Design, synthesis and evaluation of novel intercalator analogs targeting therapeutically important polymerases and folded RNA
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Polymerases and ribonucleic acids have been found to play major roles in the causation and progression of several diseases. Targeting such important therapeutic targets using small molecules can help achieve their specific recognition and inhibition thereby intercepting key stages involved in the progression of various diseases. This dissertation work is directed towards the design of intercalator based analogs that would allow specific targeting of important targets. Two such therapeutically important targets representing polymerases and RNA discussed in this dissertation are HIV-1 reverse transcriptase and miRNA-21. HIV-1 reverse transcriptase plays a key role in the viral life cycle by reverse transcribing viral RNA into replication competent cDNA via formation of a key RNA/DNA heteroduplex. Since reverse transcriptase remains in close association with this RNA/DNA heteroduplex during the reverse transcription step, targeting of this key heteroduplex could offer a unique approach to inhibit reverse transcriptase with high affinity and specificity. In this dissertation work, several bis-intercalator analogs with linkers of varying lengths and rigidities were designed, synthesized and evaluated for their inhibitory activity against reverse transcriptase. While strong intercalators such as ethidium and acridine-based analogs were able to achieve bis-intercalation, binding of such analogs appeared to be mostly driven by intercalation as opposed to the linkers connecting the two intercalators raising specificity concerns. Hence, multiple weak intercalators based bis-intercalator libraries were designed using weak intercalators such as tryptophan, naproxen and quinoxaline-2-carboxylic acid. Interesting leads were observed from tryptophan and quinoxaline-2-carboxylic acid based libraries. Tryptophan-based leads were non-promiscuous inhibitors, but unfortunately their testing against human DNA polymerase-α found these to be non-specific inhibitors of reverse transcriptase. Interesting leads were obtained from cyclic quinoxaline-2-carboxylic acid based bis-intercalator library with low μM affinities towards reverse transcriptase. Most importantly these leads were found to be both non-promiscuous and specific inhibitors of reverse transcriptase. The final portion of this dissertation details efforts towards developing a general tool for the sequence specific recognition of therapeutically important RNAs. For this effort multiple cyclic peptide based-libraries were designed, synthesized and screened against therapeutically important RNAs such as miRNA-21 using affinity selection chromatography. An important feature of these libraries was that these were devoid of any positive charges. Binding of molecules in such libraries would hence be driven by their ability to identify and map unique structural features of folded RNA such as bulges and loops.
Table of Contents
Introduction -- Inhibition of reverse transcriptase by targeting the key RNA/DNA heteroduplex -- Screening of strong intercalator based bis-intercalator libraries -- Design, synthesis and screening of multiple weak intercalator based bis-intercalator libraries -- Summary and conclusions of HIV-1 RT project -- Development of a general tool for sequence specific recognition of folded RNA using small molecules -- Design, synthesis and screening of 15 individual tetrapeptide libraries targeting binding pockets in folded RNA -- Summary and conclusions of RNA recognition project