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

Modular disordered domains autoregulate the RNA binding and annealing activities of the bacterial RNA chaperone Hfq

The RNA chaperone Hfq is a bacterial Sm protein that stabilizes small regulatory RNAs (sRNAs) and helps them bind with their mRNA targets. Hfq and sRNAs regulate how bacteria respond to stress or changes in their environment and are involved in the virulence of a wide range of pathogenic and environmentally important bacteria. Like many RNA-binding proteins, Hfq consists of both structured, and unstructured components. While the RNA binding and annealing properties of the structured Sm core of Hfq has been well characterized, the function of the disordered C-terminal domain (CTD) has remained unclear. Here I use stopped flow spectroscopy to show that the CTD of Escherichia coli and Caulobacter crescentus Hfqs are not needed to accelerate RNA base pairing as had been previously proposed, but rather are required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. This displacement allows Hfq to search among potential RNA partners and establishes a hierarchy of sRNA regulation. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and kinetics of sRNA regulation in vivo. Therefore, I propose that the Hfq CTD displaces sRNAs and annealed sRNA‧mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell. Rosetta modeling, competitive binding experiments and X-ray crystallography show that the acidic tips of E. coli and C. crescentus Hfq CTDs transiently bind the basic Sm core residues necessary for RNA annealing. The CTD tip competes against non-specific RNA binding, facilitates dsRNA release and prevents indiscriminate DNA aggregation, suggesting that this acidic peptide mimics nucleic acid to auto-regulate RNA binding to the Sm ring. The mechanism of CTD auto-inhibition I propose makes testable predictions of the function of Hfq in different bacterial genera and illuminates how Sm proteins may evolve new functions. Finally, the flexible CTDs appear to function as independent modules, enabling predictions for how Hfq homologs may act in different species of bacteria

Distribution and phasing of sequence motifs that facilitate CRISPR adaptation. Santiago-Frangos et al

Data S1. Position weight matrices of subtype I-C, I-E, I-F and II-C CRISPR leader motifs and I-F CRISPR repeat, Related to STAR methods, Table 1 and Figures 2, 3, 4, S1, S2, S5. (A) I-EA motif. (B) I-EB motif. (C) I-E IHF binding site. (D) I-E leader anchoring motif. (E) I-E Upstream motif. (F) I-F IHF binding site. (G) I-F CRISPR repeat. (H) I-F Upstream motif. (I) I-C Upstream motif. (J) II-C Upstream motif. Data S2. Summary of significant matches to motifs found in CRISPR leaders, Related to STAR methods, Table 1 and Figures 2, 3, S1, S5. (A) Significant matches to position weight matrices in all CRISPR leaders analyzed. (B) Tally of the Phyla that I-E, I-F, I-C and II-C CRISPRs originate from and relation to leader motifs. (C) IHF-regulated CRISPR loci predicted to rapidly acquire new spacers. (D) Summary of leader motifs found in Campylobacter and Neisseria spp. II-C CRISPR loci. Data S3. Analysis of I-E leader motifs found adjacent to non-canonical integration sites in vivo in E. coli, Related to STAR methods and Figures 3 and S5. (A) Identification of I-E leader motifs and repeat motifs in sequences flanking both sides of non-canonical integration sites. (B) Summary of top 25 unique integration sites across 4 biological replicates reported in Nivala et al., 2018

Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific SARS-CoV-2 Diagnostic. Santiago-Frangos et al

Table S6: Primers to generate amplicon library for SARS-CoV-2 whole genome sequencing. Related to Figure 3. Described in the ARTIC nCoV-2019 V3 Panel

Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation.

We have solved the X-ray crystal structure of the RNA chaperone protein Hfq from the alpha-proteobacterium Caulobacter crescentus to 2.15-Å resolution, resolving the conserved core of the protein and the entire C-terminal domain (CTD). The structure reveals that the CTD of neighboring hexamers pack in crystal contacts, and that the acidic residues at the C-terminal tip of the protein interact with positive residues on the rim of Hfq, as has been recently proposed for a mechanism of modulating RNA binding. De novo computational models predict a similar docking of the acidic tip residues against the core of Hfq. We also show that C. crescentus Hfq has sRNA binding and RNA annealing activities and is capable of facilitating the annealing of certain Escherichia coli sRNA:mRNA pairs in vivo. Finally, we describe how the Hfq CTD and its acidic tip residues provide a mechanism to modulate annealing activity and substrate specificity in various bacteria.SWH and BL are funded by the Wellcome Trust (200873/Z/16/Z). This work was also supported by the NIH (R01 GM120425 to SW, F31 GM123616 to JRJ, and R01 GM078221 to JJG). KF acknowledges funding by the LMU Mentoring program of the LMU Faculty of Biology

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes