Methods and kits for discovery of RNA-binding antimicrobials

Details Methods and kits for discovery of RNA-binding antimicrobials Methods and kits for discovery of RNA-binding antimicrobials

1999-12-16

2001-11-13

Myers, Carla A. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S016800, C536S023100

Reexamination Certificate

active

06316194

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the specific interactions of low molecular weight compounds with RNA. More particularly, the present invention relates to compositions, methods and kits for identifying antimicrobials and other compounds that interfere with RNA-antimicrobial interactions.
BACKGROUND OF THE INVENTION
In most biological systems, the function of RNA is often determined by the interactions between highly conserved RNA structures. In many circumstances it is desirable to develop drugs that bind RNA at sites of conserved structure to act as competitive inhibitors of the RNA function that is derived from various RNA interactions, such as those exemplified by RNA—RNA and RNA-protein interactions. These types of drugs have potential applications in a wide range of diseases including bacterial, viral, and fungal infections.
1. Use of Fluorescence to Measure Ligand Binding to RNA
A critical step in the development of RNA-binding drugs is the development of simple and robust assays that are suitable for the high throughput screening of large compound libraries developed either by combinatorial synthesis traditional medicinal chemistry approaches, or from collections of natural products.
There are many different types of assays that measure the binding of ligands to nucleic acids and utilize fluorescence resonance energy transfer (FRET) to generate a signal. FRET is caused by a change in the distance separating a fluorescent donor group from an interacting resonance energy acceptor, either another fluorophore, a chromophore, or a quencher. Combinations of donor and acceptor moieties are known as “FRET pairs”. Efficient FRET interactions require that the absorption and emission spectra of the dye pairs have a high degree of overlap. FRET is also a distance-dependent interaction which is dependent on the inverse sixth power of the intermolecular separation, making it a sensitive measurement of molecular distances (Stryer, 1978 and Selvin, 1995).
An improvement in the technology for measuring the ability of small molecules to bind to RNA is to utilize fluorescent reporters. Current methods all rely on the labeling of either the nucleic acid or the ligand with a fluorescent tag and measuring changes in fluorescence emission spectrum after binding. For example, Royer (U.S. Pat. No. 5,445,935 (issued Aug. 29, 1995)) described the use of polarization of the fluorescence emission from a labeled macromolecule, such as a DNA or RNA oligonucleotide, to assess the binding of the labeled macromolecule to a second unlabeled macromolecule, such as a protein. Similarly, (Metzger et al. (1997) have measured binding of unlabeled pep tides derived from Tat to TAR RNA by measuring quenching of the intrinsic fluorescence of the peptide after it is bound to RNA.
In another application of fluorescence polarization, Richardson and Schulman (U.S. Pat. No. 4,257,774 (issued Mar. 24, 1981)) reported a method for detecting compounds that interact with nucleic acids by inhibition of acridine orange binding to the nucleic acid which results in a change in fluorescence polarization. This method is of limited practical use because the binding of acridine is through intercalation at a wide variety of sites on double-helical structures, with the consequent result that the specificity of the assay is limited.
Wang & Rando (U.S. Pat. No. 5,593,835 (issued Jan. 14, 1997) and Wang et al., 1997) discovered that the attachment of certain fluorescent moieties to an aminoglycoside antibiotic enables the subsequent binding interaction of the antibiotic with an RNA molecule to be enhanced. They have used this property to develop quantitative screening methods and kits for RNA binding compounds. In their method the fluorescently-labeled antibiotic is bound to a pre-selected region of the target RNA, thereby forming a complex which is less fluorescent than the unbound fluorescent antibiotic because of quenching of the fluorescent moiety due to its interaction with the target RNA molecule. The complex is then mixed with a compound-to-be-tested, and the fluorescence of the antibiotic measured. The antibiotic becomes more fluorescent if the compound displaces the antibiotic in the complex and binds to the pre-selected region of the target RNA.
A general limitation to the use of a single fluorescent group on a reporter molecule is that this group has to interact directly with the RNA target in order to show alterations in its fluorescence emission spectrum. This severely limits the number of positions on the reporter that can be modified and can also alter the nature of the binding of the reporter to the RNA.
It is an object of the invention to apply FRET methodologies to measure the formation of a complex between a fluorescently-labeled antimicrobial and fluorescently-labeled RNA target. One reason why this approach has not been undertaken previously is that NMR studies have shown that of RNA-peptide complexes are in intermediate exchange, suggesting a high degree of conformational flexibility and dynamic exchange at the RNA-peptide interface (Puglisi et al., 1992; Aboul-ela et al., 1995; Brodsky & Williamson, 1997; Cai et al., 1998; De Guzman et al., 1998). These dynamic properties are, in theory, a severe hindrance to the development of FRET.
2. RNA Targets for Drug Discovery
Although RNA is often referred to as being single stranded and unstructured, most biologically active RNA molecules actually have a number of intramolecular bindings and contacts that create a wide variety of structures and folds. In RNA structures, the secondary structure is energetically the largest contributor to the overall three-dimensional fold. A primary element of secondary structure in large RNA molecules is the RNA double helix built by Watson-Crick base pairings between two regions of the RNA polynucleotide. The helical elements in RNA are typically interrupted by bulges and internal loops. In addition to disruptions of the helical structures, biologically active RNA molecules typically contain specialized loop sequences that create stable bends in RNA. Associations between single stranded regions as well as those between single stranded regions and double helices lead to structural elements creating tertiary structures. Many tertiary structural elements in RNA form recurrent motifs, such as “pseudoknots”created by the interactions of pairs of loop structures. Additional tertiary structure elements such as base triples are also commonly found in large RNA structures.
3. RNA Targets in Ribosomal RNA
Many antibiotics function by inhibiting protein synthesis, and it has become increasingly clear that many do so by acting at the level of ribosomal RNA (rRNA). The 16S rRNA of the small, 30S ribosomal subunit and the 23S rRNA of the large, 50S ribosomal subunit are both large RNAs for which there are highly refined secondary structure models. The rRNA binding sites of many different types of antibiotics have been mapped by chemical and enzymatic probing approaches. These antibiotic binding sites are localized to various subregions on the 16S and 23S rRNAs, as exemplified by those identified for the
Escherichia coli
rRNAs (FIGS.
1
and
2
). These sites include, but are not limited to, the 16S rRNA A site (FIGS.
5
and
13
), the 16S rRNA spectinomycin binding site (FIGS.
12
and
16
), the 23S rRNA L1 (or E site) (FIGS.
10
and
14
), and the 23S rRNA GTPase center (L11 binding site) (FIGS.
11
and
15
). Examples of antibiotics targeted to these sites include, but are not limited to, binding of the 16S rRNA A site by members of the aminoglycoside class, binding of the 23S rRNA L1 site (the E site) by the oxazolidinone class, and binding of the 23S rRNA GTPase center by the thiazole class.
4. RNA Mimics of Antibiotic Binding Sites
Targeting drugs against large RNAs such as the 16S rRNA (>1,400 nucleotides) and 23S rRNA (>2,700 nucleotides) can be difficult in part due to the size of the RNAs, which can hinder drug development assays. For instance, it can be difficult to produce a suitable quantity and quali

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