Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-05
2001-12-18
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06331392
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to novel methods for screening for pharmaceutical compounds, in particular those that bind to RNA sequences involved in the pathogenesis of disease or in regulation of a physiological function.
BACKGROUND OF THE INVENTION
Pharmaceuticals can be developed from lead compounds that are identified through a random screening process directed towards a target, such as a nucleic acid or a protein receptor. Large scale screening approaches can be complicated by a number of factors. First, many assays are laborious or expensive to perform. Assays may involve experimental animals, cell lines, or tissue cultures that are difficult or expensive to acquire or maintain. These considerations often place practical limitations on the number of compounds that reasonably can be screened. Thus, those employing random screening methods are frequently forced to limit their search to those compounds for which some prior knowledge suggests that the compounds are likely to be effective. This strategy limits the range of compounds tested, and many useful drugs may be overlooked.
Furthermore, the specificity of many biochemical assays may exclude a wide variety of useful chemical compounds, because the interactions between the ligand and the target are outside the scope of the assay. With such a specific assay, many potential pharmaceuticals may not be detected.
Finally, in most existing biochemical screening approaches to drug discovery, the system in question must be well-characterized before screening can begin. Consequently, biochemical screening for therapeutic drugs directed against many targets must await detailed biochemical characterization, a process that generally requires extensive research.
The present invention pertains specifically to the use of RNA targets in high-throughput screening methods for identification of useful ligands. The invention takes advantage of the existence of higher-order structures in naturally-occurring and synthetic RNA molecules. For example, RNA exists in both single stranded and helical duplex forms. These duplexes may be distorted by loops, bulges, base triples and junctions between helices. The structural diversity of RNA is far greater than that of DNA, and similar to that of proteins, making RNA a likely target for unique binding of small molecules (reviewed in Wyatt and Tinoco, 1993).
Small molecules can bind RNA with high affinity and specificity and can block essential functions of the bound RNA. The best example of such molecules are antibiotics such as erythromycin and aminoglycosides. The first suggestion that some antibiotic translation inhibitors interact specifically with RNA was the genetic mapping of resistance to kanamycin and gentamicin to the methylation of 16S RNA (Thompson et al.,
Mol. Gen. Genet
. 201:168, 1985). Erythromycin binds to bacterial RNA and releases peptidyl-tRNA and mRNA (Menninger et al.,
Mol. Gen. Genet
. 243:225, 1994). 2-DOS-containing aminoglycosides bind specifically to the structures of HIV RNA known as the RRE, block binding of the HIV Rev protein to this RNA, and thereby inhibit HIV replication in tissue culture cells (Zapp et al.,
Cell
74:969, 1993). In addition, although aminoglycosides have long been developed as translation inhibitors, they were only recently shown to bind to rRNA in the absence of proteins (Purohit and Stern, Nature 370:659, 1994; Fourmy et al.,
Science
274:1367, 1996). Hygromycin B inhibits coronaviral RNA synthesis and is thought to do so by binding to the viral RNA and blocking specifically the translation of viral RNA (Macintyre et al.,
Antimicrob. Agents Chemother
. 35:2630, 1991).
Existing assays for ligands of nucleic acids, such as, for example, methods that use equilibrium dialysis, differential scanning calorimetry, viscometric analyses, UV measurement of hyperchromic effect, or fluorescence, are often unfeasible for high-throughput applications, because of insufficient characterization, unavailability of components, expense, and/or complexity. (See, for example, Murakami et al.,
Nuc.Acids Res
. 19:4097, 1991; Walker et al.,
Nuc.Acids Res
. 24:348, 1996). Thus, prior to the present invention, random screening approaches for non-oligonucleotide ligands of RNA were limited to compounds for which some prior knowledge suggested that they might be effective. This strategy has been successful (Zapp et al., 1993), but is limited in the range of compounds that can be tested on a practical scale.
U.S. Pat. No. 5,270,163 describes the SELEX system for the identification of oligonucleotides that bind specific targets. In this system, random oligonucleotides are affinity-selected and amplified, followed by several cycles of re-selection and amplification. This method, however, is limited to screening for oligonucleotide ligands and cannot be applied in reverse, i.e., to search for non-oligonucleotide ligands that bind to nucleic acids.
U.S. Pat. No. 5,306,619 discloses a screening method to identify compounds that bind particular DNA target sequences. In this method, a test nucleic acid is constructed in which the target sequence is placed adjacent to a known protein-binding DNA sequence. The effect of test compounds on the binding of the cognate protein to the protein-binding DNA sequence is then measured. This method requires conditions in which melting of DNA hybrids and unfolding of DNA structure do not occur. RNA, by contrast, can undergo much more dramatic variations in patterns of base-pairing and overall conformation.
Thus, there is a need in the art for efficient and cost-effective high-throughput methods for random screening of large numbers of non-oligonucleotide small molecules for their ability to bind physiologically, medically, or commercially significant RNA molecules.
SUMMARY OF THE INVENTION
The present invention encompasses screening methods to identify ligands that bind any predetermined target RNA. The methods are carried out by the steps of: selecting as test ligands a plurality of compounds not known to bind to the target RNA sequence; incubating the target RNA sequence with one or more fluorescently labeled probes in the presence of a test ligand to produce a test combination, and in the absence of a test ligand to produce a control combination; measuring one or more fluorescence properties of the probe(s) to evaluate the stability and/or conformation of the target RNA in each combination; and selecting as a ligand any test ligand that causes a measurable change in at least one of the fluorescence properties of the probes in the test combination relative to the control combination. Preferably, the incubating and measuring steps are repeated with a plurality of said test ligands until a ligand that binds to the target RNA sequence is identified. Furthermore, the test and control combinations may be subjected to conditions that, in the absence of ligands (i.e., in the control combination), denature a detectable fraction of the target RNA.
In general, ligands identified by the methods of the present invention are expected to stabilize, i.e., reduce &Dgr;G° of a particular conformation of the target RNA. Stabilization may lead to a more folded conformation, a less folded conformation, or a change from a first folded conformation to a second, alternative, folded conformation. Preferably, ligands stabilize a particular conformation which inhibits the normal function of the target RNA.
Probes useful in practicing the invention include molecules which comprise fluorescent moieties whose measurable fluoresence properties, particularly polarization or anisotropy, are sensitive to the stability and/or conformation of the target RNA as reflected in the binding state of the probe. Preferably, the fluorescence anisotropy of a sample containing the probe is compared between the test and control combinations. Ligands may be identified as compounds that cause the anisotropy to increase or decrease relative to the anisotropy of a control solution.
In a preferred embodiment, the target RNA is hybridized with a complementary oligonucleotide
Arenas Jaime E.
Cload Sharon T.
Laing Lance G.
Lillie James W.
Pakula Andrew A.
Anadys Pharmaceutical, Inc.
Campbell Eggerton A.
Darby & Darby
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