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Abstract
The identification of molecules that can modulate RNA or protein function and the subsequent chemical and structural optimization to refine such molecules into drugs is a key activity in drug discovery. Here, we explored the extent to which chemical and structural differences in antisense oligonucleotides, designed as gapmers and capable of recruiting RNase H for target RNA cleavage, can affect their functional properties. To facilitate structure-activity learning, we analyzed two sets of iso-sequential locked nucleic acid (LNA)-modified gapmers, where we systematically varied the number and positions of LNA modifications in the flanks. In total, we evaluated 768 different and architecturally diverse gapmers in HeLa cells for target knockdown activity and cytotoxic potential and found widespread differences in both of these properties. Binding affinity between gapmer and RNA target, as well as the presence of certain short sequence motifs in the gap region, can explain these differences, and we propose statistical and machine-learning models that can be used to predict region-specific, optimal LNA-modification architectures. Once accessible regions in the target of interest have been identified, our results show how to refine and optimize LNA gapmers with improved pharmacological profiles targeting such regions.
Highlights
In traditional small-molecule drug discovery, large libraries of chemical compounds are screened for activity in cellular assays to identify compound hits that are active against a biological target.[1]
For antisense oligonucleotide drug discovery, we recently proposed a similar process, where diverse libraries are screened to identify oligonucleotide hits, which can be further chemically optimized until oligonucleotides with acceptable drug-like properties are identified.[4]
We designed all possible combinations of gapmers having either DNA or locked nucleic acid (LNA) at positions 2 to 5 from the end of each flank, indicated by x in Figures 1A and 1B, resulting in 28 = 256 different LNA modification architectures targeted to each region
Summary
In traditional small-molecule drug discovery, large libraries of chemical compounds are screened for activity in cellular assays to identify compound hits that are active against a biological target.[1] High diversity in shape and chemical structure between compounds has been found to increase the probability of finding such hits.[2] The compound hits identified from the initial screening are analyzed for structure-activity relationships with the target and used as structural basis for generation of new libraries These libraries are screened for a wider range of properties to identify lead molecules, which may be further optimized structurally before final selection of a clinical drug candidate.[3]. Antisense oligonucleotides (AONs) are short chemically modified oligonucleotides, designed to bind by Watson-Crick base pairing to a perfectly complementary region in an RNA target of interest and modulate its function.[5,6] We here focus on AONs designed as gapmers with a central gap region of at least six consecutive DNAs to allow the endogenous ribonuclease H1 enzyme (RNase H1) to bind to the heteroduplex between gapmer and target RNA and cleave the RNA strand.[7,8]
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