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

Genome 3′-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication

The Role of Furin in the Pathogenesis of COVID-19-Associated Neurological Disorders

Neurological disorders have been reported in a large number of coronavirus disease 2019 (COVID-19) patients, suggesting that this disease may have long-term adverse neurological consequences. COVID-19 occurs from infection by a positive-sense single-stranded RNA virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The membrane fusion protein of SARS-CoV-2, the spike protein, binds to its human host receptor, angiotensin-converting enzyme 2 (ACE2), to initiate membrane fusion between the virus and host cell. The spike protein of SARS-CoV-2 contains the furin protease recognition site and its cleavage enhances the infectivity of this virus. The binding of SARS-CoV-2 to the ACE2 receptor has been shown to downregulate ACE2, thereby increasing the levels of pathogenic angiotensin II (Ang II). The furin protease cleaves between the S1 subunit of the spike protein with the binding domain toward ACE2 and the S2 subunit with the transmembrane domain that anchors to the viral membrane, and this activity releases the S1 subunit into the blood circulation. The released S1 subunit of the spike protein also binds to and downregulates ACE2, in turn increasing the level of Ang II. Considering that a viral particle contains many spike protein molecules, furin-dependent cleavage would release many free S1 protein molecules, each of which can downregulate ACE2, while infection with a viral particle only affects one ACE2 molecule. Therefore, the furin-dependent release of S1 protein would dramatically amplify the ability to downregulate ACE2 and produce Ang II. We hypothesize that this amplification mechanism that the virus possesses, but not the infection per se, is the major driving force behind COVID-19-associated neurological disorders

Dengue Virus Nonstructural Protein 5 (NS5) Assembles into a Dimer with a Unique Methyltransferase and Polymerase Interface

<div><p>Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.</p></div

Comparison of DENV and JEV NS5 structures.

<p><b>(A)</b> Superposition of DENV and JEV NS5. DENV and JEV NS5 (PDB code 4K6M) are aligned by the MTase domains only (rmsd of 0.99 Å for 253 Cα atoms). The DENV NS5 structure is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.g002" target="_blank">Fig 2A</a>, and the JEV NS5 is in gray. With the MTase domains superimposed, the RdRp domains of DENV and JEV NS5 are related by a rotation of 102° and a ~5Å translation along the linker region (residues 262–272). A close-up view of the boxed area is shown in (B). <b>(B)</b> The linker regions in DENV and JEV NS5. Residues comprising the linker regions of DENV and JEV NS5 are shown in orange and gray, respectively. Both structures use the ²⁶⁰GTR²⁶² pivot, following which the two structures diverge. The dotted line indicates the break in the JEV NS5 linker. <b>(C)</b> Comparison of DENV and JEV NS5 structures. To emphasize the relative orientations of the MTase and RdRp domains, SAH molecules that occupy an identical binding site in both structures are shown in yellow. P113, P115 (or L115 in JEV NS5), and W121 in the MTase are implicated in viral replication and shown in magenta spheres in ribbon diagrams or magenta surfaces in surface representations. These residues in DENV NS5 are located in the dimer interface, while those in JEV NS5 are located in the domain interface.</p

Serotype–specific interactions in DENV NS5.

<p>(A) Immunofluorescence assay. DENV2 RNA encoding the wild-type (DENV2) NS5, a chimera NS5 containing DENV4 MTase, or DENV4 NS5 were transfected into BHK-21 cells, and viral replication was visualized by immunofluorescence assay using anti NS1 antibodies. (B) Serotype-specific NS5 residues. Serotype-specific residues (boxed) are conserved within one DENV serotype, but not conserved across serotypes. Please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#sec012" target="_blank">Methods</a> section for details of sequence alignment and determination of serotype-specific residues. (C) Serotype-specific residues for at least two serotypes of DENV NS5 are mapped on the 3-D structure of the DENV3 NS5 dimer. One monomer is colored in cyan (MTase domain) and yellow (RdRp domain), and the other in gray. Serotype-specific residues are colored in red and labeled. The NLS (residues 369–406) is colored in blue.</p

DENV NS5 monomer.

<p><b>(A)</b> Overall fold of the DENV NS5 monomer. The ribbon diagram of the NS5 monomer is shown looking through the canonical right hand configuration (left) and through the top of the RdRp (right). The MTase domain is shown in cyan, and the RdRp domain is colored by region (thumb, red; palm, green; fingers, blue; domain linker, orange; priming loop, yellow). SAH is shown as a space-filling model and colored by atom type. Two zinc ions are shown as gray spheres. Schematic of the NS5 domains is shown below. <b>(B)</b> Polymerase activities of NS5 proteins. Polymerase activities of NS5 proteins were measured using a subgenomic RNA as a template, as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.ref027" target="_blank">27</a>]. Lanes: 1, no polymerase; 2, NS5 RdRp domain; 3, wild-type NS5; 4, NS5-Δ2; 5, NS5-Δ6. Please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#sec012" target="_blank">Materials and Methods</a> for specific deletions. (C) Comparison of RdRp domains of DENV and JEV NS5. The RdRp domains of the two DENV (the current structure and PDB code 4V0Q) and JEV (PDB code 4K6M) are compared. The previously disordered regions including motif F (green), motif G (blue), linker (orange), and the C-terminus (red) are labeled. Since none of the DENV NS5 monomers included both motif G and the C-terminal helix, we created a model of the monomer by grafting motif G from monomer C onto monomer G, which included the C-terminal helix; the resulting model thus has both motif G and the C-terminus. Motif F in DENV NS5 is disordered and shown as a dotted line. The priming loop is shown in purple. <b>(D)</b> Stereoview of the linker between the MTase and RdRp domains. The residues 260–272 are shown in orange, and the final 2<i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> omit map for the linker region is shown as a blue mesh contoured at 1.0 σ. MTase and RdRp residues that interact with the linker are colored as in (A). Hydrogen bonds are indicated by dashed lines. <b>(E)</b> Movement of the thumb subdomain in flavivirus NS5. The RdRp domain of DENV NS5 (chain B, blue) was aligned with the JEV RdRp domain (orange) by fingers and palm subdomains (rmsd = 0.56 Å for 333 Cα atoms). The thumb subdomain of DENV NS5 is rotated by 7° around W700 (hinge residue, blue sphere) compared to the JEV thumb subdomain.</p

NS5 dimer interactions.

<p><b>(A)</b> Type I dimer. All eight NS5 monomers are involved in type I dimer interaction (AB, CD, EF, and GH). One monomer is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.g002" target="_blank">Fig 2A</a> and the other is in gray. Residues of potential physiological significance are shown as magenta (MTase) or gold (RdRp) spheres, and labeled with corresponding one-letter residue codes. The close-up view of the type I dimer interface is shown as sticks (right), and hydrogen bonds are indicated by dashed lines. <b>(B)</b> Type II dimer. The type II dimers are observed only between monomers A and F and between D and G. The close-up view of the C-terminal residues involved in the dimer interface is shown as sticks (right).</p

NS5 monomer and dimer models in the replication complex.

<p><b>(A)</b> Schematics of the NS5 monomer and dimer models. Two conformations of the MTase relative to the RdRp observed in DENV and JEV NS5 structures in the monomer model are indicated by black and gray lines. The template entry and dsRNA product exit sites on the RdRp are indicated by arrows. The MTase active site is represented by a concave region. <b>(B)</b> DENV NS5 dimer with bound RNA model. To show molecular boundaries, one monomer is shown as a ribbon diagram, and the other as a molecular surface. The MTase active site is indicated by the bound SAH (yellow) and a short RNA (orange). The single-stranded RNA was modeled in by superposition of the MTase domain in our structure and the isolated MTase domain-RNA complex (PDB code 2XBM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005451#ppat.1005451.ref045" target="_blank">45</a>].</p