Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-10
2003-07-29
Zitomer, Stephanie W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S501000, C436S512000, C436S517000, C436S002000, C436S086000, C436S144000, C436S173000
Reexamination Certificate
active
06599707
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention relates to methods of identifying hot-spot residues of a member of a receptor-ligand binding pair of interest. The invention further provides methods of using information about receptor hot-spots for a receptor-ligand pair of interest to guide the identification of compounds that functionally bind to such regions of the receptor in a manner that mimics the ligand.
2. BACKGROUND OF THE INVENTION
Protein-protein interactions such as those observed in many receptor-ligand complexes are typically mediated by large binding surfaces comprising ten to thirty contact amino acid residues on each protein of the complex (Clackson & Wells, 1995, Science 267:383-386). Recently, it has been suggested for some binding pairs that within each binding surface, the predominant contribution to the overall free energy of binding of the complex is due to only a few residues within each binding surface. (Clackson & Wells, 1995, supra).
For example, recent studies by Wells and collaborators have characterized the binding surfaces of the complex formed between human growth hormone and its cellular receptor. (Clackson & Wells, 1995, supra; Pearce et al., 1996, Biochemistry 35:10300-10307; Wells, 1996, Proc. Natl. Acad. Sci. USA 93:1-6). X-ray crystallographic studies had shown that approximately thirty amino acid side chains of the hormone contact approximately thirty amino acid side chains of the receptor. An array of mutant human growth hormone proteins and mutant receptor proteins were prepared to estimate the energetic contribution of each amino acid of the contact surface to the overall binding interaction. The study measured the binding affinities of various complexes between mutant hormones and native receptors and also between native hormones and mutant receptors.
Surprisingly, only a small number of amino acid residues within the extensive binding surfaces accounted for most of the binding energy. Overall, fewer than half of the contact residues contributed measurably to the binding interaction. Eight receptor residues and eleven hormone residues accounted for over 75% of the free energy of binding of the hormone-receptor complex. Within each group of residues on either binding pair, a limited number of “hot-spot” amino acids contributed most to the total free energy of binding. Surrounding each “hot-spot” was a group of several amino acids that contributed at lesser levels, and this group was in turn followed by a larger set of residues that contributed at even lower levels to the total free energy of binding. Most significantly, the “hot-spot” residues on the receptor binding surface directly contacted the complementary “hot-spot” amino acids on the hormone's binding surface. Subsequent studies in other systems have also shown that the total binding affinity between a receptor and a ligand is mediated mainly by small and complementary sets of “hot-spot” residues within the binding surfaces of the receptor and ligand (Wells, 1996, Science 273:449-450; Smith-Gill, 1994, Res. Immunol. 145:67-70, 1994). Some systems even suggest that multiple, non-contiguous “hot-spots” are possible.
While the methods of Wells and others can provide detailed information about residues involved in binding interactions for a variety of receptor-ligand pairs, they suffer from serious drawbacks. For example for a given receptor-binding protein pair, large panels of mutant proteins must be prepared in order to identify the thermodynamically significant or “hot-spot” residues. To do so, each residue of the receptor or ligand must be mutated individually to measure its contribution to the overall free energy of binding.
Ideally, when a three-dimensional structure of the receptor-ligand complex is available, the mutations can be limited to only those amino acids comprising the binding surfaces of each member of the pair, approximately thirty residues for each number in a typical interaction. Thus, even in such an “ideal” situation, where information about both the receptor and ligand is desired, a total of about sixty mutant proteins must be prepared (one for each residue in the binding surface of the receptor and one for each residue in the binding surface of the ligand), and the binding energy of each mutant protein for its native binding partner determined.
In a more typical situation where no three-dimensional structure is available for either binding partner, mutants must in principle be prepared for every residue of both proteins. Thus, even in an ideal situation the methods are labor-intensive and expensive. In a less than ideal situation, the time and expense of preparing the mutants and measuring binding affinities oftentimes would be comparable to that for obtaining a three-dimensional structure of the complex.
In addition, the methods do not necessarily accurately identify the hot-spot residues. Site-directed mutagenesis of individual amino acids does not simply remove an amino acid side chain from a protein. Often, mutation of a residue to, for example, an alanine, introduces local and perhaps global structural perturbations in the mutant protein. These structural changes may affect the binding interactions between the mutant protein and its ligand. As a consequence, a comparison of the binding affinities of the mutant and native proteins is unlikely to be an accurate measurement of the relative contribution that residue makes to the overall free-energy of binding. In fact, in the studies of Clackson & Wells, the sum of the apparent free energy contributions of the mutated amino acids exceeded the known free energy of binding of the native receptor-native ligand complex by a factor of two (see, Clackson & Wells, 1995, supra). Clackson & Wells concluded that the excess measured binding energy was due to mutant-induced structural changes.
Thus, it would be highly desirable to have available methods for identifying hot-spot amino acid residues for receptor-ligand pairs which do not suffer from the above-described limitations. In particular, it would be highly desirable to have available methods for identifying receptor-ligand hot-spot residues which are fast and inexpensive, and which further permit the identification of hot-spot residues for a native receptor-ligand pair without having to mutate either the receptor or ligand.
Small compounds that bind to, and thereby antagonize (antagonists) or activate (agonists) receptors of therapeutic importance are of considerable commercial value. However, to date the ability to rapidly and easily identify such small compounds, particularly those that are able to interact with protein receptors that have large polypeptide or protein ligands, is limited. In most instances, such compounds are obtained through the laborious process of rational drug design, which usually requires detailed knowledge about the three-dimensional structure of the receptor-ligand co-complex or other structure-function information. Rational drug design generally involves designing compounds based on available structure-function information on a compound-by-compound basis and screening the individual compounds in biological assays to identify those compounds which produce a desired biological activity. Usually, detailed structure-function or three-dimensional structural information for the receptor-compound complex is obtained so that the design hypotheses can be verified.
Typically, the detailed structure-function information necessary to design and assess the compounds is obtained from NMR or x-ray crystallographic studies with the receptor-ligand co-complex. Both of these technologies require large quantities of pure, co-complex, expensive, specialized equipment and highly skilled technicians, making such structural information extremely costly and time consuming to obtain. In addition, while the detailed structural information obtained from these methods can oftentimes identify those residues of each member of the binding pair that are involved in the binding interaction, neither of these methods have been used to identify the individual res
ExSAR Corporation
Pennie & Edmonds LLP
Zitomer Stephanie W.
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