Nanoscale libraries and novel drug modalities for targeting RNA methyltransferases
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Abstract
In recent years, epitranscriptomics has emerged as a rapidly evolving field of significant relevance to
biomedical research. The research primarily concerns the methylation of ribonucleic acid (RNA), a
process catalyzed by RNA methyltransferases (Mtases). Members of this enzyme family have been
implicated in a wide range of pathological conditions, including cancer, making them promising targets
for medicinal chemistry. This dissertation focused on developing and applying innovative strategies to
identify novel inhibitors of RNA Mtases. The results are divided into three distinct projects.
Project 1: Direct-to-Biology (D2B). In the first project, the D2B strategy, originally described in 2021,
was applied to two distinct subprojects. The first subproject aimed to identify fluorescent tracers suitable
for high-throughput screening (HTS) of disease-associated RNA Mtases. Two novel fluorescent tracers
were developed for a variety of RNA Mtases using a copper(I)-catalyzed azide-alkyne cycloaddition
(CuAAC) synthesis strategy. This approach enabled an HTS campaign against methyltransferase-like 1
(METTL1), resulting in the identification of (S)-crizotinib as a first-in-class inhibitor. In the second
subproject, inhibitors of Staphylococcus aureus tRNA methyltransferase D (TrmD) were identified. The
fluorescent tracer developed in the first subproject served as a starting point for a D2B-based strategy
targeting S. aureus TrmD. A CuAAC-based D2B approach led to the discovery of five promising hit
compounds. Moreover, a carbamate prodrug design was employed, yielding the first prodrug targeting
TrmD, which effectively inhibited the growth of S. aureus. Lastly, the first crystal structure of S. aureus
TrmD in complex with the active inhibitor was elucidated.
Project 2: DNA-Encoded-Library (DEL) Screening. In the second project, DEL technology was
employed to identify inhibitors of deoxyribonucleic acid (DNA) methyltransferase 2 (DNMT2) with
novel chemotypes. This approach resulted in the identification of five selective DNMT2 binders,
featuring triazin- and peptidomimetic-based core structures. Among these, only the peptidomimetic
compounds demonstrated inhibitory activity, prompting subsequent X-ray crystallographic analysis.
Structural characterization revealed an allosteric inhibition mechanism for the lead compound, which
guided a structure-activity relationship (SAR) study conducted via solid-phase peptide synthesis. The
optimized compound from this study exhibited not only enhanced binding affinity but also, as a
distinguishing feature, permeability in a parallel artificial membrane permeability assay (PAMPA).
Follow-up cellular assays confirmed that the compound effectively reduced m5C levels in tRNA and
exhibited synergistic effects in MOLM-13 cells when combined with doxorubicin.
Project 3: Novel Modalities for Medicinal Chemistry of RNA and RNA Methyltransferases. In the
third project, two novel strategies were explored to expand the repertoire of tools for targeting RNA
Mtases. The first approach focused on the degradation of METTL3 using a proteolysis-targeting chimera
(PROTAC) strategy, employing the known METTL3 inhibitor STM2457 as a recruiter ligand. Iterative
optimization across four generations led to the development of AF 151, a potent and selective METTL3
degrader. A second, conceptually distinct strategy was conducted to enable the characterization of RNAbinding
ligands and RNA modifications. This approach utilizes a mix-and-measure assay with noncovalent
RNA dyes, which facilitates the quantification of various RNA modifications, including m6A,
mediated by the METTL3/14 complex, among other applications.