Expert System Applications in Chemistry - American Chemical Society

Chapter 7

Drug Design Using a Protein Pseudoreceptor

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 27, 2015 | http://pubs.acs.org Publication Date: September 1, 1989 | doi: 10.1021/bk-1989-0408.ch007

F. A. Momany, R. Pitha, V. J . KHmkowski, and C. M . Venkatachalam Polygen Corporation, 200 Fifth Avenue, Waltham, MA 02254 The design of new pharmaceuticals depends in many cases on a detailed knowledge of the receptor pocket into which the drug binds. Structural data on receptors is seldom available, so a program was developed to computationally generate an artificial protein receptor, denoted as a "pseudo-receptor". The method uses decision algorithms to pick the residues to mutate such that a close fit around the substrate of interest is achieved. Energy minimization and molecular dynamics calculations are used to optimize the fit of the pseudo-receptor about the substrate. A n example problem is described using a hepta-peptide from the carboxyl terminal of C C K , as well as a benzodiazepine molecule which acts on the same receptor, as the substrates of interest. The method of 'receptor fit* is an important concept for the working drug design scientist. This method recognizes that the fit between drug and receptor is one of steric recognition, electrostatic complementarity, and matching of the nonpolar regions of the drug to the receptor. However, few useful methods of drug design exist which report quantitative protein-ligand interaction energies, much less give a complete description of the three dimensional structures and flexibility of the receptor or ligand necessary for binding. Several qualitative and semiquantitative procedures such as those denoted QSAR(Quantitative Structure Activity Relationships), are used in drug design, but it is difficult to see how these linear methods, which correlate the binding energy of the 0097-6156/89/0408-0082S06.00/0 o 1989 American Chemical Society

In Expert System Applications in Chemistry; Hohne, Bruce A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7. MOMANYETAL.


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ligand w i t h different p h y s i c o c h e m i c a l properties for different parts o f the l i g a n d , w i l l w o r k w e l l f o r complex flexible biological ligands. T o date, the only truly accurate description of b i n d i n g sites are f o r those systems i n w h i c h the X - r a y d i f f r a c t i o n structure o f the protein/enzyme has been determ i n e d ; there the method of binding of the substrate can be studied i n great detail. Unfortunately, the e x p l i c i t structure of receptors is i n almost a l l cases unknown. In this paper we develop a new method for finding the threed i m e n s i o n a l space that surrounds a s u b s t r a t e / l i g a n d . T h i s space, w h i c h is the c h e m i c a l equivalent o f the receptor, is represented as a p r o t e i n structure, referred herein as a "pseudo-receptor". A variety of computational tools are used to create the pseudo-receptor. A m o l e c u l a r mechanics and dynamics program, C H A R M m ( l ) , is used to calculate the energy and c o n f o r m a t i o n a l features o f the pseudo-receptor. T h e program Q U A N T A ( l ) is used to define the preliminary protein sequence, secondary structure, g r a p h i c a l l y examine molecular interactions, interface with C H A R M m , and model a m i n o - a c i d mutations i n the protein sequence. In the study reported here, the protein pseudo-receptor is first constructed as a polyalanine chain, and folded to form a pocket into which the substrate is fitted. The polyalanine chain is next mutated r e s i d u e by r e s i d u e to r e d e s i g n the s i d e - c h a i n interactions with the substrate. T h e electrostatic potential map on the substrate is used to show where the complementary charges on the protein must occur, and probe maps using hydrophobic probes show the nonpolar regions w h i c h must be i n c l u d e d by adding h y d r o p h o b i c residues i n contact w i t h specific regions of the substrate. The methodology is similar to that used to d e s i g n artificial enzymes using synthetic precursors as a framework for adding functional groups. In the pseudo-receptor m o d e l , the framework is a polypeptide and the mutations a d d the b i n d i n g stereochemistry and functionality. Future studies w i l l show how to automate the selection of residues for ideal fits about the substrate. Here we w i l l show how choices can be made for residue selections using computer graphics w h i l e e m p l o y i n g mutations and energy searching to optimize interatomic interactions.


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Materials

and Methods

The i n i t i a l c o n f o r m a t i o n of a 100 residue p o l y p e p t i d e o f polyalanine was obtained by folding the first 30 residues and last 30 residues as helices; the middle 40 residue were folded into a beta-sheet such that both helices were located on the same side of the sheet structure formed as shown i n Figure 1. The p r e l i m i n a r y structure was passed through l i m i t e d energy m i n i m i z a t i o n to remove serious contacts and appropriate alanine residues were mutated into g l y c i n e at the structural points where appropriate bends occured and where g l y c i n e could remove some strain energy. The substrates studied i n c l u d e the carboxy terminal seven residues of the gastrointestinal peptide hormone cholecystok i n i n , denoted C C K 7 , with sequence; NH2-Tyr-Met-Gly-Trp-Met-Asp-Phe-COOH, and a benzodiazepine d e r i v a t i v e ; R - 3 - ( 3 - i n d o l y l - m e t h y l ) - 5 phenyl-benzodiazepine(2),(see figure 2), w h i c h has been found to block peripheral C C K receptors. T h e l o w energy conformations of C C K 7 were studied using the B o l t z m a n J u m p conformational search method described elsewhere (3) using E C E P P 8 3 parameters (4), starting from l o w energy structures published previously (5). N e w very l o w energy structures of C C K 7 were found, which when compared graphically to the l o w energy structure of the benzodiazepine derivative using least squares f i t t i n g procedures, showed c o n s i d e r a b l e structural homology as shown i n Figure 2. The C C K 7 structure was next placed i n a bath of 81 T I P S 3 water molecules and again energy was m i n i m i z e d using the C H A R M m parameters (version 21) to ascertain its stability i n water. The resulting conformation of C C K 7 changed very little from the calculated conformation shown i n Figure 1. The C C K 7 structure was placed into the polyalanine molecule pocket such that the polar residues pointed outward toward the solvent, and the nonpolar residues pointed toward the interior, as shown i n F i g u r e 3. The mutation of the p o l y alanine was carried out using graphics to find those residues of alanine w h i c h pointed the s i d e - c h a i n m e t h y l group toward some part of the substrate molecule. F o r example, i f a sidechain pointed toward a p h e n y l group on the substrate, then that alanine was mutated using the protein contact rules (6,7)



MOMANY ET AL.


F i g u r e 1. R i b b o n structure of 100 residue p o l y a l a n i n e folded into a helix-betasheet-helix conformation to f o r m an initial pocket for C C K 7 binding.




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B Figure 2. Stick drawing of low energy structure of (A) geometry optimized S-Chloro-O'-IndolinylmethyO-SPhenyl-l,4-benzodiazepine and (B) the low energy C C K 7 conformation to show configurational similarities. Some hydrogens have been omitted from C C K 7 for ease of viewing.



MOMANY ET AL.


Figure 3. Structure of polyalanine after insertion of C C K 7 into pocket, after bending helices to enclose the pocket, and after preliminary energy m i n i m i z a t i o n .



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to test for optimum interaction. In other words, i f the contact rules suggested that a phenyl group should occur around that particular position of the substrate, then the mutation was to place a phenylalanine at that position on the polyalanine chain. S i m i l a r l y , i f a hydrogen bonding donor was required to interact with an acceptor from the substrate, then that type of mutation was made. A t each point of mutation, several different sidechain conformations were examined and the best interacting configuration was retained. If a suitable s i d e - c h a i n c o n f o r mation could not be found w i t h a reasonable binding energy and without high energy, then that mutation was discarded and a secondary choice i n residue was examined. The final mutated and energy m i n i m i z e d pseudo-receptor with the C C K 7 located inside the pocket is shown i n Figure 4. T a k i n g the resulting complex, the surface of the substrate exposed to the solvent region is covered with water and energy m i n i m i z e d . Further, after removal of the C C K 7 from the pseudo-receptor, the space previously occupied i n the now empty protein pocket is filled w i t h water molecules and energy m i n i m i z e d to obtain the energy required to remove the appropriate amount of water from the solvated receptor. Entropic contributions to the net free energy are calculated using the Einstein relationships. Results Packing around the C C K 7 T r p residue includes a T i p at position 54 i n the pseudo-receptor, Phe at position 15, He at 4 7 , and L e u at 59. The region about the Phe residue of C C K 7 includes L e u 59 and G i n 85. The A s p residue of C C K 7 forms a hydrogen bond interaction with the backbone amide, and Phe 15 forms a pocket for the T y r of C C K 7 . The packing is very compact with no holes or cavities inside the pseudo-receptor. T h e mutation effort was very efficient i n finding close contacts between a l l b i n d i n g residues and the energetics of the f u l l flexible geometry o p t i m i z a t i o n method a l l o w e d both substrate and pseudo-receptor to flex to f i t compactly and f o r m a tight binding interface. The enthalpic and entropy contributions to the b i n d i n g and stability of the pseudo-receptor and substrates w i l l be described i n more detail elsewhere. However, it is important to note that one must obtain the net free energy of the binding


MOMANY ET AL.



V a l 40

Figure 4. Configuration of sidechains of mutated pseudoreceptor w i t h C C K 7 . Residues of the pseudo-receptor w h i c h have not been mutated have been removed for v i e w i n g clarity.



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process i n order to compare these calculated values to the experimental b i n d i n g constants. T h i s requires obtaining the internal enthalpy of the substrate, protein c o m p l e x , and the solvation energy about the substrate and inside the receptor pocket i n order to get the energy required to strip off the correct amount of water from both substrate and receptor. Further, one must examine the entropic contributions to the free energy by f i n d i n g the i n t e r n a l fluctuations and translational and rotational motions of solvent, substrate and receptor when imbedded i n solvent prior to and after binding of the substrate. These values are not easy to obtain and small net free energy differences between large enthalpy energies have to be precisely calculated. Several methods for obtaining these energies are being tested, i n c l u d i n g c a r r y i n g out a l l calculations w i t h i n a box of water and carrying out dynamic simulations at room temperature for extended periods of time to achieve an e q u i l i b r i u m situation for calculation of entropy terms. T h e benzodiazepine structure was inserted into the pseudoreceptor pocket upon removal of the C C K 7 and a very good fit was achieved without modification to the pseudo-receptor. The fit of the benzodiazepine is also stereospecific, binding only i n one position and not fitting i n any other configuration. Clearly, the method o f o p t i m i z i n g the p r o t e i n structure about a substrate is capable of finding a binding site w h i c h can then be further o p t i m i z e d by adding different types o f substrates, either from sets of different agonists or from antagonists which are k n o w n to bind s i m i l a r l y , and c y c l i n g through the mutation and energetic m i n i m i z a t i o n cycles until a l l the test data is fitted. W e call this the learning cycle for the pseudo-receptor, and one can find optimal interactions for as large a data base as is deemed necessary. Once the final receptor pocket is found one can then use this to q u i c k l y screen further analogs of new structural types for binding. Conclusions A n extension to the pseudo-receptor approach is to utilize the thermodynamic perturbation method (8) to calculate approximate values for the free energy o f b i n d i n g of v a r i o u s substrates to the pseudo-receptor.



7. MOMANYETAL.


A second consequence of the pseudo-receptor approach to drug design is to consider the pseudo-receptor as the drug molecule of interest. This protein could then be synthesized and used to experimentally test for b i n d i n g to selected substrates and to subsequently be used as the drug itself. This problem w o u l d r e q u i r e a d d i t i o n a l structural m o d i f i c a t i o n to s t a b i l i z e the protein structure i n the form found to best bind the model substrate. Complete hydration models of the pseudo-receptor with substrate can be used to test for solvent stability i n these studies.

Literature

Cited

1. Q U A N T A and C H A R M m are trademarks of Polygen Corporation, Waltham, M A . 2. Evans, B.E., Rittle, K.E., Bock, M.G., DiPardo, R . M . , Freidinger, R.M.,Whitter, W.L., Gould, N.P., Lundell, G.F., Homnick, C.F., Veber, D.F., Anderson, P.S., Chang, R.S.L., Lotti, V.J., Cerino, D.J., Chen,T.B., King, P.J., Kunkel, K . A . , Springer, J.P., and Hirshfield, J., J. Med. Chem., 1987, 30, 1229. 3. Momany, F.A., Klimkowski,V.J., Pitha, R. and Venkatachalam, C.M.; "Molecular Design using Supercomputers", Video Presentation, San Diego State University and San Diego Supercomputer Center, April 20, 1988. 4. Chuman, H . , Momany, F.A., and Schafer, L . , Int. J. Peptide Protein Res. 1984, 24, 233. 5. Pincus, M.R., Carty, R.P., Chen, J., Lubowsky, J., Avitable, M . , Shaw, D., Scheraga, H.A., and Murphy, R.B., Proc. Natl. Acad. Sci. USA,1987. 84, 4821. 6. Roberts, D.C. and Bohacek, R.S., Intl. J. Pept. Prot. Res. ,1983. 21, 491. 7. Wako, H . and Scheraga, H . A . , J. Protein Chem, 1982, 1, 5. 8. Mezei, M and Beveridge, D . L . , Ann. N . Y . Acad. Sci., 1986, 482, 1. RECEIVED June 9, 1989


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Expert System Applications in Chemistry - American Chemical Society

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