Bisubstrate analogues as structural tools to investigate m⁶A methyltransferase active sites

Abstract

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m 6 A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m 6 A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m 6 A RNA MTases. The same analogues cause changes in the melting temperature of the m 1 A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m 6 A RNA MTase inhibition should aim to increase selectivity, as well as affinity.

Synthesis of adenine dinucleosides SAM analogs as specific inhibitors of SARS-CoV nsp14 RNA cap guanine-N7-methyltransferase

“…This modification presents the advantage of increasing the chemical stability of the linker and offers a possible variability of compounds due to the Nsubstitution by any chain susceptible to interact with additional protein residues (Scheme 1). It is noteworthy that bisubstrate dinucleosides with N-containing linkers have already been reported in transition state analogs of DNA methylation [15] and of RNA methylation at N6 position of adenosine [16,17]. However, till our work, none viral 2 0 O-MTase or N7-MTase have been targeted by bisubstrate adenine dinucleosides.…”

Synthesis of Triazole-Linked SAM-Adenosine Conjugates: Functionalization of Adenosine at N-1 or N-6 Position without Protecting Groups

“… ( A ) Our previous work: structure of SAM-adenosine conjugates with an alkyl linker [ 25 , 26 ]; ( B ) structure of SAM-adenosine conjugates with a 1,2,3-triazole linker synthesized in this study. …”

“…In this context, we recently described the synthesis of SAM-adenosine conjugates as first transition state analogues for m 6 A RNA MTases and their use as tools for structural study [ 25 , 26 ]. We showed that a SAM-adenosine conjugate containing a three-carbon linker tethering the analogue of SAM to the N-6 atom of the adenosine binds the bacterial RNA MTase RlmJ with a conformation close to the real transition state.…”

METTL3 inhibitors for epitranscriptomic modulation of cellular processes

“…9 To date, there are no cell-permeable inhibitors of METTL3/14 with a disclosed chemical structure except for the universal nucleoside analogue sinefungin, which inhibits most methyltransferases and is not selective for METTL3. 28,29 Therefore, all published studies of the role of m 6 A in cancer have relied on the knockdown/overexpression of the writers, erasers, and readers. In contrast to this approach, using small molecule inhibitors preserves the function of the target enzyme to act as a scaffold for protein-protein interactions that would otherwise be disrupted by RNAi, thus enabling discrimination between the enzymatic and structural roles.…”