Methods for targeting RNA molecules

Details Methods for targeting RNA molecules Methods for targeting RNA molecules

2000-02-29

2004-02-03

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300, C536S024310, C536S024320

Reexamination Certificate

active

06686148

ABSTRACT:

BACKGROUND
This application relates to the field of identification of compounds active on RNA and of target sites on the RNA.
The wealth of information gained from the study of the human genome has allowed identification of many genes whose inappropriate expression results in disease. To exploit this information, several technologies have been developed to specifically target gene expression. Modulating the expression of such genes allows treatment of the disease. In particular, the strategy of modulating gene expression by targeting RNA has spawned several new classes of rationally designed therapeutics (e.g., ribozymes, DNAzymes, and antisense oligonucleotides). However, to date, these technologies have had very limited clinical success. One of the reasons for the limited success is due to the difficult pharmacological barriers these macromolecules must overcome.
The pharmacological treatment of disease is one of the signal advances in medicine in the twentieth century. Modern methods of small molecule development have also proven highly useful in allied fields such as veterinary medicine and pest eradication in agriculture. An ever increasing number of diseases can be ameliorated by appropriate drug therapy, reflecting increasing knowledge of disease pathophysiology and improved methods for conducting clinical research. As the sequence of the human (and other) genomes is solved and the molecular understanding of disease advances it will increasingly be possible to select optimal targets for therapeutic attack. Thus while many current drug treatments act by poorly understood mechanisms, and/or provide only symptomatic relief, increasingly it should be possible to reverse disease pathophysiology and provide more specific and potent therapeutic relief. To achieve this goal, however, will require improved methods of drug development. In particular, the development of drug targeting strategies that enable highly specific targeting of selected genes or gene products will be of great use.
The information provided and references cited herein are provided solely to assist the understanding of the reader, and is not admitted to be prior art to the present invention.
SUMMARY OF THE INVENTION
The present invention provides certain advantageous methods for developing biologically active molecules. The invention includes a new method for targeting RNA with small molecules, where the small molecule makes contact with both a cellular protein and a cellular RNA. The effect of the tripartite complex is to increase the affinity of the complex beyond what could be achieved alone with the small molecule and the RNA. This addresses what is believed to be the major limitation of current attempts to target RNA with small molecules, the lack of specific high affinity binding of small molecules to RNAs, which are much more flexible than proteins. This is a generic method for inhibiting any cellular RNA, as described in the disclosure, including, but not limited to allele specific inhibition or inhibition based on targeting to identified regions of RNA secondary structure where that secondary structure differs between allelic forms.
The invention also provides a method for identifying RNA targets for allele specific therapy. These methods have application in at least two major areas: (1) autosomal dominant diseases, or diseases of excess gene dosage, where elimination of one mRNA (but not both) would have a therapeutic effect and (2) cancer and other diseases with LOH where allele specific therapy will provide a differential effect on disease tissue compared to normal tissue. Such use of allele specific therapy has been described in Housman, U.S. Pat. No. 5,702,890, issued Dec. 30, 1997 and Housman et al., U.S. patent application No. 09/045,053, filed Mar. 19, 1998 which are hereby incorporated by reference in their entireties including drawings. Targeting of different alleles can, for example, involve the use of protein-small molelcule complexes as described below, but is not limited to such complexes. For example, different alleles can be targeted with small molecules or with oligonucleotide inhibitors.
The present invention provides a unique strategy for targeting cellular RNAs. This new method overcomes the limitations of current approaches with respect to affinity and specificity. The invention entails formation of a trimolecular complex involving the target RNA, a small molecule, and a cellular protein. The recruitment of a specific cellular protein to the RNA:small molecule complex has the advantage of substantially increasing the area of binding due to contacts between the protein and the RNA, and thereby increase the affinity of the complex beyond what could be achieved alone with the small molecule and the RNA by cooperative binding generally involving electrostatic, hydrogen bonding, and hydrophobic interactions. These favorable binding interactions will increase the affinity of the complex beyond what could be achieved with a small molecule and RNA.
The present invention also provides a new class of RNA therapeutic targets that will provide the basis for inhibition, e.g., allele specific inhibition, by small molecules, and methods for identifying such targets in candidate RNAs. The basis of the invention is the novel observation that single nucleotide polymorphisms in mRNAs are associated with altered secondary structures. These structural differences, not previously known to be associated with normal allelic variation, provide scope for allele specific small molecule inhibition of RNA. Also described are methods for producing and identifying small molecule inhibitors of RNAs.
Thus, in a first aspect the invention provides a method for identifying a potential small molecule inhibitor, where the inhibition involves the induction of formation of a complex of a cellular RNA, a small molecule inhibitor, and a cellular protein. The potential small molecule inhibitor includes a first and a second moiety joined by a linker. The method involves identifying a first moiety which binds to at least a portion of a selected cellular protein; and also identifying a second moiety which binds to at least a fragment of a cellular RNA. Such joint binding indicates that the small molecule is a said potential small molecule inhibitor. The inhibitory ability of the small molecule can be tested by standard methods, e.g., testing the level of protein synthesis from a target mRNA. Clearly, the identifications of the moieties can be performed in either order. The method can also involve selection of a linker or provision of a standard linker, e.g., based on prior screening. In certain cases the linker need not be separate from the first or second moiety, but may be a part of one or both moieties.
In this context, the term “potential small molecule inhibitor” refers to a small molecule which binds to both a selected protein and a target RNA but for which the ability to inhibit a biological activity (e.g., reduce the rate or amount of protein translation from an mRNA) of the RNA has not yet been tested. Following confirmation of such inhibitory characteristic, the small molecule can be referred to as a “small molecule inhibitor” or “inhibitor”.
The term “small molecule” refers to a compound which has a molecular mass equal to or less than 5000 Daltons (5 kD), preferably less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is highly preferred that a small molecule have a molecular mass equal to or less than 700 Da.
The term “inhibitor” refers to a compound that reduces to a statistically significant extent at least one activity of a reference compound. In the context of this invention, an inhibitor generally reduces an activity of an RNA, most commonly, but not limited to, reducing or eliminating the rate and/or extent of translation of an mRNA, reducing or eliminating processing of a pre-mRNA to produce a mature RNA; reducing or eliminating transport of an mRNA from nucleus to cytoplasm, or reducing or eliminating a regulatory function of an RNA molecule. Prefer

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