this is the simple fact that the experimental proof that a new compound possesses those attributes which will provide the required value to some customer can only be obtained from tests for those attributes on target insect species under realistic conditions. It seems unlikely to me that biochemical or physiological research will have a significant impact on this part of the product discovery process in the foreseeable future. A more plausible role for this research 0097-6156/8 7/0356-0316S06.00/0 © 1987 American Chemical Society
Novel Structures
Known Therapeutic Agents
Target Site Biochemistry
Structural Info on Target
Natural Products
Ligand-Receptor Mechanism
Figure 1. An i n s e c t i c i d e discovery process.
2° Attribute Testing & Optimization
Models for Synthesis
Toxicological Consequences of Target Modification
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seems to be the f a c i l i t a t i o n of the discovery of new early leads through some knowledge of biochemical target s i t e s within the pest insect· Target s i t e research l o g i c a l l y begins with the selection of an appropriate target s i t e . A l i k e l y source of clues to new target s i t e s comes from studying the mode of action of an i n s e c t i c i d a l compound discovered in the in vivo evaluations, but targets can also be selected based on a thorough knowledge of physiology. There are many factors which one might consider in the selection of a target, but I have included a few that I think are p a r t i c u l a r l y important. Some knowledge of the t o x i c o l o g i c a l consequences of target s i t e modification i s c r u c i a l . If i n h i b i t i n g a particular receptor or enzyme by 5% results in a desirable toxic e f f e c t , that target i s more sensitive to manipulation than one that requires 9 5 % i n h i b i t i o n before in vivo a c t i v i t y i s observed. At least some rudimentary information on the biochemistry of the target s i t e i s h e l p f u l , since the development of e f f i c i e n t binding and functional assay systems i s c e r t a i n l y f a c i l i t a t e d by background information. The a v a i l a b i l i t y of chemical models provides the clues on which to base i n i t i a l synthesis. Thus known toxins or drugs represent starting points for the chemist and tools for the testing of new assays by the biologist. Once a biochemical target has been selected, in v i t r o assays can be developed which measure both the binding of ligands and the functional i n t e g r i t y of the target. These assays provide a c t i v i t y information at the target s i t e without the complicating influences of penetration, metabolism, and excretion. Of course, comparing the in v i t r o and in vivo results i s the f i r s t step i n identifying and solving these toxicokinetic problems. The in v i t r o assays also provide the foundation for the study of target s i t e biochemistry. I think the key issue here i s the need to c o l l e c t biochemical information about the target s i t e that i s useful to the synthesis chemist. For example, the molecular weight, number of subunits, or even amino acid sequence of a receptor i s not p a r t i c u l a r l y h e l p f u l , while the location and identity of functional groups adjacent to a p a r t i c u l a r toxin binding site and the nature of the chemical interaction between the ligand and receptor which r e s u l t s in receptor activation give clues to the design of potential i n s e c t i c i d e s . Ihus the interaction between the chemist and the target s i t e biochemist tends to focus on the design of various chemical probes to gain t h i s information. I w i l l conclude t h i s introduction by stating the obvious. It seems clear to me that biochemical, p h y s i o l o g i c a l , and neurochemical research has much to contribute to the product discovery process, but the success of t h i s approach i s c r u c i a l l y dependent on the close i n t e r d i s c i p l i n a r y interactions that I have indicated in the f i g u r e . T. Narahashi: Could you give us some examples of insecticide development based on t h i s scheme? M. E. Schroeder: I don't think there are any commercial i n s e c t i c i d e s whose discovery or development was s i g n i f i c a n t l y impacted by in v i t r o t e s t i n g . A l l that I know about were discovered through the t r a d i t i o n a l screening process. However, as you know, i t i s becoming more and more d i f f i c u l t to discover new classes of i n s e c t i c i d e s . So I think that in v i t r o testing and input from biochemists that provide models for synthesis based on biochemical
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and physiological information w i l l play an increasingly important r o l e in the product discovery process i n the future. D. W. Gammon: One example where in v i t r o testing has had an impact i s in the case of the bicycloorthocarboxylate esters which were designed to attack GABA receptors based on the known mode of action of the bicyclophosphates. An in v i t r o assay was used to optimize the potency of these compounds at a receptor s i t e in a process that was p a r a l l e l to the in vivo t e s t i n g . M. A. Brown: Even in t h i s case, however, the o r i g i n a l observation was the ill vivo t o x i c i t y of these compounds. In t h i s sense, the bicycloorthocarboxylates are more of an example of in v i t r o optimization of an existing in vivo lead. R. M. Hollingworth: If you want an example, i t i s d i f f i c u l t to pick one from the a g r i c u l t u r a l area, but i t i s my understanding that one of the most important recent drugs in the U.S., Tagamet, was discovered through a receptor screening process. Also, another major recent drug, the antihypertensive, c a p t o p r i l , was designed s p e c i f i c a l l y to i n h i b i t the angiotensin converting enzyme based on a knowledge of the substrate s p e c i f i c i t y and mechanism of t h i s enzyme. So we do not have to look too far a f i e l d to find commercial success stories that came through an in v i t r o approach. M. E. Eldefrawi: Did we hear in t h i s symposium about a possible product? I am referring to Dr. Usherwood s work on toxins from spider venoms. Does he think that t h i s might be an example of a possible product starting from in v i t r o assays? 1
P.N.R. Usherwood: It i s possible that a product may arise from this research but I think i t i s a long way o f f yet. However, we are i n danger of missing the point about the role of i n v i t r o assays i n pesticide research and discovery. One talks of random screening having been^the way forward i n the past, and that there i s no evidence, past.or present, that i n v i t r o screening has any r e a l value i n the discovery of i n s e c t i c i d e s . However, even with so-called random screening r a t i o n a l decisions are made about which compounds should be tested. Biologists working with i n v i t r o preparations are now i n j e c t i n g additional ideas into such screening programmes. Hopef u l l y , i n due course, this added input w i l l make i t s present f e l t . I don't see this as a different process or as a new way of proceeding towards product discovery. I see i t simply as a contemporary development of t r a d i t i o n a l procedures. 4
R. M. Hollingworth: I think we spend too much time trying to j u s t i f y the top l i n e of what you have in Figure 1. I think i t should be self-evident that t h i s i s another l e v e l or type of knowledge that feeds into the product discovery process. I t ' s obvious that we must do t h i s . I would l i k e to see us talk more about how we can best integrate and use t h i s approach to enhance i n s e c t i c i d e discovery. K. R. Jennings: One type of contribution can be to aid in the pre-selection of chemistry. We have recently introduced the amidinohydrazones which were developed by American Cyanamid for
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cockroach and f i r e ant c o n t r o l . The o r i g i n a l lead came from a drug synthesis program. Frequently we see that compounds made for another program show a c t i v i t y against i n s e c t s ; so a knowledge of other areas such as vertebrate pharmacology can p o s i t i v e l y impact product discovery. F. Matsumura: There are c e r t a i n l y other such examples. One that comes to mind i s the carbamate i n s e c t i c i d e s which came o r i g i n a l l y from a natural product model. Although I think i t i s true that every 10 or 20 years someone w i l l discover a completely new structure with a new mode of action such as Merck has done with the avermectins, there may come a time when we run out of r e a l l y good models for synthesis. Then we may want to come back to some of the older but proven targets such as GABA receptors for the design of new i n s e c t i c i d e s . M. E. Eldefrawi: I would l i k e to d i r e c t a related question to Scott Sieberth as the chemist on the panel. You have heard about the action of cyclodienes on GABA receptors from several groups at t h i s symposium. Would you consider investigating the p o s s i b i l i t y of making a non-chlorinated, e a s i l y biodegradable "cyclodiene"? Would that be a viable kind of product? S. M. Sieburth: Sure. Anything that has demonstrated appropriate i n t r i n s i c a c t i v i t y i s f a i r game for further work. Once you know that cyclodienes i n t e r f e r e with GABA receptors, i t ' s a l o g i c a l step to investigate other compounds which interfere with t h i s target with some expectation of getting similar types of a c t i v i t y . The bicycloorthocarboxylates are an example. On the other hand, one might worry about the l i k e l i h o o d of cross-resistance occurring since resistance to cyclodienes i s documented. D. M. Soderlund: I agree that further work on established targets i s worthwhile. The fact that we have a group of compounds which act on a given target i s a conceptual lead of considerable value as opposed to a target that has hypothesized but not proven t o x i c o l o g i c a l s i g n i f i c a n c e . Now, considering the potential negative aspects of preselected resistance to, say, pyrethroids diminishing the value-of the sodium channel as a target, we should keep i n mind that Jeff Bloomquist working in Tom M i l l e r ' s lab, and now others, have shown that the dreaded kdr target s i t e resistance to pyrethroids does not modify the sodium channel s e n s i t i v i t y to a l l toxins acting there. So there i s a great deal to be learned about overcoming broad target s i t e resistance by understanding existing target s i t e interactions. I think t h i s emphasizes the idea that just because you have a group of compounds which act i n some i l l - d e f i n e d way on a target, i t does not mean that one cannot find other chemical means to attack the same target successfully. F. Matsumura: An important contribution of t h i s more basic research i s i d e n t i f y i n g areas on which we can focus our synthesis e f f o r t . We know that the sulfonylurea herbicides i n h i b i t an enzyme that i s important in the synthesis of amino acids in plants but not mammals. Now i t i s turning out that several new classes of herbicides attack plants at the same s i t e . Another example are the new s t e r o l
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i n h i b i t o r fungicides. We know now that i n h i b i t i o n of ergosterol synthesis i s very f u n g i c i d a l . Other enzymes in t h i s pathway may be important targets for further synthesis. By i d e n t i f i c a t i o n of these c r i t i c a l areas of biochemistry, we can focus synthesis e f f o r t in what may be more f r u i t f u l areas. unknown: Another use of t h i s sort of system i s in the i d e n t i f i c a t i o n of compounds from natural sources where there i s often such a small quantity of compound available that you would never see a c t i v i t y in more t r a d i t i o n a l whole animal or insect assays. The test system doesn't have to be a single receptor or enzyme. The kind of thing I have in mind i s that we would never have discovered one of our a n t i b i o t i c s i f we had not had a b a c t e r i a l c e l l wall assay. The fermentation mixture i t s e l f has no antibacterial activity. A. E. Lund: That i s something that i s not even on t h i s chart ( F i g . 1), and perhaps should be. It i s c e r t a i n l y being used by companies today. Screening fermentation broths or crude extracts from plants to discover new a g r i c u l t u r a l chemicals can be done using t r a d i t i o n a l kinds of in vivo assays or one can screen for s p e c i f i c in v i t r o a c t i v i t i e s . It i s , of course, c r u c i a l in the l a t t e r case that one c a r e f u l l y i d e n t i f y the a c t i v i t y of i n t e r e s t . Once t h i s i s accomplished, one can go about designing an assay to detect the a c t i v i t y in very small samples. K. R. Jennings: A further point on natural products concerns the p u r i f i c a t i o n and i d e n t i f i c a t i o n of the active component. It i s advantageous i f not necessary in most cases to have a rapid in ° assay that can give r e s u l t s on active fractions i n a matter of hours using very small quantities of compound. Otherwise, of course, you loose too much of your sample at each bioassay. So a good in v i t r o assay i s c e r t a i n l y important for successful p u r i f i c a t i o n and structural i d e n t i f i c a t i o n . This i s what we did at the American Cyanamid Research Center in Princeton, when we p u r i f i e d methyllycaconitine (an i n s e c t i c i d a l plant alkaloid active on insect n i c o t i n i c receptors) from Delphinium plants. As reported by Dr. Chalmers in the Tuesday afternoon session, we were able to employ the in v i t r o cholinergic receptor binding assay to follow the active material present in small quantities on TLC plates. Results were available in a matter of hours. This type of strategy could expedite p u r i f i c a t i o n s of active materials from other natural sources. Also, knowing the mode of action of the i n s e c t i c i d a l component at an early stage f a c i l i t a t e d our decision to purify the active material. v i t r
A. T. Eldefrawi: I would l i k e to bring attention to something else that in v i t r o binding assays can provide, and that i s a measure of target s e l e c t i v i t y . If we do the same kinds of assays with both insect and mammalian preparations we can learn what kind of structures within a large group of drugs we should study. Let me give you an example. If you look at the n i c o t i n i c acetylcholine receptor, you may find 30-40 drugs which have similar potency in vertebrates and invertebrates. However, there may be one or two to
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which the insect target i s much more s e n s i t i v e . This gives us a clue to zero in on for further investigation. This comparative i n v i t r o approach can be useful in detecting s e l e c t i v e compounds for development as i n s e c t i c i d e s . M. E. Schroeder: Another related area where basic research may have an impact on product discovery i s in the support of optimization once lead compounds are i d e n t i f i e d . In t h i s case the in v i t r o assay can t e l l the chemist i f the modifications that he i s making are increasing or decreasing the a c t i v i t y at the target s i t e . I think most chemists would l i k e to know whether changes i n i n s e c t i c i d a l a c t i v i t y of a series of compounds are due to changes in penetration or metabolism or due to changes in the inherent a c t i v i t y . S. M. Sieburth: I agree that in v i t r o assays should be a component of any optimization scheme. It i s also helpful to have a side-by-side comparison of vertebrate and invertebrate assays to give some clue as to target s i t e s e l e c t i v i t y even though we recognize that multiple factors w i l l ultimately determine the whole animal s e l e c t i v i t y . M. A. Brown: I think one of the key problems that we face in trying to discover a new i n s e c t i c i d e and that pharmacologists probably face to a lesser extent i s the problem of s e l e c t i v i t y . After compounds have been shown to be active at a particular target s i t e , the key problem frequently i s not to increase potency but to find proper s e l e c t i v i t y . I think i t i s f a i r to say that most commercially-available i n s e c t i c i d e s owe there s e l e c t i v i t y to s e l e c t i v e metabolism. This i s a feature of compounds that can only be i d e n t i f i e d , r e l i a b l y , in whole animal assays. A. E. Lund: One part of me l i k e s very much the idea of using i n v i t r o assays to hint at s e l e c t i v i t y between insects and mammals. However the problem that frequently arises i f one t r i e s to use such assays to guide synthesis i s a marked discomfort in making synthesis decisions based on these r e s u l t s . If one l i m i t s synthesis to an area of chemistry with target s i t e s e l e c t i v i t y , one i s haunted by the concern that the most selective compound in vivo i s one that i s less s e l e c t i v e on the target s i t e because i t possesses some t o x i c o k i n e t i c v u l n e r a b i l i t y i n mammals. On the other hand, compounds which are inactive on the mammalian target may ultimately be l e s s safe because of a deleterious action on some t o t a l l y unrelated system in the mammal. So the discrepancies between s e l e c t i v i t y measured in vivo and in v i t r o cloud our a b i l i t y to make synthesis decisions with much confidence. Perhaps i f one continually compares i n vivo and in v i t r o r e s u l t s with a group of compounds, you can minimize these e r r o r s . K. R. Jennings: I think t h i s i s an important point with a c o r o l l a r y that concerns target s e l e c t i o n . It i s not necessarily true that target s i t e s common to insects and mammals w i l l r e s u l t s in non-selective agents. Therefore i t i s unwise to r u l e out a l l targets which have been e v o l u t i o n a r i l y conserved. Again the example I w i l l give i s our amid ino hydra zone s which Dr. Hollingshaus working in our group has shown to i n h i b i t electron transport in
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mitochondria. This i s a highly conserved pathway across a l l l i v i n g organisms. So i f you were looking at t h i s process a_ p r i o r i , one might rule i t out as too conserved to allow for s e l e c t i v i t y and yet these compounds show very good s e l e c t i v i t y . So I think that while i t i s important to look at pharmacological differences between insects and mammals, we should keep our eyes open to the fact that there are other factors involved in determining s e l e c t i v i t y . R. Neumann: It seems to me somehow naive to talk about the insect or the mammalian receptor. I think the movement of companies away from screening houseflies to screening market-relevant insects indicates a widespread b e l i e f that target s i t e s and d e t o x i f i c a t i o n mechanisms d i f f e r widely between insect species. Therefore i t i s very d i f f i c u l t to relate conclusions about receptor systems i n cockroaches, locusts or some other model system to the economic " r e a l world". Unknown: When you assay compounds that are active in some in v i t r o system and they are inactive in vivo, how often do you assay those insects to determine i f the enzyme or receptor was, i n f a c t , affected in the whole animal? M. E. Schroeder: The answer i s very r a r e l y . In order to j u s t i f y the major research e f f o r t required to go after a biochemical target, one needs to determine the t o x i c o l o g i c a l consequence of i n h i b i t i n g that target. It i s one thing to i n h i b i t a GABA-chloride channel complex i n v i t r o and another to correlate that i n h i b i t i o n with i n vivo t o x i c i t y . I think that bridge must be made before you can gain r e a l confidence in your target. D. W. Gammon: This has been done though. The example of GABA transaminase I reported on yesterday i s a case where we could gain some insight into the r e l a t i v e s e n s i t i v i t y of t h i s enzyme as an i n s e c t i c i d e target, and allow a decision to be made on future synthesis. People have, of course, been doing these experiments for a long time trying to measure enzyme a c t i v i t i e s i n v i t r o and i n vivo. It i s often the only way to determine i f a given target s i t e i s important. In the case of receptor binding, one can use radiolabeled ligands to do similar experiments. I believe Jeff Lawrence i s in the audience; perhaps he would comment. L. J. Lawrence: We t r i e d for a while to demonstrate involvement o f GABA receptors i n deltamethrin poisoning i n r a t s by dosing the rats in vivo, removing the brain and assaying for the number of TBPS-binding s i t e s s t i l l available. We could never demonstrate complete i n h i b i t i o n of these s i t e s or i n h i b i t i o n to the extent that one would have expected from the i n v i t r o studies. We could show some i n h i b i t i o n , but i t s extent was too variable for detailed s t r u c t u r e - a c t i v i t y studies. I think that making the step from the in v i t r o to in vivo situation i s d i f f i c u l t due to various toxicokinetic complications. D. M. Soderlund: There i s one very simple and important barrier to the success of these experiments. Unless the target site i s covalently modified, you w i l l never be able to show a clear 1:1
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c o r r e l a t i o n between what happens during e q u i l i b r a t i o n of some dose at a target s i t e in vivo and subsequent i s o l a t i o n and preparation of some in v i t r o system. If you have a reversible insecticide-target i n t e r a c t i o n , the minute you begin preparing the tissue you start a l t e r i n g the d i s t r i b u t i o n of the toxicant by destroying the equilibrium with the receptor s i t e . unknown: Most of our commercial i n s e c t i c i d e s today are nerve poisons with r e l a t i v e l y quick k i l l . I think that i f you are familiar with some of the insect neurobiology conferences which have occurred over the past five years or so, there i s much more attention being given to insect neurohormones and neuromodulators — things which may not act l i k e neurotransmitters but have longer term effects on insect behavior and reproduction. I think these are areas which can be exploited to the detriment of the insect even though you do not see dead insects i n 24 hours. You may e f f e c t s u f f i c i e n t crop protection to r e a l i z e a yield increase at the end of your growing season. This has a much larger impact on how we look for a c t i v i t y of compounds, because most of our screens look for mortality in 24 or at most 72 hours. I think the b i o l o g i s t s and biochemists have to give feedback to the people doing the screens so that they can structure their evaluation program to pick up a c t i v i t i e s that are i n t e r f e r i n g with modulatory s i t e s that may give the same crop protection as an organophosphate or pyrethroid. R. M. Hollingworth: That i s a very important point. We seriously underestimate the impact on insect populations of compounds which have deleterious e f f e c t s on every l i f e stage of the insect but are not spectacular on any one of them. Current screens are not going to pick those up with any great f i d e l i t y . I was very interested to see Dr. Khowles l i f e table approach to the action of formamidines which impact the insect at every point from the egg, through the l a r v a , to the adults and their reproduction. Even a r e l a t i v e l y small e f f e c t at each l i f e stage can add up to excellent economic control of the o v e r a l l population, p a r t i c u l a r l y over more than one generation. Accepting t h i s approach and developing appropriate screens i s a degree of sophistication that we have not yet attained, but I hope we w i l l be able to use i t in the future. 1
R. Neumann: Shouldn't we discuss de novo design a l i t t l e ? I suspect that most i f not a l l chemicals that have been synthesized have been properly evaluated in vivo. Most of the basic research that i s done i s to just prove why compounds are active. What I think industry hopes to get out of basic research i s ideas on how to design new chemicals de novo. I wonder i f anyone would l i k e to comment on how the research that we have been hearing about the l a s t two and one-half days could contribute to de novo design. I must say that I am s t i l l somewhat surprised that new compounds have not been designed to attack targets that are so well known. There are at least a h a l f a dozen compounds that i n t e r f e r e with the sodium channel, but s t i l l we are completely unable to design anything de novo that can act on that target. Even in such a well known and researched system, our knowledge that can be exploited for de novo synthesis seems to be very l i m i t e d .
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R. M. Hollingworth: This i s a very promising area. In fact, progress i s being made. One area of great current promise i s the design of compounds to i n h i b i t photosystem II in plants. The plastaquinone binding s i t e has been defined by biochemical genetic and crystallographic techniques. The amino acid sequence and three dimensional structure are known, and t h i s information now i s being used to synthesize photosynthesis i n h i b i t o r s which are active and novel. I believe i t w i l l not be many more years, perhaps, u n t i l we are at t h i s point with the acetylcholine receptor. We already have a good deal of information from Torpedo electropiax on the molecular nature of the receptor. There are detailed models of the active s i t e . There i s some relevant information available on the associated ion channel as a second target area on the receptor complex. I think we have to keep i n mind, though, that you have to have t h i s very exact information in order to design compounds, and i t just i s n ' t there in most cases. But l e t ' s be optimistic and hopeful about i t for the future. Again, I think we w i l l suffer i f we i n s i s t on precedents and on seeing proofs of probable success before the f r u i t on t h i s particular tree has ripened. Α· E. Lund: I agree with you completely that there i s new information accumulating a l l the time that i s getting us closer to de novo design. Certainly the growing application of molecular genetic techniques to neurobiology promises to provide much of the needed information. I think, however, there i s something else one can do along these l i n e s that i s a l i t t l e more conventional and that puts us closer to design without waiting for molecular b i o l o g i s t s and x-ray crystallographers to hand us the t o t a l picture. It should be possible to study the chemistry of the interaction between small organic molecules and a protein target that can reveal certain physico-chemical c h a r a c t e r i s t i c s of the t a r g e t . We might then design molecules with the appropriate complementary c h a r a c t e r i s t i c s . The tools needed for t h i s research include two r e l i a b l e assays — one to measure the binding of a ligand to a s p e c i f i c s i t e on the protein, and a second to measure the functional consequence of that i n t e r a c t i o n . One also needs to construct a series of chemical probes with group s p e c i f i c reagents or a f f i n i t y l a b e l s at various locations around the known ligand. One then can begin to map out certain features of the topography o f the binding s i t e . With the aid of computer modeling to help the chemist v i s u a l i z e new p o s s i b i l i t i e s , t h i s new information allows the development of s t r u c t u r e - a c t i v i t y hypotheses that can be tested using a conventional SAR approach. This process can r e s u l t i n a p a r a l l e l development of iii v i t r o active compounds and a structure-topography which d r i f t s more and more into focus. The ultimate aim of t h i s approach, as I see i t , i s to step away from known chemistry and to design a molecule that has the c h a r a c t e r i s t i c s (overall shape, l i p o p h i l i c i t y , electronic configuration, etc.) needed to f i t the hypothesized s i t e but actually uses d i f f e r e n t chemistry. Perhaps I shouldn't c a l l t h i s process de novo design, since i t i s r e a l l y an extension of a conventional s t r u c t u r e - a c t i v i t y approach, but chemical design based on an important input of structural information on the target site i s c e r t a i n l y involved. RECEIVED
September 25, 1987
