METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes.

The methylase METTL3 is the writer enzyme of the N6 -methyladenosine (m6 A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose-dependent reduction in the m6 A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m1 A, m6 Am , m7 G), suggesting selectivity of the developed compound towards other RNA methyltransferases.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir.

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

Atomistic and Thermodynamic Analysis of N6-Methyladenosine (m6A) Recognition by the Reader Domain of YTHDC1.

N6-Methyladenosine (m6A) is the most frequent modification in eukaryotic messenger RNA (mRNA) and its cellular processing and functions are regulated by the reader proteins YTHDCs and YTHDFs. However, the mechanism of m6A recognition by the reader proteins is still elusive. Here, we investigate this recognition process by combining atomistic simulations, site-directed mutagenesis, and biophysical experiments using YTHDC1 as a model. We find that the N6 methyl group of m6A contributes to the binding through its specific interactions with an aromatic cage (formed by Trp377 and Trp428) and also by favoring the association-prone conformation of m6A-containing RNA in solution. The m6A binding site dynamically equilibrates between multiple metastable conformations with four residues being involved in the regulation of m6A binding (Trp428, Met438, Ser378, and Thr379). Trp428 switches between two conformational states to build and dismantle the aromatic cage. Interestingly, mutating Met438 and Ser378 to alanine does not alter m6A binding to the protein but significantly redistributes the binding enthalpy and entropy terms, i.e., enthalpy-entropy compensation. Such compensation is reasoned by different entropy-enthalpy transduction associated with both conformational changes of the wild-type and mutant proteins and the redistribution of water molecules. In contrast, the point mutant Thr379Val significantly changes the thermal stability and binding capability of YTHDC1 to its natural ligand. Additionally, thermodynamic analysis and free energy calculations shed light on the role of a structural water molecule that synergistically binds to YTHDC1 with m6A and acts as the hub of a hydrogen-bond network. Taken together, the experimental data and simulation results may accelerate the discovery of chemical probes, m6A-editing tools, and drug candidates against reader proteins.

Hitting a Moving Target: Simulation and Crystallography Study of ATAD2 Bromodomain Blockers.

Small molecule ligand binding to the ATAD2 bromodomain is investigated here through the synergistic combination of molecular dynamics and protein crystallography. A previously unexplored conformation of the binding pocket upon rearrangement of the gatekeeper residue Ile1074 has been found. Further, our investigations reveal how minor structural differences in the ligands result in binding with different plasticity of the ZA loop for this difficult-to-drug bromodomain.

Small-Molecule Inhibitors of METTL3, the Major Human Epitranscriptomic Writer.

The RNA methylase METTL3 catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to the N6 atom of adenine. We have screened a library of 4000 analogues and derivatives of the adenosine moiety of SAM by high-throughput docking into METTL3. Two series of adenine derivatives were identified in silico, and the binding mode of six of the predicted inhibitors was validated by protein crystallography. Two compounds, one for each series, show good ligand efficiency. We propose a route for their further development into potent and selective inhibitors of METTL3.

Selectively Disrupting m6A-Dependent Protein-RNA Interactions with Fragments.

We report a crystallographic analysis of small-molecule ligands of the human YTHDC1 domain that recognizes N6-methylated adenine (m6A) in RNA. The 30 binders are fragments (molecular weight < 300 g mol-1) that represent 10 different chemotypes identified by virtual screening. Despite the structural disorder of the binding site loop (residues 429-439), most of the 30 fragments emulate the two main interactions of the -NHCH3 group of m6A. These interactions are the hydrogen bond to the backbone carbonyl of Ser378 and the van der Waals contacts with the tryptophan cage. Different chemical groups are involved in the conserved binding motifs. Some of the fragments show favorable ligand efficiency for YTHDC1 and selectivity against other m6A reader domains. The structural information is useful for the design of modulators of m6A recognition by YTHDC1.

Flexible Binding of m6A Reader Protein YTHDC1 to Its Preferred RNA Motif.

N6-Methyladenosine (m6A) is the most prevalent chemical modification in human mRNAs. Its recognition by reader proteins enables many cellular functions, including splicing and translation of mRNAs. However, the binding mechanisms of m6A-containing RNAs to their readers are still elusive due to the unclear roles of m6A-flanking ribonucleotides. Here, we use a model system, YTHDC1 with its RNA motif 5'-G-2G-1(m6A)C+1U+2-3', to investigate the binding mechanisms by atomistic simulations, X-ray crystallography, and isothermal titration calorimetry. The experimental data and simulation results show that m6A is captured by an aromatic cage of YTHDC1 and the 3' terminus nucleotides are stabilized by cation-π-π interactions, while the 5' terminus remains flexible. Notably, simulations of unbound RNA motifs reveal that the methyl group of m6A and the 5' terminus shift the conformational preferences of the oligoribonucleotide to the bound-like conformation, thereby facilitating the association process. The binding mechanisms may help in the discovery of chemical probes against m6A reader proteins.

Ligand retargeting by binding site analogy.

The DNA-repair enzyme MutT homolog 1 (MTH1) is a potential target for a broad range of tumors. Its substrate binding site features a non-catalytical pair of aspartic acids which resembles the catalytic dyad of aspartic proteases. We hypothesized that inhibitors of the latter might be re-targeted for MTH1 despite the two enzyme classes having different substrates and catalyze different reactions. We selected from the crystal structures of holo aspartic proteases a library of nearly 350 inhibitors for in silico screening. Three fragment hits were identified by docking and scoring according to a force field-based energy with continuum dielectric solvation. These fragments showed good ligand efficiency in a colorimetric assay (MW <300 Da and IC50<50µM). Molecular dynamics simulations were carried out for determining the most favorable interaction patterns. On the basis of the simulation results we evaluated in vitro seven commercially available compounds, two of which showed submicromolar potency for MTH1. To obtain definitive evidence of the predicted binding modes we solved the crystal structures of five of the 10 inhibitors predicted in silico. The final step of hit optimization was guided by protein crystallography and involved the synthesis of a single compound, the lead 11, which shows nanomolar affinity for MTH1 in two orthogonal binding assays, and selectivity higher than 2000-fold against its original target (BACE1). The high rate of fragment-hit identification and the fast optimization suggest that ligand retargeting by binding site analogy is an efficient strategy for drug design.

A Reader-Based Assay for m6A Writers and Erasers.

We have developed a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay to measure the catalytic activity of N6-methyladenosine (m6A) methyltransferases and demethylases. The assay detects m6A modifications using the natural m6A-binding proteins (m6A readers). The reaction product or substrate m6A-containing RNA and the m6A reader protein are fluorescently labeled such that their proximity during binding initiates Förster resonance energy transfer (FRET). We show that our HTRF assay can be used for high-throughput screening, which will facilitate the discovery of small-molecule modulators of m6A (de)methylases.

Fragment-Based Design of Selective Nanomolar Ligands of the CREBBP Bromodomain.

Novel ligands of the CREBBP bromodomain were identified by fragment-based docking. The in silico discovered hits have been optimized by chemical synthesis into selective nanomolar compounds, thereby preserving the ligand efficiency. The selectivity for the CREBBP bromodomain over other human bromodomain subfamilies has achieved by a benzoate moiety which was predicted by docking to be involved in favorable electrostatic interactions with the Arg1173 side chain, a prediction that could be verified a posteriori by the high-resolution crystal structure of the CREBBP bromodomain in complex with ligand 6 and also by MD simulations (see Xu, M.; Unzue, A.; Dong, J.; Spiliotopoulos, D.; Nevado, C.; Caflisch, A. Discovery of CREBBP bromodomain inhibitors by high-throughput docking and hit optimization guided by molecular dynamics. J. Med. Chem. 2015, DOI: 10.1021/acs.jmedchem.5b00171).