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PROTEINS: Structure, Function, and Genetics 1129- 34 (1991) Fun ctionality Ma ps of Binding Sites: A Multiple Copy Simu ltaneous Search Met ho d Andrew Miranker and Martin Karpl us Committee on Higher Degrees in Biophysics and Department o f Chemistry, Harvard University, Cambridge, Massachusetts 02138 ABSTRACT A new method is proposed f o r determining energetically favorable positions an d orientations for functional groups on the surface of proteins with known three-dimen- sional structure. From 1,000 to 5,000 copies of a functional group ar e randomly placed in the site an d subjected to simultaneous energy min- imization and/or quenched molecular dynam- ics. The resulting functionality maps of a pro- tein receptor site, which can take account of its flexibility, can be used fo r th e analysis of pro- tein ligand interactions an d rational drug de- sign. Application of the method to the sialic acid binding site of the influenza coat protein, hemagglutinin, yields functional group minima that correspond with those of the ligand in a cocrystal structure. Key words: drugdesign, ligand-binding, he- magglutinin, functional groups, MCSS INTRODUCTION The design o f ligands tha t bind strongly to key regions of biologically important molecules (e.g., en- zyme active sites, receptor proteins) so as to inhibit or alter their activity is th e essence of drug design. Rational approaches to this problem are still limited.' In t hi s report, we p ropos e a method based on energy minimizatiodquenched molecular dy- namics for the determination of energetically favor- able positions o f functional groups in t he binding sites of proteins with known three-dimensional struct ure. The method can aid in t he design o f mol- ecules that incorporate such functional groups by modification of known ligands or de nouo construc- tion. To determine and characterize the local minima o f a functio nal grou p in t he force field o f a protein, mult iple copies o f selected functional group s are irst distributed in the binding site of interest. En- ergy minimization of these copies by molecular me- chanics o r quenched dyna mics yields the distinct lo- cal minima. The neighborhood of these minima can then be explored by a grid search or by constrained minimization. An essential element o f th e approach is the classical time dependent Hartree (TDH) approximation,' which can be used to simulta- 0 1991 WILEY-LISS, INC. neously minimize or quench many identical groups in the force field of a single protein or of a protein with multiple cop ies o f important residues. The pro- posed method provides a useful supplement to more standard visual (graphical) approaches3 and grid search techniques.*~~ METHOD Implementation of the multiple copy simulta- neous search method (MCSS) requires a choice of functional groups and a molecular mechanics model for them. Groups must be simple enough to be easily characterized and manipulated (3-6 atoms, few or no dihedral degrees of freedom), yet comple x enough to approximate the steric and electrostatic interac- tions that the functional group would have in bind- ing to the site o f interes t. An appropriate set is one in which most organic molecules can be described as a col lection o f such group^.^ This includes fragments such as acetonitrile, methanol, acetate, methyl am- monium, dimethyl ether, methane, and acetalde- h~de.~ thers have used functionality descriptions with fewer at oms: or a spherical approximation to a multi-atom An application o f such an atom- based grid search t o th e docking o f molecules b y sim- ulated annealing has been described re~ently.~ Whereas energetically favorable positions for these smaller fragments are simpler to calculate , such po- sitions ar e likely to be less useful be cause they may be sterically unattaina ble for a larger ligand. More - over , in th e design o f ligands it is important t o have orientational, as well as positional, information about possibl e functional group binding sites. In the present application, parameters for the functional groups were chosen fr om the polar hydro- gen se t o f CHARM M 20, augmented a s necessary b y information from ab initio calculations. This set tr eat s aliphatic hydrogens as par t of the "extended" carbon atom t o which they a re bonde d; polar hydro- gens are treated explicitly. A 7.5A shifted non- bonded cu toff and a con stant dielectric o f one were used. The exact choice (e.g., distance dependant di- ReceivedNovember 28,1990; evision accepted February 19 , 1991. Addre ss reprint re quests to Dr. Martin Karplus, Department o f Chemist ry, Har vard Unive rsity, 12 Oxford St., Cambridge, MA 02138.
