Part I: enhancing repair of oxidate DNA damage with small molecule activators of MTH1; part II: development of 2'OH acylating probes for detection of RNA-small molecule interactions and cellular delivery of mRNA
Keywords: Chemistry.

Chemical tools have been developed to modulate biomolecules including protein and RNA, often for therapeutic implications. Small molecule leads are optimized to improve target activity, selectivity, and pharmacological properties by reiterative synthesis of structural analogs and biological assessment. While a small molecule drug aims to affect one therapeutic target, it can cause unintended biological effects, so understanding a drug's off-target profile is critical to better understand its pharmacology. In addition to small molecules, RNA is a powerful therapeutic modality and chemical modifications are necessary to optimize RNA for therapeutic purposes. This work encompasses syntheses and applications of chemical tools for DNA repair enzyme activation, drug-RNA interaction profiling, and mRNA modifications for cellular delivery. Chapter 1 describes the development of small molecule activators for the DNA repair enzyme MTH1. Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. In this chapter, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-dGTP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogs of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 μM and 410 ± 60% at 5 µM. The compounds are found to increase activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks. This work has been reprinted with permission from ACS Chem. Biol. 2022, 17, 2074-2087. Copyright 2022 American Chemical Society. Chapter 2 describes the design and synthesis of chemical probes for profiling interactions of small molecule drug and RNA. Off-target interaction of small molecule drugs with the transcriptome is underexplored. A known RNA binder, SMN-C5, was conjugated to a linker with an acylating group for covalent modification of interacting RNA, and clickable azide group for pull down. Treatment of the probe enriched target RNA SMN2, and unmodified drug effectively impeded its enrichment. Acylation was driven by drug affinity and the linker design was applied to small molecule drugs for profiling in the transcriptome. Chapter 3 describes exploration of 2'OH acylation tools for cellular delivery of mRNA. N-(3-azidopropyl)-N-methylglycine derived acylating reagent (PMG-N3) and N, N-dimethylglycine derived acylating reagent (DMG) were explored to stabilize mRNA, decorate mRNA with delivery agents Arg8 and Tri-GalNAc, and recover functionality in cellular environment. Both PMG-N3 and DMG were not adequately efficient in deacylation ("uncloaking"). Also, Tri-GalNAc modifications along poly A tail was not sufficient for cellular delivery. Future efforts will investigate second generations of DMG and PMG-N3 acylating reagents with more efficient uncloaking, their capability of stabilizing mRNA, and varied levels of Arg8 and Tri-GalNAc labeling necessary for cellular delivery