Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-02-22
2003-11-25
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S007100, C435S007200, C536S022100, C536S023100, C536S024300
Reexamination Certificate
active
06653067
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed in general to the areas of detecting and measuring molecular interactions. In particular, the present invention pertains to the identification of ligand binding sites in a biomolecule, preferably using mass spectral analysis.
BACKGROUND OF THE INVENTION
Drug discovery has long been one of the most important areas of pharmaceutical research. New or improved drugs are constantly in demand for the treatment of both established and emerging health threats. Drug discovery has evolved from what was, several decades ago, essentially random screening of natural products, into a scientific process that not only includes the rational and combinatorial design of large numbers of synthetic molecules as potential bioactive agents, such as ligands, agonists, antagonists, and inhibitors, but also includes the identification, mechanistic, and structural characterization of their ligand targets, which may be polypeptides, proteins, or nucleic acids. These key areas of drug design and structural biology are of tremendous importance to the understanding and treatment of disease. However, significant hurdles need to be overcome when trying to identify or develop high affinity ligands for a particular biological target. These include the difficulty surrounding the task of elucidating the structure of targets and targets to which other molecules may be bound or associated, the large numbers of compounds that need to be screened in order to generate new leads or to optimize existing leads, the need to dissect structural similarities and dissimilarities between these large numbers of compounds, correlating structural features to activity and binding affinity, and the fact that small structural changes can lead to large effects on biological activities of compounds.
There are numerous facets to the drug discovery process including not only the identification of potential drug targets, but the determination of the structural and electronic bases of target-drug interactions. Knowledge of target structure has been the basis for rational approaches to drug design, and accordingly a number techniques for the structural elucidation of biologically interesting targets have been developed. For instance, techniques and instrumentation are readily available for the sequencing of proteins and nucleic acids. Presently however, sequencing reveals only primary structure, leaving secondary and tertiary structure to be deduced from theory and physiochemical properties of the molecule. In addition, there are some classes and structures of biopolymeric targets that are not amenable to sequencing efforts.
Another approach to structural elucidation of drug targets and their complexes, resolving some of the deficiencies of sequencing, involves X-ray crystallography. This powerful technique allows for the determination of secondary and tertiary structure of biopolymeric targets and can reveal drug binding sites. As with all techniques, however, it also has limitations. For instance, the data obtained from X-ray crystallography of macromolecules is limited to the quality of crystals being analyzed. Further, crystallization of biopolymers is well known to be extremely challenging, difficult to perform at adequate resolution, and is often considered to be as much an art as a science. Although the wealth of structural information provided by a crystal structure is profound, X-ray crystallography is unable to reveal true insight into the solution-phase, and therefore the biologically relevant, structures of the targets and complexes of interest.
A method that is particularly adept at pinpointing the site of ligand binding in a polypeptide molecule involves systematic site-directed mutagenesis coupled with ligand binding assays. This method is referred to as “alanine scanning” because of the preferred use of alanine variants in the ligand binding assays. Other amino acid substitutions, however, are possible. By systematically replacing each residue in a polypeptide with alanine, a set of mutant proteins can be prepared and assayed by quantitative ligand binding analysis. Changes in ligand binding affinities (K
D
) for a particular mutation points to certain residues involved in ligand binding. Alanine scanning has been used to map several human biological receptors such as human growth hormone receptor (Cunningham et al.,
Science
, 1989, 244,1081), insulin-like growth factor-1 receptor (Mynarcik et al.,
J. Biol. Chem
., 1997, 272, 18650), seratonin 5HT
3
receptor (Yan et al.,
J. Biol. Chem
., 1999, 274, 5537), and receptor for urokinase-type plasminogen activator (G{dot over (a)}rdsvoll et al.,
J. Biol. Chem
., 1999, 274, 37995). Similarly, cysteine scanning has been used to map a transmembrane span within prostaglandin transporter (Chan et al.,
J. Biol. Chem
., 1999, 274, 25564).
Polynucleotides also have been studied using site specific chemical modifications for the study of macromolecular structure and function. For instance, phosphorothioate substitutions in RNA molecules have implicated regions involved in binding metal ions and contacting with other proteins (Ruffner, et al.,
Nucleic Acids Res
., 1990,18,6025; Chanfreau, et al.,
Science
, 1994, 266, 1383; Jeoung, et al.,
Nucleic Acids Res
., 1994, 22, 3722; Michels, et al.,
Biochemistry
, 1995, 34, 2965; Kufel, et al.,
RNA
, 1998, 4, 777; Milligan, et al.,
Biochemistry
, 1989, 28, 2849; and Schnitzer, et al.,
Proc. Natl. Acad. Sci. USA
, 1997, 94, 12823). In some cases, phosphorothioate substitutions may cause substantial structural changes in RNA at places remote from the substitution (Smith, et al.,
Biochemistry
, 2000, 39, 5642), but this is most likely peculiar of RNA having complex secondary structure, as the structure of phosphorothioate-modified DNA/RNA duplexes are very similar to that of their unmodified counterparts (Bachelin, et al.,
Nat. Struct. Biol
., 1998,5,271 and Gonzalez, et al.,
Biochemistry
, 1994, 33, 11062).
Relatively recent progress in the area of mass spectrometry (MS) has allowed this analytical method to play an increasingly important role in drug discovery. Certain advances now allow the detection of large biomolecules and their non-covalent complexes with small molecules. Not only are MS techniques capable of preserving such weak molecular interaction and resolving biomolecules and their complexes, it is fully capable of quantitatively measuring their amounts, allowing for accurate measurement of ligand binding affinities.
Particularly suited for the analysis of biomolecules, electrospray ionization mass spectroscopy (ESI-MS) has been used to study biochemical interactions of biopolymers such as enzymes, proteins and macromolecules such as oligonucleotides and nucleic acids and carbohydrates and their interactions with their ligands, receptors, substrates or inhibitors (Bowers et al.,
Journal of Physical Chemistry
, 1996, 100, 12897-12910; Burlingame et al.,
J. Anal. Chem
., 1998, 70, 647R-716R; Biemann,
Ann. Rev. Biochem
., 1992, 61, 977-1010; and Crain et al.,
Curr. Opin. Biotechnol
., 1998, 9,25-34). While interactions that lead to covalent modification of biopolymers have been studied for some time, one of the most significant developments in the field has been the observation, under appropriate solution conditions and analyte concentrations, of specific non-covalently associated macromolecular complexes that have been promoted into the gas-phase intact (Loo, Mass Spectrometry Reviews, 1997, 16, 1-23; Smith et al.,
Chemical Society Reviews
, 1997, 26, 191-202; Ens et al., Standing and Chemushevich, Eds.,
New Methods for the Study of Biomolecular Complexes, Proceedings of the NATO Advanced Research Workshop
, held Jun. 16-20, 1996, in Alberta, Canada, in
NATO ASI Ser., Ser. C
, 1998, 510, Kluwer, Dordrecht, Netherlands).
A variety of non-covalent complexes of biomolecules have been studied using ESI-MS and reported in the literature (Loo,
Bioconjugate Chemistry
, 1995, 6, 644-665; Smith et al.,
J. Biol. Mass Spectrom
., 1993, 22, 493-501; Li et al.,
J. Am. C
Griffey Richard H.
Hofstadler Steven A.
Swayze Eric E.
Boswell Herb
Isis Pharmaceuticals , Inc.
Legaard Paul K.
Riley Jezia
Tarbet Kenneth H.
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