Methods and kits for discovery of RNA-binding compounds

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

1999-06-03

2003-06-03

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S005000, C435S091100, C536S022100

Reexamination Certificate

active

06573045

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 compounds that interfere with RNA-protein or RNA-ligand interactions and thereby interfere with RNA function.
BACKGROUND OF THE INVENTION
In most biological systems, the maturation, transport, stability and expression of RNA is closely regulated by the interactions between highly conserved regulatory RNA sequences and proteins. In many circumstances it is desirable to develop drugs that bind RNA at sites of regulatory protein binding and act as competitive inhibitors of the RNA-protein interaction. These types of drugs have potential applications in a wide range of diseases including viral, bacterial and fungal infections and chronic diseases such as cancer and autoimmune disease.
1. RNA Targets for Drug Discovery
Although RNA is often referred to as being single stranded and unstructured, most biologically active RNA molecules actually fold back on themselves to create a wide variety of structures. In RNA structures, the secondary structure is energetically the largest contributor to the overall three-dimensional fold. The main secondary structure element 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 and 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.
Regulatory proteins seldom target fully-double stranded regions of RNA, which like A-form DNA forms a comparatively rigid structure in which most functional groups that distinguish between the bases are inaccessible. Instead, RNA-binding proteins generally target functional groups where bulges, loops and hairpins disrupt the regular RNA helix and open up the major groove for interaction with protein side chains. Regulatory proteins usually interact with these bulges, loops and hairpins that disrupt the regular RNA helix. These secondary structure elements provide the structural diversity needed for protein recognition as well as for drug development.
2. RNA Targets in HIV
A great deal of the present knowledge about the structure of RNA targets and their recognition by RNA binding proteins has come from studies of two human immunodeficiency virus (HIV) regulatory proteins Tat, the trans-activator protein and Rev, the regulator of virion expression. The two proteins play complementary roles in the virus life cycle. Tat stimulates transcription from the viral long terminal repeat (LTR), whereas Rev is required for the efficient export from the nucleus of the late mRNAs encoding the structural proteins of the virus.
Tat and Rev both exert their effects through specific cis-acting viral RNA regulatory sequences. Tat activity requires the trans-activation-responsive region (TAR), an RNA regulatory element of 59 nucleotides (nt) located immediately downstream of the initiation site for transcription (Cullen, 1990; Karn et al., 1995). Because of its position in the HIV genome, each viral mRNA carries a copy of TAR at its 5′-end.
TAR RNA forms a highly stable, nuclease-resistant, stem-loop structure. Point mutations, which disrupt base-pairing in the upper TAR RNA stem invariably, abolish Tat-activated transcription. In 1989 Dingwall et al. provided the first demonstration that recombinant Tat expressed in
Escherichia coli
could bind specifically to bases in the stem of the TAR RNA (Dingwall et al., 1989). The binding site for Tat includes a U-rich trinucleotide bulge found in the upper stem of TAR RNA.
Extensive mutagenesis, chemical probing and peptide binding studies have defined the key elements required for TAR recognition by Tat and have shown that the Tat binding site surrounds a UCU bulge located near the apex of TAR (Calnan et al., 1991a; Weeks & Crothers, 1991; Delling et al., 1992; Churcher et al., 1993; Hamy et al., 1993; WO92/02228). Tat interacts with the first uridine of the bulge, U23, while the other residues in the bulge act predominantly as spacers and may be replaced by other nucleotides, or even by non-nucleotide linkers (Churcher et al., 1993). Tat recognition requires two base-pairs in the stem above the U-rich bulge, G26·C39 and A27·U38 (Weeks & Crothers, 1991; Delling et al., 1992; Churcher et al., 1993). Two base pairs below the bulge, A22·U40 and G21·C41, also make significant contributions to Tat binding. Critical phosphate contacts involve phosphates P21, P22, and P40, which are located below the bulge on both strands (Calnan et al., 1991b; Hamy et al., 1993; Pritchard et al., 1994).
In addition to binding the Tat protein, TAR is able to form complexes with short peptides containing an arginine-rich sequence derived from residues 48 to 57 of Tat (Cordingley et al., 1990; Weeks et al., 1990; Calnan et al., 1991a; Weeks & Crothers, 1991; Churcher et al., 1993). However, compared to the Tat protein, short arginine-rich peptides bind with reduced affinity and, in certain cases, also have a significantly reduced binding specificity (Churcher et al., 1993). For example, short basic region peptides (such as the ADP-3 peptide, residues 48 to 72) show less than a 2-fold difference in affinity between wild-type TAR RNA and TAR sequences carrying the mutations U23→C and G26·C39→C·G mutations (Churcher et al., 1993). Similarly, some basic peptides display a high affinity for any duplex RNA sequences that carries one or more bulged residues. Monomeric complexes between peptides and TAR are only obtained when TAR is truncated and the competing binding sites normally present on the lower stem are removed (Weeks et al., 1990; Weeks & Crothers, 1991; Churcher et al., 1993). By contrast, peptides such as ADP-1 (residues 37 to 72, see
FIG. 3
) that contain residues from the conserved “core” region of lentivirus trans-activators, have a higher affinity for TAR RNA and are able to discriminate between TAR RNA mutants with a specificity which more closely resembled that of the Tat protein itself (Churcher et al., 1993).
Recent NMR studies of TAR RNA demonstrate that the accessibility of the critical functional groups recognized by Tat and basic peptides is enhanced by rearrangement of the bulge region (Puglisi et al., 1992; Aboul-ela et al., 1995; Aboul-ela et al., 1996). This refolding process involves one of the arginine side chains present in the basic binding domain of the Tat protein (Puglisi et al., 1992; Aboul-ela et al., 1995). In the presence of the arginine, the stacking of the bulged residues U23 on A22 and C24 on U23 is disrupted and A22 becomes juxtaposed to G26. This creates a binding pocket where the guanidinium and (—NH groups of the arginine are placed within hydrogen-bonding distance of G26-N7 and U23-04, respectively. The conformational change in TAR RNA also repositions the P22, P23 and P40 phosphates, which provide energetically important contacts with Tat (Pritchard et al., 1994).
The Tat-TAR interaction therefore provides a clear example of the ‘indirect readout’ of nucleic acid sequences through recognition of backbone phosphates. The importance of arginine binding pocket in TAR for Tat binding is confirmed by the observation that the mutations that produce the most severe reductions in TAR activity involve G26 and U23 and disrupt the intermolecular interactions that are responsible for the folding transition (Weeks & Crothers, 1991; Churcher et al., 1993).
Rev activity requires a second

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