Number of publications per year since 2006 associating bioactivities with a subset of three PAINS: Rhodanine analogs, curcumins, and resveratrol.

<p>A) All PubMed-indexed journals. B) All PLOS journals. Searches were conducted using “compound name” and publication years 2006–2017 as keywords and for (B) triaged for articles associating bioactivities with these compounds.</p

Filters for PAINS and promiscuous compounds.

<p>Filters for PAINS and promiscuous compounds.</p

Representation of MeV H head domains complexed with soluble Slam receptor based on the coordinates reported by Hashiguchi and colleagues [<b>31</b>].

<p>Slam moieties (dark green) and covalently linked H dimers (cyan and light purple) in the tetrameric arrangement are highlighted. Receptor binding is proposed to trigger a significant reorganization of the non-covalent dimer-dimer interface (form I versus form II <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>). In the original X-ray analysis, form II was observed when an additional L482R mutation was introduced into MeV H. This mutation was found to enhance SLAM-dependent fusion and also appeared in a clinical MeV isolate of the D1 genotype <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>. Structural renderings were prepared as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat-1002058-g001" target="_blank">Figure 1</a>. Dotted lines highlight the dimer–dimer intersection. Hypothetical positions of the H stalk domains are marked in the side view representations.</p

Schematic of bi-molecular H complementation to explore the organization of the physiological complex.

<p>(Left panel) Overview of previously identified functional domains in H, responsible for interaction with F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lee1" target="_blank">[34]</a>, receptor binding <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Corey1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Patterson1" target="_blank">[61]</a>, or required for F triggering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Corey2" target="_blank">[53]</a>. For simplicity, an H dimer is shown representing form I as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>. (Right panel) Co-expression of H variants defective in individual functions in all possible combinations restores F fusion promotion activity through trans-complementation of functionality <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Brindley1" target="_blank">[32]</a>. Structural renderings were generated as outlined for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat-1002058-g001" target="_blank">Figure 1</a>.</p

Measles virus fusion model.

<p>(Left panel) Model representation of the MeV envelope glycoprotein prefusion hetero-oligomer. The H and F complexes are aligned in a staggered head configuration in which the F head is thought to stand in contact with the H stalk <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Prussia1" target="_blank">[57]</a>. (Middle and right panels) Hypothetical dissociation model of F triggering. Upon binding to the cellular receptor, H and F dissociate, resulting in triggering of major conformational changes in metastable prefusion F. Refolding into the stable postfusion conformation is considered to occur through a series of intermediate conformations, including a hypothetical pre-hairpin intermediate <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lamb2" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin1" target="_blank">[56]</a>. Likely, refolding of multiple F complexes is required to open a fusion pore and enable viral entry. For improved clarity, MeV H is represented as a single tetramer, and F as a single trimer in the hetero-oligomeric fusion complex. More than one F trimer may interact, however, with each individual H tetramer. The insert shows an enlarged representation of proposed lipid mixing intermediates. As F refolds, first the outer membranes are thought to fuse, creating a lipid stalk. Membrane merger is then thought to advance through hemifusion to pore formation. For clarity, F complexes have been eliminated from the lipid mixing representations. Structural renderings are based on original crystal structures (form I H head domains as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Hashiguchi1" target="_blank">[31]</a>), homology models of MeV F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Lee2" target="_blank">[55]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Plemper3" target="_blank">[58]</a> based on coordinates reported for pre- and post-fusion PIV5 and PIV3 F, respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin1" target="_blank">[56]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Yin2" target="_blank">[59]</a>, or hypothetical structural models (F pre-hairpin intermediate). H stalk domains are modeled in an assumed α-helical configuration <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Paal1" target="_blank">[33]</a>. High-resolution structural models were aligned at the level of the transmembrane domain (viral envelope) and then morphed into low resolution images using the Sculptor (resolution 12, voxel size 3) package <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002058#ppat.1002058-Birmanns1" target="_blank">[60]</a>.</p

Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens

Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose–response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase

Receptor-induced conformational change in H detected by mAb αH-1347.

<p>(A) Reactivity to mAb αFLAG and αH-1347 of standard (H-wt) or headless (Hstalk) attachment proteins in the presence or absence of F-wt. After addition of the secondary antibody, MFI values were recorded by flow cytometry. H/F co-immunoprecipitation with different anti-CDV-H mAbs. (B) Cell surface assessment of H protein interaction with cleaved F-proteins. To stabilize H/F complexes, transfected Vero cells were treated with the membrane non-permeable cross-linker DTSSP and subsequently lysed with RIPA buffer. Complexes were then immunoprecipitated (IP) with anti-CDV-H mAb 3734 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref061" target="_blank">61</a>] and protein G-Sepharose beads treatment. Proteins were boiled and subjected to immuoblotting using a polyclonal anti-CDV-F antibody [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref071" target="_blank">71</a>] to detect F antigenic materials (coIP). Co-IP F proteins were detected in comparison with F proteins present in cell lysates prior to IP by immuoblotting using the same anti-F antibody (TL, total lysate; F₀, uncleaved F protein; F₁, cleaved membrane-anchored F subunit). The specific mAb used for the immunoprecipitation step is indicated at the top of the gel. (C) Similar to (B) but with cell extracts obtained from Vero cells transfected with headless H and F. (D) Effect of receptor treatment on mAb αH-1347’s H-binding activity (at 4°C or 37°C). Vero cells expressing H-wt were co-cultured with Vero-SLAM, Vero-Nectin-4 or Vero cells together with mAb αH-1347. After addition of the secondary antibody, MFI values were recorded by flow cytometry. Means ± S.D. of data from three independent experiments performed in triplicates are shown.</p

Models of paramyxovirus membrane fusion activation.

<p>(A) Summary of the putative mechanism of membrane fusion activation for morbilliviruses (carrying the “spacer” module in the H-stalk). In the first panel, the cartoon represents one H-wt tetramer (in blue) assuming the auto-repressed state not yet bound to its cognate receptor (<i>i</i>.<i>e</i>. SLAM; represented by black ovals anchored in the target plasma membrane). The associated trimeric fusion protein (F) in the pre-fusion conformational state is represented in green. Because the “4-heads-down” configuration determined for NDV HN [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref036" target="_blank">36</a>] contains elements that fit with the proposed pre-receptor-bound locked H-structure, we arbitrarily illustrated H in this conformation. In this state, the two “lower” H-heads are partially covering the “spacer” stalk microdomain (green box) where the mAb αH-1347 epitopes also locate. These “head-to-spacer” contacts are critical in stabilizing the auto-repressed H state. Because the H-stalk “spacer” module maps membrane-distal from the candidate F-binding/activation regulatory segment (red box), H/F assembly can occur even prior to receptor binding. In the second panel, both accessible “upper” heads engage with the receptor. Conversely, distance and/or physical constraints may prevent efficient receptor binding to the “lower” head units. In the third panel, because both monomeric head units within each dimers may assemble into structurally stable complexes (or may require some adjustment prior to achieve stable dimeric units [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref075" target="_blank">75</a>]), the contact of the “upper” heads with the receptor leads to a rearrangement of the dimers that automatically relocates the “lower” heads “away” from the stalk (achieving a putative “heads away” structural intermediate). This step thus disrupts the critical “head-to-spacer” contacts and leads to the “de-activation” of the auto-repressed state. In the fourth panel, the stalk is now free to refold into the F-triggering competent state. The latter includes “structural flexibility” or “opening” of the central section. In the fifth panel, upon H-mediated activation, F achieves the pre-hairpin intermediate structural state bridging the viral envelope and the target cell plasma membranes (color-coded in yellow and orange, respectively). Basics of this model was recently hypothesizes for CDV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref068" target="_blank">68</a>] and MeV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref048" target="_blank">48</a>] (referred to as “safety catch” in the latter study). (B) Summary of the putative mechanism of membrane fusion activation of PIV5 and NDV (expressing attachment protein with short “spacerless” stalks). In the first panel, the pre-receptor-bound state of HN is represented in the “4-heads-down” configuration not yet bound to its cognate receptor (<i>i</i>.<i>e</i>. sialic acid; represented by red spheres attached to membrane bound molecules). As a major difference with morbillivirus attachment proteins, HN-stalks do not carry the analogous “spacer” segment that locate membrane-distal to the F-binding/activation sites (red box). Consequently, the two backfolding dimeric head units directly cover the F-binding/activation sites, which prevent HN/F assembly prior to receptor engagement. In the second panel, both accessible “upper” heads engage with the receptor. In the third panel, the contact of the HN-Heads with the receptor triggers a large-scale conformational change that switches the heads from the “down” to the “up” configuration. The latter structural state implies a tetrameric assembly of the heads positioned above the stalk. Consequently, the F-binding/activation sites are unmasked, which in turn allow for HN/F interactions and subsequent “induced fit” mechanism that ultimately lead to F-triggering. In the fourth panel, F achieves the pre-hairpin intermediate structural state bridging the viral envelope and the target cell plasma membranes (color-coded in yellow and orange, respectively). This model is referred to as the “stalk-exposure/induce fit” model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref040" target="_blank">40</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref046" target="_blank">46</a>].</p

Investigation of the mAb αH-1347 to H stoichiometry required for membrane fusion inhibition.

<p>(A) Cartoon representation of the CDV H-wt tetramer and derived “headless” variant in a putative pre-F-triggering state. The reported I98A mutation shown to abrogate F-triggering without impairing H/F interaction is also shown in the full length H-wt protein [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref017" target="_blank">17</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.ref064" target="_blank">64</a>]. (B) Representation of the different hetero-oligomeric assemblies that may emerge from H-98A and H-stalk co-expressing cells. (C-E) Syncytium formation assay. Fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-98A and F-wt, or headless H and F-wt, or H-I98A and headless H and F in the presence (+) of absence (-) of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (F-H) Quantitative fusion assay. The fusion promotion efficiency of each H/F combinations was determined as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004880#ppat.1004880.g001" target="_blank">Fig 1B</a>. Means ± S.D. of data from three independent experiments performed in triplicates are shown.</p

Inhibition of CDV-mediated viral-cell and cell-cell fusion by the anti-CDV-H mAb-1347.

<p>(A) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt and CDV F-wt (A75/17 strain) in the presence of absence of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (B) Quantitative fusion assay. Vero-cSLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the F-wt and H-wt-expressing vectors and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. (C) Virus neutralization assay. A total of 100 infectious units of recA75/17^red was incubated with the indicated dilution of antibody for 1 h at 37°C. The virus-antibody mixtures were then added to 3h on Vero cells, overlaid with agar-containing medium and further incubated for 72 h at 37°C. Cell entry efficiency was determined by counting the number of red fluorescent syncytia induced by recA75/17^red. (D) Effect of mAbs on H/SLAM binding efficiency. Vero cells were transfected with H-wt. Prior to treatment with soluble HA-tagged cSLAM molecules, mAbs were added as indicated, and SLAM-binding activity was calculated as the ratio of mean fluorescence intensities obtained with an anti-HA polyclonal Ab values (staining for sol. cSLAM) normalized to the levels obtained with the anti-FLAG mAb (staining for H). Values recorded for H-wt/cSLAM-binding efficiency in the absence of the mAb were set at 100%. Wt: wild type, α: monoclonal antibody, sSLAM: soluble version of cSLAM. Means ± S.D. of data from three independent experiments in triplicate are shown.</p