Selective manipulation of the epitranscriptome could
be beneficial
for the treatment of cancer and also broaden the understanding of
epigenetic inheritance. Inhibitors of the tRNA methyltransferase DNMT2,
the enzyme catalyzing the S-adenosylmethionine-dependent
methylation of cytidine 38 to 5-methylcytidine, were designed, synthesized,
and analyzed for their enzyme-binding and -inhibiting properties.
For rapid screening of potential DNMT2 binders, a microscale thermophoresis
assay was established. Besides the natural inhibitors S-adenosyl-l-homocysteine (SAH) and sinefungin (SFG), we
identified new synthetic inhibitors based on the structure of N-adenosyl-2,4-diaminobutyric acid (Dab). Structure–activity
relationship studies revealed the amino acid side chain and a Y-shaped
substitution pattern at the 4-position of Dab as crucial for DNMT2
inhibition. The most potent inhibitors are alkyne-substituted derivatives,
exhibiting similar binding and inhibitory potencies as the natural
compounds SAH and SFG. CaCo-2 assays revealed that poor membrane permeabilities
of the acids and rapid hydrolysis of an ethylester prodrug might be
the reasons for the insufficient activity in cellulo.
Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations.
Developing methyltransferase inhibitors is challenging,
since most
of the currently used assays are time-consuming and cost-intensive.
Therefore, efficient, fast, and reliable methods for screenings and
affinity determinations are of utmost importance. Starting from a
literature-known fluorescent S-adenosylhomocysteine
derivative, 5-FAM-triazolyl-adenosyl-Dab, developed for a fluorescence
polarization assay to investigate the histone methyltransferase mixed-lineage
leukemia 1, we herein describe the applicability of this compound
as a fluorescent tracer for the investigation of DNA-methyltransferase
2 (DNMT2), a human RNA methyltransferase. Based on these findings,
we established a microscale thermophoresis (MST) assay for DNMT2.
This displacement assay can circumvent various problems inherent to
this method. Furthermore, we optimized a screening method via MST
which even indicates if the detected binding is competitive and gives
the opportunity to estimate the potency of a ligand, both of which
are not possible with a direct binding assay.
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