Effect of Small Molecules on Nucleic Acid Stability and Improvements to RNA Structure Prediction

Table of Contents

Introduction
RNA Structural Motifs Involved in Binding
Targeting the Ribosome

References

Abstract

Nucleic acids have proven to be viable targets for small molecule drugs. While many examples of such drugs are detailed in the literature, only a select few have found practical use in a clinical setting. These currently employed nucleic acid targeting therapies suffer from either debilitating off-target side effects or succumb to a resistance mechanism of the target. The need for new small molecules that target nucleic acids is evident. However, designing a novel drug to bind to DNA or RNA requires a detailed understanding of exactly what binding environments each nucleic acid presents. In an effort to broaden this knowledge, the work presented in this thesis details the binding location and affinity of known and novel nucleic acid binding small molecules with targets ranging from simple RNA secondary structure all the way to the complex structure of ribosomal RNA. Specifically, it is shown that the anthracycline classes of antineoplastics prefer to bind at or near mismatch base pairs in both physiologically relevant iron responsive element RNA hairpin constructs as well as DNA hairpin constructs presenting mismatched base pairs. Also characterized in this thesis is a novel class of topoisomerase II / histone deacetylase inhibitor conjugates that display a unique affinity for DNA over RNA. Finally, the novel class of macrolide-peptide conjugates, known as peptolides, is shown to retain potent translation inhibition of the prokaryotic ribosome and identification of a novel binding site for the anthracycline class of drugs and the characterization of the two novel drug designs presented in this thesis will undoubtedly aid in the effort to design and discover new molecules that aim for nucleic acid targets.

Keywords: Messenger RNA, DNA, Ribosomal RNA, SAR

Introduction

RNA Structural Motifs Involved in Binding

In recent decades, nucleic acids have been shown to be not merely the scribes of the genetic and proteomic code, but they have also been revealed to play crucial roles in regulating genes and gene products via interaction with a variety of ligands including other nucleic acids, proteins, metabolites, cofactors, enzymes, and small molecules (1-11). Prior to this realization, proteins were the main targets of a medicinal chemistry campaign to identify novel small molecules with therapeutic potential. With the successful sequencing of the human genome, hopes were high that all proteins in the human body could be identified and subsequently many new ones could become the target for the treatment of diseases. This, however, has not panned out as only 15% of the human proteome has been deemed drugable and only 207 proteins are targeted by current FDA approved small molecule drugs (12, 13). Many of these clinically successful drugs share the trait of mimicking a protein partner in a protein-protein interaction as their primary mode of action (14, 15). Due to the large surface area and relative complexity of these interactions, it has proven to be a very difficult endeavor to target proteins with small molecules for therapeutic application. This has emphasized the importance of being able to target the source of the protein product related to a disease state, the DNA and RNA encoding it, as a means of treatment. There have been relatively few successful cases of directly targeting DNA in a clinical setting. The most notable success stories would be the alkylating antineoplastic agents such as nitrogen mustards, nitrosoureas, and alkyl sulfonates as well as the cisplatin and anthracycline families of chemotherapeutics (16-19). With an even more underwhelming representation is the effort to develop treatments exploiting RNA as the target, excluding of course ribosomal RNA-targeting antibiotics. While RNAi holds great promise as a means to target specific nucleic acid sequences, the difficulty in delivering the small RNA constructs to the target has hindered its application in a clinical setting (20-21). Efforts are concurrently underway by multiple labs to develop not only a clear understanding of how nucleic acids react and bind to small molecules drugs, but also to identify and characterize novel nucleic acid binders in both a high-throughput and a rationally-driven design basis (22-24).

In order to facilitate this search for novel nucleic acid targeting small molecules, a thorough understanding of what a nucleic acid binding site has to offer to a small molecule is needed. This can be gleaned by examining the secondary and tertiary structures formed by doth DNA and RNA and by considering several examples of drugs that have successfully pinpointed nucleic acids in a clinical setting. DNA and RNA share a similar structure in that they both form a duplex of phosphate backbone linked nucleosides stitched together in a helical arrangement by base pairs between adjacent, anti-parallel strands. However, due to the unique sugar pucker restrictions of RNA owing to its additional 2` hydroxyl group, duplex RNA in a physiological environment presents a much different helical structure in three dimensions than DNA (Figure 1.1). The typical B-form DNA found inside the cell presents a wide yet shallow major groove and a narrow but deep minor groove.

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Figure 1.1 A) Crystal structure of the typical B-DNA helix. B) Crystal structure of typical A-RNA helix. (25, 26)

The width of the major groove allows for easy access to the hydrogen bond participant and pi-stacking rich core of the DNA duplex by small molecules. The deep and narrow nature of the minor groove presents a hydrophobic and densely electronegative rift for small electropositive molecules. The binding of daunorubicyn (DAU), a member of the anthracycline class of anti-tumor drugs that also includes doxorubicin (DOX), to B-DNA clearly illustrates the two main non-covalent binding modes of small molecules to a nucleic acid target: minor groove binding and intercalation (Figure 1.2).

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Figure 1.2) A) General skeleton of the anthracycline class of drugs; DAU X=H, DOX X=OH.

B) Crystal structure of two molecules of DAU (sphere) bound to B-DNA helix (stick) (27).

The flat, conjugated ring system of the aglycone moiety of DAU slips snuggly in between adjacent base pairs of the DNA helix (Figure 1.2B). It is this favorable intercalation that has been attributed to the anthracycline family’s strong binding preference for DNA (28-36). Further stabilizing the interaction, the amino sugar moiety is presented to the minor groove where it binds firmly via electrostatic interactions (27).

An example of a covalent small molecule interaction with DNA can be found with the chemotherapeutic cisplatin (Figure 1.3). Cisplatin is a platinum containing molecule that upon administration looses one of its chlorine atoms to water displacement. This water molecule, also an excellent leaving group, allows the platinum atom to readily bind to bases in the DNA helix, preferably G residues. The second chlorine atom is subsequently displaced, causing an intrastrand cross link between adjacent G residues. This crosslinking causes a distortion of the helix (Figure 1.3B) that is recognized by the DNA repair pathway but cannot be undone, thus signaling cell death. From these examples it is clear that while DNA is certainly druggable, its confined nature in the cell, wound tightly around histone complexes in the nucleus, has made it difficult to directly target with but a few successful clinical applications. Even the success stories such as anthracyclines and cisplatin are plagued by systemic toxicity and off-target side effects, thus leaving much room for improvement.

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Figure 1.3 A) Structure of Cisplatin B) Crystal structure of cisplatin (Pt is pink sphere near center) covalently bound to a B-DNA helix. Figure adapted from (37).

Conversely, RNA is ubiquitous in both the nucleus and cytoplasm and readily accessible to small molecule invaders of the cell. However, a survey of RNA’s secondary structuring highlights a dissimilar topology to that of DNA described above. With its fixed C-3` endo ribose pucker, RNA presents an A-form helix inside the cell (Figure 1.1B). Unique to the A-form helix are its distinct groove characteristics. The major groove of A-RNA is wider than the minor groove, but recent results have indicated that it is too narrow to accommodate small molecule binding (38). This is unfortunate since the major groove is rich with small molecule luring functional groups along the Hoogsteen edges of the base pairs (39). The minor groove of the A-form RNA helix is both shallow and narrow, thus appearing to be a less alluring interaction site for a small molecule ligand than the inviting grooves of the B-DNA helix.

The seemingly constrained and minor groove-inaccessible conformation of the A-RNA duplex helix is not indicative of its ability to form complex tertiary structures. In fact, RNA has the gymnastic ability to form some of the most complex binding motifs found inside the cell (39). It owes this flexibility to several key differences from its DNA counterpart. DNA for the most part exists as a duplex of individual strands. This dictates that the DNA always be paired to its compliment in what tends to be a very strict, canonical Watson-Crick base pairing fashion. In contrast, RNA exists almost entirely as a single stranded entity, save of course for its time spent paired with its molecular role model in a DNA:RNA hybrid. As a result of its inherent single strandedness, RNA is left to fold back upon itself in a complimentary fashion. Without a strict compliment for the RNA strand, very unique and complex structures arise from the lone RNA strand searching for its lowest entropic energy state with only itself, water, and a few metal ions to rely on. Some of the most commonly observed secondary structures found to be conserved in molecular recognition in an RNA-ligand binding event are presented in Figure 1.4.

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Figure 1.4 Some of the conserved and unique secondary structures found in RNA-ligand interactions. A) hairpin loop B) single base bulge C) mismatch bulge D) multi stem junction

Despite its limiting features as a strict A-form helix, the building blocks presented in Figure 1.4 are just a few of the features that RNA employs to engineer incredibly complex secondary and tertiary structure. The hairpin loop allows the RNA strand to make sharp turns back onto itself, the bulges allow for symmetrical and asymmetrical disregard of any unruly bases not wanting to pair, and the multi-way junction allows multiple, distinct regions of functionality to bud and branch out of the same single strand of RNA (39, 40). Another factor contributing to the structural plasticity of RNA is the high incidence and tolerance of mismatched base pairs. Unlike DNA, RNA does not have a housekeeping repair pathway to clean up mismatched pairs. While a base mismatch in DNA has the possibility to corrupt the genome and must be corrected swiftly, the mismatch pair in RNA is employed as a point of flexibility in the tertiary structure. In fact, mismatch pairs are often also a conserved feature in RNA recognition motifs (41). The base-pair mismatch distorts the shape of the A-RNA helix. This distortion kinks and partially unwinds the otherwise inaccessible helix, thus widening the major groove and opening up an electronegative pocket offering an inviting home to any number of small molecule binders.

An elegant example of all of these highly emancipating structural features coming together to form a macromolecule important to all known life can be found in the structure of transfer RNA (tRNA). When the crystal structure of yeast tRNAPHE was solved for the first time in 1974 it revealed an incredible number of structural features made from only a 76-nt long RNA strand (42, 43). As crystallographic resolution improved over the coming decades, even more structural characteristics were gleaned from the similar E. coli tRNAPHE (Figure 1.5).

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Figure 1.5 Cyrstal structure of tRNAPHE flaunting its many structural motifs unique to RNA (44).

In this single, relatively short tRNA there exist a number of secondary and tertiary interactions serving to hold the oligo in exactly the correct orientation to recognize and selectively deliver charged amino acids to the ribosome for incorporation into the nascent polypeptide chain. Some of these interactions include non-canonical base pairs, hairpins, 10 modified bases, and a complex three way stem junction held together by a number of base-triple pairs (44). Additionally, there are a number of water molecules and divalent Mg2+ ions helping to stabilize tertiary interactions (44). It is truly remarkable to recognize just how many special building blocks were taken from the RNA engineering tool kit to create and conserve an RNA motif capable of recognizing its binding partner with such high fidelity.

An equal marvel of RNA structural complexity and ligand recognition can be found in the enzymatically active class of RNAs known as ribozymes. Prior to the discovery of the first ribozymes, enzymatic ability had been relegated solely to protein enzymes. It was the discovery of this class of self-cleaving RNA structures that opened the doors for the RNA world theory; it was now possible for RNA to possess characteristics of both the chicken and the egg (the encoder of heredity and the enzymatic catalyst) in the long-lived debate over which came first in the origin of life, RNA or DNA. It was the complex tertiary structures observed in crystal structures of tRNA that led to the hypothesis that this was indeed feasible as early as the late 1960’s. Proof did not come until the late 1970’s via work by Thomas Cech involving a self-splicing RNA fragment and work by Sydney Altman on the discovery of the auto-catylytic RNase P (45-47). Cech and Altman shared the 1989 Nobel Prize in Chemistry for their contribution to the discovery that RNA was not just a molecule of heredity, but that it was more importantly a biocatalyst (48). Ribozymes have been shown to be capable of several types of enzymatic reactions without the need for cofactors such as adenosine triphosphate (ATP). The most common of these enzymatic capabilities is the cleavage of its own phosphodiester backbone via a transeserification reaction, as found in, for example, the hammerhead ribozyme (HHR) family (Figure 1.6A). The structural characteristics of HHR ribozymes are unique and have been found to be conserved in one form or another in all three boughs of the tree of life (49). A close look at the secondary and tertiary structure of HHRs again highlights the complexity attainable by the ribonucleic acid polymer (FIG 1.6B&C).

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