Ligand enrichment among top-ranking hits is a key metric of molecular docking. To avoid bias, decoys should resemble ligands physically, so that enrichment is not simply a separation of gross features, yet be chemically distinct from them, so that they are unlikely to be binders. We have assembled a directory of useful decoys (DUD), with 2950 ligands for 40 different targets. Every ligand has 36 decoy molecules that are physically similar but topologically distinct, leading to a database of 98,266 compounds. For most targets, enrichment was at least half a log better with uncorrected databases such as the MDDR than with DUD, evidence of bias in the former. These calculations also allowed forty-by-forty cross docking, where the enrichments of each ligand set could be compared for all 40 targets, enabling a specificity metric for the docking screens. DUD is freely available online as a benchmarking set for docking at http://blaster.docking.org/dud/.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

A procedure to determine the electrostatic parameters has been developed for a polarizable empirical force field based on the classical Drude oscillator model. Atomic charges and polarizabilities for a given molecule of interest were derived from restrained fitting to quantum-mechanical electrostatic potentials (ESP) calculated at the B3LYP/ cc-pVDZ or B3LYP/aug-cc-pVDZ levels on grid points located on concentric Connolly surfaces. The determination of the atomic polarizabilities requires a series of perturbed ESP maps, each one representing the electronic response of the molecule in the presence of a background charge placed on Connolly surfaces primarily along chemical bonds and lone pairs. Reference values for the partial atomic charges were taken from the CHARMM27 additive all-atom force field, and those for the polarizabilities were based on adjusted Miller's ahp atomic polarizability values. The fitted values of atomic polarizabilities were scaled to reflect the reduced polarization expected for the condensed media and/or to correct for the systematic underestimation of experimental molecular polarizabilities by B3LYP calculations. Following correction of the polarizabilities, the atomic charges were adjusted to reproduce gas-phase dipole moments. The developed scheme has been tested on a set of small molecules representing functional moieties of nucleic acids. The derived electrostatic parameters have been successfully applied in a preliminary polarizable molecular dynamics simulation of a DNA octamer in a box of water with sodium counterions. Thus, this study confirms the feasibility of the use of a polarizable force field based on a classical Drude model for simulations of biomolecules in the condensed phase.

Molecular docking is the most practical approach to leverage protein structure for ligand discovery, but the technique retains important liabilities that make it challenging to deploy on a large scale. We have therefore created an expert system, DOCK Blaster, to investigate the feasibility of full automation. The method requires a PDB code, sometimes with a ligand structure, and from that alone can launch a full screen of large libraries. A critical feature is self-assessment, which estimates the anticipated reliability of the automated screening results using pose fidelity and enrichment. Against common benchmarks, DOCK Blaster recapitulates the crystal ligand pose within 2 Å rmsd 50−60% of the time; inferior to an expert, but respectrable. Half the time the ligand also ranked among the top 5% of 100 physically matched decoys chosen on the fly. Further tests were undertaken culminating in a study of 7755 eligible PDB structures. In 1398 cases, the redocked ligand ranked in the top 5% of 100 property-matched decoys while also posing within 2 Å rmsd, suggesting that unsupervised prospective docking is viable. DOCK Blaster is available at .

DNA methylation is involved in gene regulation in eukaryotes (1), protection of ''self'' DNA in prokaryotes (2), and it plays a role in the etiology of some cancers (3-5). For chemical modification of the target base being methylated to occur, as catalyzed by methyltransferases, it is necessary for the base to be flipped out of the DNA double helix (Fig. 1A) and into the enzyme's active site (6, 7). Base flipping is also important for other enzymes that chemically alter DNA, including DNAmismatch repair enzymes, such as uracil glycosylase and endonucleases (8). Base flipping may even be linked to early events in the opening and unwinding of DNA for transcription and replication processes (9, 10). Understanding the mechanism by which an enzyme can facilitate base flipping is, therefore, an important first step in elucidating more complex enzymecatalyzed DNA processing involved in transcription and replication.The cytosine 5-methyltransferase from the HhaI bacterium (M.HhaI) is the most studied enzyme among the DNA methyltransferases. The ternary complex of M.HhaI with DNA and the cofactor product S-adenosylhomocysteine (SAH) was the first system in which the structural phenomenon of base flipping was observed (11). Based on the this crystal structure, it was suggested that flipping of the target cytosine (target C) occurs via the minor groove of the DNA duplex. In uracil glycosylase, on the other hand, experimental evidence indicates that flipping occurs via the major groove (12, 13). Recent theoretical studies indicate that, in solution, flipping of the target C base out of either the minor or major groove of DNA have similar energetic barriers (14, 15). In addition, it is known that M.HhaI stabilizes the flipped conformation of the target C in the ternary complex (16). In the present work, the energetic consequences and structural events associated with base flipping in the presence of M.HhaI are studied to develop an atomistic structural model of enzymatic facilitation of the flipping process.Structural information available from experimental studies on base flipping in M.HhaI is mostly restricted to the endpoint states because of the higher energies and resulting short lifetimes of the intermediate states (17-19). Computational methods based on molecular dynamics (MD) simulations can access these structural intermediates and identify accompanying energetic changes at an atomic level of detail (20). However, the current standard of MD simulations does not allow access to the millisecond time scales at which these events occur (21,22). To overcome this limitation, the method of umbrella sampling (23) was applied in conjunction with MD simulations to drive flipping of the base out of the DNA double helix. In this approach, an external (i.e., umbrella) potential (24) is included in an MD simulation to maintain the base in high energy regions of the flipping free-energy surface not normally sampled. In the present work, the umbrella potential was based on a previously developed pseudodihedral angle, x, that allows...

Recent studies have established the involvement of the fat mass and obesity-associated gene (FTO) in metabolic disorders such as obesity and diabetes. However, the precise molecular mechanism by which FTO regulates metabolism remains unknown. Here, we used a structure-based virtual screening of U.S. Food and Drug Administration–approved drugs to identify entacapone as a potential FTO inhibitor. Using structural and biochemical studies, we showed that entacapone directly bound to FTO and inhibited FTO activity in vitro. Furthermore, entacapone administration reduced body weight and lowered fasting blood glucose concentrations in diet-induced obese mice. We identified the transcription factor forkhead box protein O1 (FOXO1) mRNA as a direct substrate of FTO, and demonstrated that entacapone elicited its effects on gluconeogenesis in the liver and thermogenesis in adipose tissues in mice by acting on an FTO-FOXO1 regulatory axis.

Virtual database screening allows for millions of chemical compounds to be computationally selected based on structural complimentary to known inhibitors or to a target binding site on a biological macromolecule. Compound selection in virtual database screening when targeting a biological macromolecule is typically based on the interaction energy between the chemical compound and the target macromolecule. In the present study it is shown that this approach is biased toward the selection of high molecular weight compounds due to the contribution of the compound size to the energy score. To account for molecular weight during energy based screening, we propose normalization strategies based on the total number of heavy atoms in the chemical compounds being screened. This approach is computationally efficient and produces molecular weight distributions of selected compounds that can be selected to be (1) lower than that of the original database used in the virtual screening, which may be desirable for selection of leadlike compounds or (2) similar to that of the original database, which may be desirable for the selection of drug-like compounds. By eliminating the bias in target-based database screening toward higher molecular weight compounds it is anticipated that the proposed procedure will enhance the success rate of computer-aided drug design.