AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Structural and Biochemical Insights into the Peptidoglycan Hydrolase Domain of FlgJ from Salmonella typhimurium.

FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis

Independent inhibition of the polymerase and deubiquitinase activities of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein.

BACKGROUND:The Crimean-Congo hemorrhagic fever virus (CCHFV) is a segmented negative-sense RNA virus that can cause severe human disease. The World Health Organization (WHO) has listed CCHFVas a priority pathogen with an urgent need for enhanced research activities to develop effective countermeasures. Here we adopted a biochemical approach that targets the viral RNA-dependent RNA polymerase (RdRp). The CCHFV RdRp activity is part of a multifunctional L protein that is unusually large with a molecular weight of ~450 kDa. The CCHFV L-protein also contains an ovarian tumor (OTU) domain that exhibits deubiquitinating (DUB) activity, which was shown to interfere with innate immune responses and viral replication. We report on the expression, characterization and inhibition of the CCHFV full-length L-protein and studied both RNA synthesis and DUB activity. METHODOLOGY/PRINCIPLE FINDINGS:Recombinant full-length CCHFV L protein was expressed in insect cells and purified to near homogeneity using affinity chromatography. RdRp activity was monitored with model primer/templates during elongation in the presence of divalent metal ions. We observed a 14-mer full length RNA product as well as the expected shorter products when omitting certain nucleotides from the reaction mixture. The D2517N mutation of the putative active site rendered the enzyme inactive. Inhibition of RNA synthesis was studies with the broad-spectrum antivirals ribavirin and favipiravir that mimic nucleotide substrates. The triphosphate form of these compounds act like ATP or GTP; however, incorporation of ATP or GTP is markedly favored over the inhibitors. We also studied the effects of bona fide nucleotide analogues 2'-deoxy-2'-fluoro-CTP (FdC) and 2'-deoxy-2'-amino-CTP and demonstrate increased inhibitory effects due to higher rates of incorporation. We further show that the CCHFV L full-length protein and the isolated OTU domain cleave Lys48- and Lys63-linked polyubiqutin chains. Moreover, the ubiquitin analogue CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of DUB activity does not affect elongation of RNA synthesis, and inhibition of RNA synthesis does not affect DUB activity. Both domains are functionally independent under these conditions. CONCLUSIONS/SIGNIFICANCE:The requirements for high biosafety measures hamper drug discovery and development efforts with infectious CCHFV. The availability of full-length CCHFV L-protein provides an important tool in this regard. High-throughput screening (HTS) campaigns are now feasible. The same enzyme preparations can be employed to identify novel polymerase and DUB inhibitors

Structural description of <i>St</i>FlgJ_{GH(151–316)}.

<p>A) Cartoon diagram of the FlgJ GH domain from <i>Salmonella typhimurium</i> (<i>St</i>FlgJ_{GH(151–316)}) (blue) is shown with the catalytic glutamate (E184; orange), within the active site groove. Circled is the incomplete β-hairpin, with missing residues 223–229, that forms half of the active site groove. B) All molecules within the crystal structure were found to have their active sites blocked <i>in trans</i> by the C-terminal α-helix (red) of a neighbouring symmetry mate (grey) (conserved catalytic residue E184 is orange). C) <i>In trans</i> blockage of the GH domain active site of <i>Sp</i>FlgJ (grey carbons) by the C-terminus of a symmetry mate (green carbons) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref021" target="_blank">21</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref023" target="_blank">23</a>]. D) <i>In trans</i> blockage of the GH domain active site of Auto (grey carbons) by the N-terminal inhibitory helix of a symmetry mate (magenta carbons) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref002" target="_blank">2</a>]. Structural models were generated using PyMol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref028" target="_blank">28</a>].</p

Structural comparison of GH73 enzymes.

<p>A superposition of <i>St</i>FlgJ_{GH(151–301)} (PDB: 5DN5; green), Auto (PDB: 3FI7; orange)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref002" target="_blank">2</a>] and <i>Sp</i>FlgJ (PDB: 3VWO; yellow). β-hairpin residues (E223 for <i>St</i>FlgJ_{GH(151–301)}, E156 for Auto, and E224 for <i>Sp</i>FlgJ) are shown on each β-hairpin. Also superposed is the structure of <i>St</i>FlgJ_{GH(151–316)} (PDB: 5DN4; blue). Superposition using the DaliLight server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref029" target="_blank">29</a>] showed, for Auto: C_α rmsd of 3.3 Å over 140 equivalent positions to <i>St</i>FlgJ_{GH(151–301)}; <i>Sp</i>FlgJ: C_α rmsd of 2.3 Å over 140 equivalent positions to <i>St</i>FlgJ_{GH(151–301)}.</p

Crystal packing of <i>St</i>FlgJ_{GH(151–301)}.

<p>A) Packing of asymmetric units (ASUs) within the crystal structure of the C-terminally truncated mutant, <i>St</i>FlgJ_{GH(151–301)}. Each ASU is comprised of three copies of <i>St</i>FlgJ_{GH(151–301)}. One ASU is coloured green with three neighbouring ASUs coloured grey for clarity. B) The β-hairpin of each monomer of each ASU packs identically against the active site of a monomer within a neighbouring symmetry related ASU (grey) within the crystal structure. This packing imparts order to β-hairpin of each monomer while it partly occluding the active site of each copy of the protein in the crystal (conserved active site residue E184 shown in orange).</p

Crystallographic data and model refinement statistics.

<p>Crystallographic data and model refinement statistics.</p

Spatial conservation of the N- and C-termini of GH73 enzymes.

<p>A superposition of <i>St</i>FlgJ_{GH(151–316)} (PDB: 5DN4; blue) with Auto (PDB: 3FI7; orange) and FlgJ from <i>Sphingomonas</i> sp., <i>Sp</i>FlgJ (PDB: 2ZYC; yellow), reveal that the N- and C-terminal regions of the enzymes are spatially conserved. Auto and <i>Sp</i>FlgJ share high structural similarity to <i>St</i>FlgJ_{GH(151–316)} with RMSD values of 2.2 Å and 3.6 Å, respectively. For Auto and <i>Sp</i>FlgJ, 133 and 154 C_α positions were aligned to <i>St</i>FlgJ_{GH(151–316)}, respectively. All superpositions were generated using the DaliLight server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149204#pone.0149204.ref029" target="_blank">29</a>].</p

Superpositions of the viral OTU proteases with yeast OTU1 and one another.

<p>Superpositions of yeast OTU1 (3BY4) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-Messick1" target="_blank">[57]</a> with (<b>A</b>) CCHFV OTU (3PT2) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-James1" target="_blank">[23]</a>, RMSD: 1.8 Å over 112 residues, (<b>B</b>) EAV PLP2 (4IUM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-vanKasteren2" target="_blank">[26]</a>, RMSD: 2.8 Å over 69 residues, and (<b>C</b>) TYMV PRO (4A5U) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-Robin1" target="_blank">[27]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-Lombardi1" target="_blank">[28]</a>, RMSD: 1.4 Å over 76 residues. Superpositions of the yeast OTU1-Ub complex with (<b>D</b>) the CCHFV OTU-Ub complex and (<b>E</b>) the EAV PLP2-Ub complex, highlighting the difference in the orientation of Ub between the two viral OTU domains versus the eukaryotic yeast OTU1 domain. The Ub that is complexed with yeast OTU1 is depicted in yellow, while the Ub complexed with CCHFV OTU or EAV PLP2 is depicted in orange. (<b>F</b>) Superposition of EAV PLP2 and TYMV PRO, RMSD: 2.5 Å over 53 residues. (<b>G</b>) Close-up of the active site region (boxed) of the superposition depicted in <b>F</b>. Side chains of the catalytic cysteine (Cys270 and Cys783 for EAV PLP2 and TYMV PRO, respectively) and histidine (His332 and His869 for EAV PLP2 and TYMV PRO, respectively) residues are shown as sticks, as well as the active site Asn263 for EAV PLP2. The backbone amide group of Asp267 likely contributes to the formation of the oxyanion hole in the active site of EAV PLP2, yet a functionally equivalent residue is absent in TYMV PRO. The Gly266 and Gly268 residues flanking Asp267 in EAV PLP2 are depicted as sticks as well, for clarity. Note the alternative orientation of the active site cysteine residue of TYMV PRO which, unlike EAV PLP2, was not determined in covalent complex with an Ub suicide substrate. All alignments were generated using the PDBeFOLD server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-Krissinel1" target="_blank">[64]</a>, and thus the reported RMSD values differ from those reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat-1003894-t001" target="_blank">Table 1</a>, in which the DALI server was used. The yeast OTU1, CCHFV OTU, and EAV PLP2 domains were all crystallized in complex with Ub, which has been removed in panels <b>A</b>, <b>B</b>, <b>C</b>, <b>F</b>, and <b>G</b> for clarity. All images were generated using PyMol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003894#ppat.1003894-DeLano1" target="_blank">[60]</a>. RMSD, root-mean-square deviation.</p