The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N-methyladenosine (mA) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor -adenosylmethionine (SAM).

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on mRNA in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM).

Structure-Based Design of Inhibitors of the mA-RNA Writer Enzyme METTL3.

Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, -methyladenosine (mA). METTL3 is the catalytic subunit that binds the co-substrate -adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The mA modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay.

Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity.

The cellular prion protein PrP mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrP (GDL) can also initiate neurotoxicity by inducing an intramolecular R -H hydrogen bond ("H-latch") between the α2-α3 and β2-α2 loops of PrP . Importantly, GDL that suppresses the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways.

1,4,9-Triazaspiro[5.5]undecan-2-one Derivatives as Potent and Selective METTL3 Inhibitors.

-methyladenosine (mA) is the most frequent of the 160 RNA modifications reported so far. Accumulating evidence suggests that the METTL3/METTL14 protein complex, part of the mA regulation machinery, is a key player in a variety of diseases including several types of cancer, type 2 diabetes, and viral infections. Here we report on a protein crystallography-based medicinal chemistry optimization of a METTL3 hit compound that has resulted in a 1400-fold potency improvement (IC of 5 nM for the lead compound () in a time-resolved Förster resonance energy transfer (TR-FRET) assay).

METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes.

The methylase METTL3 is the writer enzyme of the N -methyladenosine (m 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 m 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.

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

N6-Methyladenosine (mA) 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 mA 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.

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 N 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.

Selectively Disrupting mA-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 (mA) in RNA. The 30 binders are fragments (molecular weight < 300 g mol) 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 -NHCH group of mA.

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

-Methyladenosine (mA) 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 mA-containing RNAs to their readers are still elusive due to the unclear roles of mA-flanking ribonucleotides.

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state.

Structure-based drug design identifies polythiophenes as antiprion compounds.

Prions cause transmissible spongiform encephalopathies for which no treatment exists. Prions consist of PrP(Sc), a misfolded and aggregated form of the cellular prion protein (PrP(C)). We explore the antiprion properties of luminescent conjugated polythiophenes (LCPs) that bind and stabilize ordered protein aggregates.

Evolutionary conserved Tyr169 stabilizes the β2-α2 loop of the prion protein.

Experimental evidence indicates that the primary structure of the β2-α2 loop region (residues 165-175) in mammalian prion proteins (PrP) influences the conversion from the cellular species (PrP(C)) to the β-sheet-rich aggregate. Here, we captured the transition of the β2-α2 loop from 310-helical turn to β turn by unbiased molecular dynamics simulations of the single-point mutant Y169G. Multiple conformations along the spontaneous transition of the mutant were then used as starting point for sampling of the free-energy surface of the wild type and other single-point mutants.

Structural Analysis of the Binding of Type I, I1/2, and II Inhibitors to Eph Tyrosine Kinases.

We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out).

Structured water molecules in the binding site of bromodomains can be displaced by cosolvent.

Bromodomains are α-helical bundles of approximately 110 residues that recognize acetylated lysine side chains mainly on histone tails. Bromodomains are known to play an important role in cancer and inflammation, and as such, significant efforts are being made to identify small-molecule inhibitors of these epigenetic reader proteins. Here, explicit solvent molecular dynamics (MD) simulations of two bromodomains (BAZ2B and CREBBP) are used to analyze the water molecules that seem to be conserved at the bottom of the acetyl-lysine binding site in most crystal structures of bromodomains.

Does bromodomain flexibility influence histone recognition?

Bromodomains are protein modules that selectively recognize histones by binding to acetylated lysines. Here, we have carried out multiple molecular dynamics simulations of 20 human bromodomains to investigate the flexibility of their binding site. Some bromodomains show alternative side chain orientations of three evolutionarily conserved residues: the Asn involved in acetyl-lysine binding and two conserved aromatic residues.

Optimization of inhibitors of the tyrosine kinase EphB4. 2. Cellular potency improvement and binding mode validation by X-ray crystallography.

Inhibition of the tyrosine kinase erythropoietin-producing human hepatocellular carcinoma receptor B4 (EphB4) is an effective strategy for the treatment of solid tumors. We have previously reported a low nanomolar ATP-competitive inhibitor of EphB4 discovered in silico by fragment-based high-throughput docking combined with explicit solvent molecular dynamics simulations. Here we present a second generation of EphB4 inhibitors that show high inhibitory potency in both enzymatic and cell-based assays while preserving the appealing selectivity profile exhibited by the parent compound.

Discovery of tyrosine kinase inhibitors by docking into an inactive kinase conformation generated by molecular dynamics.

Several small molecules that bind to the inactive DFG-out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG-out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type-A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening.

How Does Darunavir Prevent HIV-1 Protease Dimerization?

The drug Darunavir (DRV) is a potent inhibitor of HIV-1 protease (PR), a homodimeric essential enzyme of the AIDS virus. Recent experimental data suggest that DRV is able to prevent dimerization of HIV-1 PR, which, together with its high affinity for the mature enzyme, has been linked to the high genetic barrier to the development of viral resistance. The mechanism of dimerization inhibition and the binding mode(s) of DRV to monomeric HIV-1 PR are unknown.

High-throughput virtual screening lead to discovery of non-peptidic inhibitors of West Nile virus NS3 protease.

The non-structural 3 protease is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus infections. In this chapter, we discuss in detail the computational methods used in the previous two docking campaigns which lead to the discovery of non-peptidic low micromolar inhibitors. Not only an X-ray structure but also an alternative conformation generated from molecular dynamic simulations is used in the in silico screening.

Hydrogen bonding penalty upon ligand binding.

Ligand binding involves breakage of hydrogen bonds with water molecules and formation of new hydrogen bonds between protein and ligand. In this work, the change of hydrogen bonding energy in the binding process, namely hydrogen bonding penalty, is evaluated with a new method. The hydrogen bonding penalty can not only be used to filter unrealistic poses in docking, but also improve the accuracy of binding energy calculation.

The free energy landscape of small molecule unbinding.

The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM.

PARP1 ADP-ribosylates lysine residues of the core histone tails.

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites.