High affinity, sequence-selective recognition of RNA by synthetic molecules is increasingly recognized as a key strategic goal for the production of novel therapeutics and biochemical probes (1
). The importance of this area is accentuated by the ever-increasing pace of discovery of new RNA sequences with biochemically important (and therefore potentially biomedically important) functions. Among the many recent advances in this area is the recognition that many non-coding RNA (ncRNA) elements present in the eukaryotic RNAome play a direct role in controlling cellular processes and disease (2
). Human diseases believed to have an ncRNA origin include spinocerebellar ataxia, fragile X-syndrome, diabetes mellitus, myoclonus epilepsy and the myotonic dystrophies (DM) (3
). However, to date only a relatively small number of compounds have been reported that bind specific RNA sequences and elicit a desired biological response. For these reasons, expanding the pool of sequence-selective RNA-targeted synthetic molecules presents a critically important but under-examined challenge in chemical biology.
The DM are central examples of a growing family of RNA-mediated diseases (3–5
). Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy, affecting ~1 in 8000 people (6
). An autosomal dominant inherited disease, DM1 results from a CTG repeat expansion (CTGexp
) in the 3′-UTR of the DM protein kinase gene (DMPK
) on chromosome 19q. The expanded CTG is transcribed into long CUGexp
repeat mRNA. These RNA repeats sequester RNA-binding proteins such as the MBNL (muscleblind-like) family of splicing regulators, retaining them in the nucleus as foci. This in turn leads to misregulated alternative splicing, or spliceopathy. Myotonic dystrophy type 2 (DM2) is caused by an unstable expansion of a CCTG repeat in intron 1 of the cellular nucleic acid-binding protein gene (CNBP
, also known as the zinc finger protein 9 gene or ZNF9
) on chromosome 3q. Transcription produces toxic mRNA containing hundreds to thousands of CCUGexp
. Like CUGexp
, these are also sequestered into foci, and deplete MBNL1 protein from the affected cell (7
). Currently, there is no pharmaceutical therapy for either DM1 or DM2. However, the molecular understanding of these diseases suggests that displacement of MBNL1 from its CUGexp
binding sites constitutes an attractive strategy for developing therapies targeting DM (8
). Experiments in animal models support this hypothesis. For example, a morpholino antisense oligonucleotide (CAG-25) complementary to CUGexp
RNA was able to hybridize with the CUGexp
RNA in vitro
, and displace MBNL1. Intramuscular injection of CAG-25 in a transgenic DM1 mouse model partially restored chloride channel-1 (Clcn-1) protein expression, and DM1 symptoms lessened (10
). The high cost and challenging pharmacological properties of oligonucleotide-based drugs suggest, however, that alternative approaches to targeting CUGexp
RNA are of value.
In the absence of well-defined rules guiding the design of sequence-selective RNA-binding molecules [such as those developed by Dervan et al.
) for DNA recognition], the question arises as to how one successfully addresses the binding problem. Several years ago, a number of groups, including ours, developed the concept of Dynamic Combinatorial Chemistry (DCC) as a ‘rapid prototyping’ method for testing binding hypotheses and facilitating the identification of compounds with novel architectures and properties (12
). Since that time, DCC has evolved in many directions, demonstrating its potential as a method for the identification of receptors for small-molecule analytes, catalysts, new materials, sensors and a broad range of compounds able to bind protein and nucleic acid targets. However, to our knowledge no DCC-derived hit compound has either directly or through analog production resulted in a structure with in vivo
In 2008, we reported the first non-nucleic acid-based compounds (1
is a representative structure) capable of binding CUGexp
RNA and competitively inhibiting CUGexp
-MBNL1 binding in vitro
). This work relied on a resin-bound form of DCC, termed RBDCC, that we developed to facilitate the identification of sequence-selective DNA (14
) and RNA (13
) binding compounds. Several groups have subsequently demonstrated elegant and structurally varied approaches to binding CUGexp
). This recent upsurge of interest highlights the fact that DM1 and DM2 RNAs are important therapeutic targets, as well as valuable model systems for testing hypotheses regarding the factors influencing selectivity and affinity in RNA recognition. Despite these advances, demonstration of the restoration of MBNL1 activity in vivo
by cell-permeable, highly selective CUGexp
RNA binders remains an important goal.
RBDCC hit compound 1
() and related molecules identified in our initial work provided a useful demonstration of feasibility, and set the stage for building toward a compound that would be suitable for further evaluation in the biological context. To accomplish that goal, we anticipated that replacing the disulfide bridge with an olefin bioisostere would not have a dramatic impact on affinity, based on results from parallel efforts in our lab targeting an RNA sequence involved in regulating −1 ribosomal frameshifting in HIV (20
). Since disulfides are easily reduced in the cytoplasm, replacing the disulfide with an olefin or alkane would facilitate cellular studies. Second, molecules containing hydrocarbon bridges of varied length would allow us to examine the effect of linker length and configuration on binding ability and selectivity. Third, we wished to explicitly examine the importance of the amino acid sequence order. Finally, as quinolines are known intercalators, at least in the DNA-binding context (21
), we hypothesized that increasing the pi surface area of this group would enhance affinity. In this regard, we were surprised to discover that despite the vast amount of research conducted into the nucleic acid recognition properties and biological activity of acridine derivatives, including the use of several acridines in humans as antimicrobials (22
) and chemotherapeutic agents (23
), we are only aware of one mention of the closely related benzo[g]quinoline heterocycle (i.e. 2
, ) in the nucleic acid recognition literature (24
). Thus, synthesizing and testing derivatives incorporating this moiety would constitute the first examination of this heterocycle in the RNA binding context.
Hit compound 1 identified via RBDCC and molecules (2–11) synthesized in this work.