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

A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system

Leishmania major protein disulfide isomerase as a drug target : Enzymatic and functional characterization.

International audienceLeishmaniasis is a major health problem worldwide and tools available for their control are limited. Effective vaccines are still lacking, drugs are toxic and expensive, and parasites develop resistance to chemotherapy. In this context, new antimicrobials are urgently needed to control the disease in both human and animal. Here, we report the enzymatic and functional characterization of a Leishmania virulence factor, Leishmania major Protein disulfide isomerase (LmPDI) that could constitute a potential drug target. LmPDI possesses domain structure organization similar to other PDI family members (a, a', b, b' and c domains), and it displays the three enzymatic and functional activities specific of PDI family members: isomerase, reductase and chaperone. These results suggest that LmPDI plays a key role in assisting Leishmania protein folding via its capacity to catalyze formation, breakage, and rearrangement of disulfide bonds in nascent polypeptides. Moreover, Bacitracin, a reductase activity inhibitor, and Ribostamycin, a chaperone activity inhibitor, were tested in LmPDI enzymatic assays and versus Leishmania promastigote in vitro cultures and Leishmania amastigote multiplication inside infected THP-1-derived macrophages. Bacitracin inhibited both isomerase and reductase activities, while Ribostamycin had no effect on the chaperone activity. Interestingly, Bacitracin blocked in vitro promastigote growth as well as amastigote multiplication inside macrophages with EC(50) values of 39 μM. These results suggest that LmPDI may constitute an interesting target for the development of new anti-Leishmania drugs

Fyn phosphorylates ISRAA.

<p>(A) Direct phosphorylation of ISRAA by the Fyn protein was assayed using different concentrations of mouse Fyn in the presence of 1 μM concentrations of rISRAA and the rISRAA-Y102A mutant. The mutation of the Y₁₀₂ residue significantly affects the kinase activity of Fyn at concentrations of 0.5 μg/ml (**<i>p</i><0.001) and 1.5 μg/ml (*<i>p</i><0.01). (B) The kinase reaction mixtures were analyzed via western blotting using an anti-phosphotyrosine antibody (PY-20); lane 1: recombinant Fyn + rISRAA-Y102A, lane 2: recombinant Fyn + rISRAA. Only rISRAA shows phosphorylated tyrosine content.</p

Pull-down assay shows that ISRAA binds to Fyn.

<p>Bead-immobilized rISRAA pulled down from Expi293F lysates was used as bait for EL4 cell lysates. Western blot analysis using a monoclonal anti-Fyn antibody did not reveal any detectable Fyn protein in EL4 lysates incubated with beads alone (lane 2). The Fyn protein (59 KDa) was specifically detected in EL4 whole cell lysates (lane 3) and pulled down specifically with rISRAA immobilized beads (lane 4).</p

Cell transfection and rISRAA expression.

<p>Expi293F cells were transfected with p-SELECT-zeo-ISRAA and selected with zocin, and p-SELECT-zeo-HGh was used as a positive control for expression. After 48 hours of transfection, the cell lysates and supernatants were analyzed via western blotting with an anti-His6 antibody. HGh was strongly expressed by Expi293F cells in both cell pellets (lane 3) and supernatants (lane 5). Expi293F cells strongly expressed rISRAA in an intracellular form at the expected molecular weight (15 KDa) (lane 2), and no rISRAA was detected in the supernatants (lane 4).</p

ISRAA-Fyn docking prediction.

<p>(A) SH2 domain structure. P-Tyr-containing peptides have two docking sites in the Fyn SH2 domain: a phospho-Tyr binding pocket located between the central anti-parallel βC-sheet and the α-helix αA and a hydrophobic binding pocket near the EF loop. Computational docking of the ISRAA model with the Fyn SH2 domain shows specific binding of Y₁₀₂ to the p-Tyr binding pocket (B), where Y₁₀₂ is located 3.5 A from R₁₅₆ (C). An unconventional binding mode is observed for L₁₀₈ (pTyr+6), which is located in a binding pocket between the βD-sheet and the BC loop (D) that is different from the classic pocket. In the entire figure, the Fyn-SH2 domain is represented in gray, and the ISRAA peptide (Y₁₀₂TEVSTL) is represented in green.</p

rISRAA affects early cell activation.

<p>(A) MTT Cell Activation Assay; T-cell activation by anti-CD3 in the presence and absence of 1 μM soluble rISRAA was measured after 24 h of stimulation of mouse splenocytes. ISRAA significantly downregulates early T-cell activation in comparison to anti-CD3 treatment alone (***<i>p</i>< 0.0001). (B) Western blotting analysis of time-dependent Tyr 416 phosphorylation in Src-family kinases. Treatment of splenocytes with 1 μM rISRAA clearly downregulates the phosphorylation of an activation tyrosine in the SFK members Src (59 KDa) and Fyn (59 KDa). Total Fyn and Src analysis showed equivalent amounts of the proteins in all samples. Untreated cells show equivalent amounts of the actin protein and stable Tyr-416 phosphorylation at all time points.</p

Pull-down of rISRAA.

<p>rISRAA was pulled down from Expi293F lysates after transfection using cobalt beadsExpi293F. The beads were boiled and analyzed for rISRAA binding via western blotting with (A) an anti-His antibody (lane 2) and (B) a monoclonal anti-rISRAA antibody (lane 2) (Genescript). Both antibodies detected a 15 KDa protein, which was shown to bind and be pulled down in satisfactory amounts. No cross-reaction with beads incubated with mock-transfected Expi293F lysates was observed for either antibody Expi293F(lane 3 in A and B).</p

A high-throughput turbidometric assay for screening inhibitors of Leishmania major protein disulfide isomerase.

International audienceThe use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity