A Procedure for Preparing Models of Receptor Sites D. Eric Waiters,' Robert A. P e a r l ~ t e i n and , ~ C. Peter Krimmel New Sweetener Research Group and Department of Medicinal Chemistry Searle Research and Development, 4901 Searle Parkway, Skokie, IL 60077
a
Chemists have a t their disposal wide variety of models for representine chemical structures. However. there has not been-a satisfactory way of preparing models of receptor (binding) sites except when the receptor is a macromolecule that has been isolated, characterized, and studied by X-ray crystallography. When receptor site topography has been inferred on the basis of structure-activity data, the results have sometimes been represented as two-dimensional "cartoon"-type sites (Figs. 1;4, and 6, adapted from refs. 1 5 ,are examples). I t is often difficult to represent irregular threedimensional surfaces adequately with such pi&res. This report describes a straightforward procedure for constructing three-dimensional models of receptor sites using spacefilling models of active compounds as templates. The purposes of modeling receptor sites are (1)to rationalize observed structure-activity data and (2) t o allow predictions about the fit of potential new compounds in advance of their synthesis. Two-dimensional representations are a first step in this direction, but they are often not sufficiently detailed to be useful in a predictive manner. Materials and Methods
The receptor models may be constructed from materials that can lw~shaprdand forked around space-filling models of appropriate compounds. Plastics such as I'olyform'* (purchasrd from Rolvan Mfx. CO..P.O. Uox 555.. Menomonee Falls, WI 53051jare rigrd a t ;oom temperature, but when heated to 70 'C thev become flexible. These olastics are routinely used in orthopedic medicine for constructing splints and supports. A sheet of the plasticcan he heated in a water bath fo& few minutes and thkn shaped around spacefilling models. The plastic is easily cut with scissors while i t is warm. Modification of the model may be done by heating small regions of the plastic in hot water or with a heat gun. Extra pieces of plastic can he added by heating the edges to he joined or with solvent-based glue. An alternative material for model building is heavy-duty aluminum foil. While aluminum foil models are more fragile, they are substantially less expensive. We have found it useful to increase the rigidity of the foil by laminating several thicknesses together with a spray adhesive of the type used in photo mounting. I t is also possihle to laminate a thinlayer of paper onto the aluminum, to provide a surface that can be marked upon easily. Receptor models may be built by forming the modeling material around space-filling molecular models such as Corey-Pauling-Koltun (CPK) models. Space-filling molecular models of active compounds can he placed in calculated or experimentally determined conformations, or conformationally restricted analogs may he used. The receptor model is made by forming the modeling material around the spacefilling model. In addition to providing a model of theshape of thereceptor site, such a model can be used to locate possible binding sites such as hydrogen bond donors and acceptors, ionic groups, charge-transfer sites, and hydrophobic regions. ~~
These sites may be mapped onto the model using markers, paints, or other means. Discusdon. I t is usually not sufficient to build the model around a single active compound; the fit of a range of compounds should be examined. In this way, the model can be modified such that it can accommodate all active com~oundsin a series. In addition, it should exclude inactive compounds (eitheron the basis of bad steric tit or because of unfavorable interactions with postulated binding sites). The implicit assumption in this approach is that all compounds tested are acting a t the same-site; if this were not the case, the model produced would be of little value. The usefulness of these kinds of models can be demonstrated in a comparison of several published models of the receptor site for aspartic acid-based sweeteners such as aspartame (1,ref. 6). Figures 1,4, a n d 6 show three published models of this receptor site. Most models proposed for this receptor assume three major features of the molecule are involved in binding of these comoounds to their recentor: the ionic carboxylaie and amino Goups of the aspartic;esidue, and hydrophobic group attached to the aspartic acid.
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' Author to whom CorresDondenceshould be addressed,
* Present address: Collegeof ~harmacy.The University of lllinoisat
Chicago. Chicago, IL 60612.
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Figure 1. Sweet recepta madel proposed by Temussl and ca-wwkers (1-3. Figure 4. Sweet receptor model proposed by van der Heijden and co-workers
(4).
Figure 2. Threedimensional sweet receptor Model 1, based on the model proposed by Temussi and co-workers(1-3). showingthe fit of aCPK model of aspaname (I).
Figure 3. Threedimensional sweet receptor Model 1, as described for Figure 2, using a skeletal model of aspartame (1) to facilitate viewing the fit of the back face of lhe molecule into the receptor site.
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The receptor site proposed by Temussi and co-workers (I3) is shown in Figure 1. This model, based on a proposed "active conformation" of aspartame (I), has some threedimensional character, and i t attempts to portray the size limitations of both the large hydrophobic group and the ester group of aspartame. Positively charged and negatively charged binding sites were postulated to bind to the carboxylate and amino groups, respedivelv, of asoartame. Fieure 2 shn,ws the same s?tr (;'iewed from tl;e oppo&re fare) acmodeled in thermoplastic (Model 1). with a C P K model of a s ~ a r tame (1) in the site. The carboxylate group (lower right) is seen to be near the positively charged binding site, and the protonnted a m i n o g i u p (lower left; is nrxt tothe negatively charged hindingsite. The hydrophobic region is at the top of the figure. In E'igure3, rhesame rereptor site model is shown with a skeletal model of aspartame. Framework Molecular Models. I'rentire-Hall. were cur to the same scale as CPK - -models: 1.25 cm = 1A. The use of skeletal models permits viewing of the fit of the underside of the molecule to the receptm site. In this case, i t is possible to view the pocket into which themethvl ester fits (center of firmre): there is not sufficient room in this pocket to accomod&e the less sweet ethyl ester. Model 2, illustrated in Figure 4, was suggested by van der Heijden and co-workers (4). I t was built around a different conformation of aspartame (based on a reinterpretation of the experimental and computational data of ref. I ) . This model also attempts to illustrate the spatial relationships among the three functional groups thought to be required for sweetness. A thermoplastic model of this site is shown in Figure 5 (cut away to show the fit of the aspartame molecule). Again, complementary charged binding sites have been mapped onto the model. The hydrophohic site in this model is a t the upper left of the figure, and the methyl ester pocket is a t the upper right. The third model (Fig. 6), showing yet another conformation of aspartame-type sweeteners, was proposed by Iwamura (5) on the basis of a quantitative structure-activity study of more than 200 aspartic acid-derived sweeteners. This study indicated an important role for the amide NH group in receptor binding. I&unura's model focused primarilv on the size and shape of the hydrophohic hinding site, using Verloop's Sterimul steric parameters (7). The conformation illustrared for the aspartic acid portion of the moleshown in Fieure 7. cule was rhosen arbitrarih. Model :%. encompasses the hydrophobic end of the binding site; it was built on the basis of the dimensions found to be optimal in Iwamura's QSAR study. The dimension labeled "W,," in Figure 6 appears a t the lower front portion of the model in
r gar? 7 Tnree-a men9 ona suet.! receptor Model 3, oasea on tne mooel p'(lP05e-Y by uanbra (3, show ng the fil of a CPK m e 1 of aspaname ( I ) Figure 5. Threedimensional sweet receptor Model 2, based on me modei proposed by van der Heijden and co-workers (4). showing the fit of a CPK model of aspartame (1). The front face of the receptor rncdel was cut away to show the fit of the aspartame molecule.
Fltaof Molecular Models of Sweet Compounds (1-4) and NonSweet Compounds (5-7) Into Receptor Slte Models 1-3 Model 1
Model2
Model 3
1 2 3 4
ti
flt
no ti
no fit no fit fit
fit fit fit fit
5 6 7
no fit no fit
no fit no fit fit
fit fit fit
Compound Sweet
Non-sweet
fit fit
no fit
shows the results for the fitof the seven compounds into the three-dimensional models. Not surprisingly, aspartame (1) fits readily into all three models. However, only Model 3 has a hydrophobic site large enough to accommodate the fenchyl ester arouo of the intenselv sweet comoound 2. The aromatic groupof the anilidp 3 cannot uccupy t'he hydrophobic site of .Model 2 without icriousls distortinr" the amidc hond. Among the non-sweet analogs, only the first model correctly excludes all three compounds. I t can be seen that each of these models has its shortcomings when tested three-dimensionally with a series of compounds. The use of these physical models facilitates the process of model development and refinement. Conclusion
I t is evident from these three receptor models that tbreedimensional lieand-receotor interactions cannot be adequately or completely represented in two dimensions. Thermoplastic or aluminum foil models oermit readv visualizarion of these three-dimensional interactions. ~l;ese modpls ulsoofier the particular ndvanrare of allowinr: one to test the fit of designed molecules prior t i their synthisis, so that the most promising synthetic targets can be identified.
Figure 6. Sweet receptor modei proposed by iwamura (5).
Figure 7; "W,z"is a t the upper right, and "WU? is on the rear face of the model. These three models were evaluated usine CPK models of ~four sweet compounds (1-4) and three non-sweet analogs (57). D-Tryptophan (4) was included with the aspartic acidderived sweeteners on the basis of Schiffman's study (a), which showed that aspartame and D-tryptophan cross-adapt and, therefore, may occupy the same receptors. The table ~
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Acknowledgment
The authors thank Gail Crutcher, Glenda Rose, Gertrude Smith, Linda Walters, and members of the New Sweetener Research Group for materials and helpful discussions. Literature Cned Letj, F.: ~ancredi.~.; ~ e m w iP. , A ; ~ ~ n i o t o J. , ' ~~ .r n e rchm. . sac. 1976.98.6669, ( 2 ) Temus8i.P.A.: Lelj,F.;Tancredi,T. J.Med.Chrm. 1978,21,1151 (1)
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(3) Temussi, P. A.; Leij, F.: Tancrodi, T.;Csatiglione Moreui. M. A.; P a a m . A. Inf.J. Quan~umChem. 1984.26.88s. ( 4 ) van der Heijden, A,; Brulisei, L. B. P.; Peer,H. G. Fond Chem. 1918.3, m. ( 5 ) Iwsmura. H.J. Mod. Chem. 1981,24.572.
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(6) Mazur,R. H.: Sehlatter,J.M.;Goldkamp,A. H.J.Amer. Chern.Soe. 1969.91.26e4 (7) Verlmp. A.: Hwgenstraaten, W.: Tipker, J.Drug Design 1976,7,165. (8) Schiffman, S. S.; C a b , H.: Lindley, M. G. Phormocol. Blocham. Behouior 1981, 15, 377.
