Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-30
2003-09-09
Wessendorf, T. D. (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S005000, C435S004000, C435S091500, C435S091500, C435S091500, C435S091500, C530S350000, C530S324000, C530S325000, C530S330000
Reexamination Certificate
active
06617114
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to methods of identifying drugs which can mediate the biological activity of a target protein.
Protein Binding and Biological Activity
Many of the biological activities of the proteins are attributable to their ability to bind specifically to one or more binding partners (ligands), which may themselves be proteins, or other biomolecules.
When the binding partner of a protein is known, it is relatively straightforward to study how the interaction of the binding protein and its binding partner affects biological activity. Moreover, one may screen compounds for the ability of the compound to competitively inhibit the formation of the complex, or to dissociate an already formed complex. Such inhibitors are likely to affect the biological activity of the protein, at least if they can be delivered in vivo to the site of the interaction.
If the binding protein is a receptor, and the binding partner an effector of the biological activity, then the inhibitor will antagonize the biological activity. If the binding partner is one which, through binding, blocks a biological activity, then an inhibitor of that interaction will, in effect, be an agonist.
The residues whose functional groups participate in the ligand-binding interactions together form the ligand binding site, or paratope, of the protein. Similarly, the functional groups of the ligand which participate in these interactions together form the epitope of the ligand.
In the case of a protein, the binding sites are typically relatively small surface patches. The binding characteristics of the protein may often be altered by local modifications at these sites, without denaturing the protein.
While it is possible for a chemical reaction to occur between a functional group on a protein and one on a ligand, resulting in a covalent bond, protein ligand binding normally occurs as a result of the aggregate effects of several noncovalent interactions. Electrostatic interactions include salt bridges, hydrogen bonds, and van der Waals forces.
What is called the hydrophobic interaction is actually the absence of hydrogen bonding between nonpolar groups and water, rather than a favorable interaction between the nonpolar groups themselves. Hydrophobic interactions are important in stabilizing the conformation of a protein and thus indirectly affect ligand binding, although hydrophobic residues are usually buried and thus not part of the binding site.
Peptides have been found to bind proteins at the same sites as those by which the proteins interact with other proteins, macromolecules and biologically significant substances e.g. nucleic acids, lipids and enzyme substrates. The first examples of this property were in the publications of several groups who showed that there is a single peptide binding site on the biotin binding protein streptavidin. This is the same site responsible for biotin binding and these peptides compete with biotin for binding to this site (Biochemistry 34: 15430-15435 (1995) Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities, L. B. Giebel, R. T. Cass, D. L. Milligan, D. C. Young, R. Arze & C. R. Johnson; Gene 128: 59-65 (1993) An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets, B. K. Kay, N. B. Adey, Y. S. He, J. P. Manfredi, A. H. Mataragnon & D. M. Fowlkes; Nature 354: 82-4 (1991) A new type of synthetic peptide library for identifying ligand-binding activity Septou, et al.; Proc Natl Accad Sci USA 92: 5426-5430 (1995) Library of libraries: approach to synthetic combinatorial library design and screening of “pharmacophore” motifs, I. Saggio and R. Laufer; Biochem J 293 (Pt 3): 613-6 (1993) Biotin binders selected from a random peptide library expressed on phage, I. Saggio & R. Laufer). Many other examples exist, for instance Smith demonstrated that peptides displayed on phage which bound to ribonuclease S had a specific consensus motif and that these PLs were antagonistic to ribonuclease activity, implying that the peptides and the RNA were bound by the same ligand binding site (Gene 128: 37-42 (1993) A ribonuclease S-peptide antagonist discovered with a bacteriophage display library, G. P. Smith, D. A. Schultz & J. E. Ladbury). Another example is from the binding of peptide ligands to cell surface integrins (Biochemistry 34: 3948-3955 (1995) Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries, J. M. Healy, O. Murayama, T. Maeda, K. Yoshino, K. Sekiguchi & M. Kikuchi; J Cell Biol 124: 373-80 (1994) Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library, E. Koivunen, B. Wang & E. Ruoslahti). Peptides obtained in this way clearly mimic natural protein:protein interactions as in the case for the proteins MDM2 and p53 (Bottger et al. Identification of novel mdm2 binding peptides by phage display, Oncogene, 13:2141-7 (1996)). However, it has not hitherto been appreciated that this phenomenon is sufficiently common so that it might be exploited in identifying inhibitors of the interaction of a protein with an unknowing binding partner. Nor have others explained just how to take advantage of this phenomenon for that purpose.
Traditional Drug Screening
In traditional drug screening, natural products (especially those used in folk remedies) were tested for biological activity. The active ingredients of these products were purified and characterized, and then synthetic analogues of these “drug leads” were designed, prepared and tested for activity. The best of these analogues became the next generation of “drug leads”, and new analogs were made and evaluated.
Both natural products and synthetic compounds could be tested for just a single activity, or tested exhaustively for any biological activity of the interest to the tester. Testing was originally carried out in animals, later, less expensive and more convenient model systems, employing isolated organ, tissue, or cell cultures, membrane extracts or purified receptors, were developed for some pharmacological evaluations.
These methods have many disadvantages. Many of these approaches require large amounts of chemical compound to test, especially testing in whole animals and isolated organs. Since the quantity of a given compound within a collection of potential medicinal compounds is limited, this requires one to limit the number of screens executed.
Also, it is inherently difficult to establish structure/activity relationships (SAR) among compounds tested using whole animals, isolated organs and cultured cells. This is because the actual molecular target of any given compound's action may be quite different from that of other compounds scoring positive in the assay. By testing a battery of compounds on a very specific target, one can correlate the action of various chemical residues with the quantitative activity and use that information to focus ones search for active compounds among certain classes of compounds or even direct the synthesis of novel compounds having a composite of the properties shared by the active compounds tested.
Another disadvantage to whole animal, organ and cell based screening is that certain limitations may prevent an active compound from being scored as such. For instance, an inability to pass through the cellular membrane may prevent a potent inhibitor, within a tested compound library, from acting on the activated oncogene ras and giving a spurious negative score in a cell proliferation assay. However, if it were possible to test ras in an isolated system, that potent inhibitor would be scored as a positive compound and contribute to the establishment of a relevant SAR. Subsequent, chemical modifications could then be carried out to optimize the compound structure for membrane permeability.
The overwhelming disadvantage to the receptor based methods for screening compounds is that they require a priori knowledge about the activity of receptor and its biologi
Fowlkes Dana M.
Frelinger Jeffrey A.
Hyde-Deruyscher Robin Parish
Kay Brian K.
Cooper Iver P.
Karo Bio AB
Wessendorf T. D.
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