Evaluation of the TRF immunoassays using Syphilis Qualification Panel (QSS701).

<p>^a Members of the panel are manufactured from human serum or plasma, as provided by the panel supplier. Five members are formulated with various reactivities of Syphilis. Non-reactive member was formulated from Syphilis non-reactive pools.</p><p>^b Results were provided by the panel supplier. R, reactive; NR, non-reactive.</p><p>^c Values indicate S/Co ratios obtained in this study, using the in-house TRF immunoassays with 10 min and 1 h incubation times. Samples with S/Co values <1.0 are designated as negative (−) and those with values ≥1.0 are designated as positive (+).</p

Evaluation summary of the TRF immunoassays using the in-house serum sample panel.

<p>^a Results expressed as ‘number of positive samples with the indicated assays’/‘total number of samples’. ‘+/−’ indicates that indeterminate result was obtained with the respective assay; ‘ND’ indicates that the test was not done.</p

Design, expression and purification of the r-antigens used in the study.

<p>(A) The r-Tp15-17-47 antigen is a chimeric in-frame fusion construct containing thioredoxin, 6x His tag (indicated by the asterisk), followed by the membrane proteins (Tp15, Tp17 and Tp47) of Tp stitched together with flexible tetra-glycyl (G₄) linkers (presented with zig-zag lines). In r-Bio-Tp15-17-47, the BAP is inserted in-frame to generate the <i>in vivo</i> biotinylated version of the antigen. (B) SDS-PAGE analysis of r-Tp15-17-47 and r-Bio-Tp15-17-47 antigens. Aliquots of total cell lysates of <i>E. coli</i> harboring the r-p15-17-47 and r-Bio-p15-17-47 antigen constructs (<i>uninduced</i>: lanes marked ‘U’; <i>induced</i>: lanes marked ‘I’), and aliquots of the affinity-purified dialyzed and soluble antigens (lanes marked ‘P’), were electrophoresed on denaturing gels and visualized by Coomassie staining. Protein size markers were run in lane ‘M’; their sizes (in kDa) are shown on the left. The arrows shown denote the bands of purified proteins. The asterisk denotes the position of biotin ligase enzyme.</p

Results from the non-conclusive primary and their respective follow-up samples mostly from category #3 and #4 in Table 1.

<p>^a Results obtained from the primary samples using the three reference assays, and both the TRF immunoassays (with 10 min and 1 h incubation times), as indicated. All the primary samples are from category #3 and #4, as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. ‘Enzg.’ indicates Enzygnost syphilis EIA. ‘+’, ‘−’ and ‘+/−’ indicate positive, negative and indeterminate results, respectively, as obtained with the mentioned assays.</p><p>^b Results obtained from the thirteen follow-up samples of their respective primary samples using Enzygnost syphilis, TPHA, VDRL and I-L (Inno-Lia Syphilis score line immunoassay) as reference assays, and both the TRF immunoassays (with 10 min and 1 h incubation times), as indicated. Ten out of 13 samples are from category #3 and #4, as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. Each primary sample had either one or none follow-up sample. ‘NA’ indicates that a follow-up sample was not available to us.</p><p>sample belongs to category #2 as divided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>.</p><p>Sample not tested with TRF immunoassays in this study.</p><p>*The antibody titer obtained in TPHA assay is shown in parentheses. ‘ND’, indicates that the test was not done.</p><p>^c Syphilis status indicates whether the person has, or has had syphilis previously, and is based upon the results of previous or follow-up samples and on the available clinical data. ‘N’ indicates a negative and ‘P’ indicates a positive status for syphilis. ‘?’ indicates that true syphilis status is unknown.</p><p>sample from a new-born baby, with borderline level of maternal antibodies (data not shown).</p

Evaluation of the TRF immunoassays using Syphilis Mixed Titer Performance Panel (PSS202).

<p>^a Assays performed using commercial kits as indicated. Results were provided by the panel supplier. RPR, rapid plasma reagin; ATA, anti-<i>Treponema</i> antibody; S/Co, signal to cutoff ratio; EIA, enzyme immunoassay; TPPA, <i>Treponema pallidum</i> particle agglutination assay; TPHA, <i>Treponema pallidum</i> haemagglutination assay; Neg, negative; R, reactive; NR, non-reactive. RPR results are endpoint dilutions. S/Co ratios ≥1.0 are considered reactive.</p><p>^b Values indicate S/Co ratios obtained in this study, using the in-house TRF immunoassays with 10 min and 1 h incubation times. Samples with S/Co values <1.0 are designated as negative (−) and those with values ≥1.0 are designated as positive (+).</p

Design and evaluation of the in-house TRF immunoassays.

<p>(A) Schematic representation of the in-house TRF immunoassay. The design of both the TRF immunoassays was same, except for different incubation times and tracer amounts. The numbers represent the following: (1) microtiter well surface, (2) streptavidin, (3) r-Bio-p15-17-47, (4) serum anti-Tp IgG antibody, (5) serum anti-Tp IgM antibody, and (6) r-p15-17-47 coated on Eu³⁺ chelate-doped nanoparticles. (B) Scatter plot with the S/Co values of all serum samples (n = 311) analyzed in this study, using the in-house TRF immunoassays with 1 h (<i>x</i> axis) and 10 min (<i>y</i> axis) incubation times. Different symbols represent positive (+) or negative (-) serum samples, either from commercial panels based on the results provided by the panel supplier, or from six different categories (#1 – #6) of the in-house samples based on their reactivities with the reference assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084050#pone-0084050-t001" target="_blank">Table 1</a>. Symbols for different kind of samples are as the following: PSS202 (+), filled blue triangles; PSS202 (-), empty blue triangles; QSS701 (+), filled cyan triangles; QSS701 (-), empty cyan triangle; samples of category #1, filled red circles; samples of category #2, filled green circles; samples of category #3, filled blue circles; samples of category #4, filled magenta circles; sample of category #5, filled olive square; samples of category #6 (except one sample), empty red circles; and exceptional sample of category #6 which gave positive results with in-house TRF immunoassays, black star. Dashed vertical and horizontal lines represent the cutoffs (at S/Co = 1) for the two TRF immunoassays.</p

DataSheet1_Optimizing drug discovery for snakebite envenoming via a high-throughput phospholipase A2 screening platform.docx

Snakebite envenoming is a neglected tropical disease that causes as many as 1.8 million envenomings and 140,000 deaths annually. To address treatment limitations that exist with current antivenoms, the search for small molecule drug-based inhibitors that can be administered as early interventions has recently gained traction. Snake venoms are complex mixtures of proteins, peptides and small molecules and their composition varies substantially between and within snake species. The phospholipases A2 (PLA2) are one of the main pathogenic toxin classes found in medically important viper and elapid snake venoms, yet varespladib, a drug originally developed for the treatment of acute coronary syndrome, remains the only PLA2 inhibitor shown to effectively neutralise venom toxicity in vitro and in vivo, resulting in an extremely limited drug portfolio. Here, we describe a high-throughput drug screen to identify novel PLA2 inhibitors for repurposing as snakebite treatments. We present method optimisation of a 384-well plate, colorimetric, high-throughput screening assay that allowed for a throughput of ∼2,800 drugs per day, and report on the screening of a ∼3,500 post-phase I repurposed drug library against the venom of the Russell’s viper, Daboia russelii. We further explore the broad-spectrum inhibitory potential and efficacy of the resulting top hits against a range of medically important snake venoms and demonstrate the utility of our method in determining drug EC50s. Collectively, our findings support the future application of this method to fully explore the chemical space to discover novel PLA2-inhibiting drugs of value for preventing severe pathology caused by snakebite envenoming.</p

Table1_Snakebite drug discovery: high-throughput screening to identify novel snake venom metalloproteinase toxin inhibitors.XLSX

Snakebite envenoming results in ∼100,000 deaths per year, with close to four times as many victims left with life-long sequelae. Current antivenom therapies have several limitations including high cost, variable cross-snake species efficacy and a requirement for intravenous administration in a clinical setting. Next-generation snakebite therapies are being widely investigated with the aim to improve cost, efficacy, and safety. In recent years several small molecule drugs have shown considerable promise for snakebite indication, with oral bioavailability particularly promising for community delivery rapidly after a snakebite. However, only two such drugs have entered clinical development for snakebite. To offset the risk of attrition during clinical trials and to better explore the chemical space for small molecule venom toxin inhibitors, here we describe the first high throughput drug screen against snake venom metalloproteinases (SVMPs)—a pathogenic toxin family responsible for causing haemorrhage and coagulopathy. Following validation of a 384-well fluorescent enzymatic assay, we screened a repurposed drug library of 3,547 compounds against five geographically distinct and toxin variable snake venoms. Our drug screen resulted in the identification of 14 compounds with pan-species inhibitory activity. Following secondary potency testing, four SVMP inhibitors were identified with nanomolar EC50s comparable to the previously identified matrix metalloproteinase inhibitor marimastat and superior to the metal chelator dimercaprol, doubling the current global portfolio of SVMP inhibitors. Following analysis of their chemical structure and ADME properties, two hit-to-lead compounds were identified. These clear starting points for the initiation of medicinal chemistry campaigns provide the basis for the first ever designer snakebite specific small molecules.</p

Image2_Snakebite drug discovery: high-throughput screening to identify novel snake venom metalloproteinase toxin inhibitors.tif

Snakebite envenoming results in ∼100,000 deaths per year, with close to four times as many victims left with life-long sequelae. Current antivenom therapies have several limitations including high cost, variable cross-snake species efficacy and a requirement for intravenous administration in a clinical setting. Next-generation snakebite therapies are being widely investigated with the aim to improve cost, efficacy, and safety. In recent years several small molecule drugs have shown considerable promise for snakebite indication, with oral bioavailability particularly promising for community delivery rapidly after a snakebite. However, only two such drugs have entered clinical development for snakebite. To offset the risk of attrition during clinical trials and to better explore the chemical space for small molecule venom toxin inhibitors, here we describe the first high throughput drug screen against snake venom metalloproteinases (SVMPs)—a pathogenic toxin family responsible for causing haemorrhage and coagulopathy. Following validation of a 384-well fluorescent enzymatic assay, we screened a repurposed drug library of 3,547 compounds against five geographically distinct and toxin variable snake venoms. Our drug screen resulted in the identification of 14 compounds with pan-species inhibitory activity. Following secondary potency testing, four SVMP inhibitors were identified with nanomolar EC50s comparable to the previously identified matrix metalloproteinase inhibitor marimastat and superior to the metal chelator dimercaprol, doubling the current global portfolio of SVMP inhibitors. Following analysis of their chemical structure and ADME properties, two hit-to-lead compounds were identified. These clear starting points for the initiation of medicinal chemistry campaigns provide the basis for the first ever designer snakebite specific small molecules.</p

Image1_Snakebite drug discovery: high-throughput screening to identify novel snake venom metalloproteinase toxin inhibitors.tif

Snakebite envenoming results in ∼100,000 deaths per year, with close to four times as many victims left with life-long sequelae. Current antivenom therapies have several limitations including high cost, variable cross-snake species efficacy and a requirement for intravenous administration in a clinical setting. Next-generation snakebite therapies are being widely investigated with the aim to improve cost, efficacy, and safety. In recent years several small molecule drugs have shown considerable promise for snakebite indication, with oral bioavailability particularly promising for community delivery rapidly after a snakebite. However, only two such drugs have entered clinical development for snakebite. To offset the risk of attrition during clinical trials and to better explore the chemical space for small molecule venom toxin inhibitors, here we describe the first high throughput drug screen against snake venom metalloproteinases (SVMPs)—a pathogenic toxin family responsible for causing haemorrhage and coagulopathy. Following validation of a 384-well fluorescent enzymatic assay, we screened a repurposed drug library of 3,547 compounds against five geographically distinct and toxin variable snake venoms. Our drug screen resulted in the identification of 14 compounds with pan-species inhibitory activity. Following secondary potency testing, four SVMP inhibitors were identified with nanomolar EC50s comparable to the previously identified matrix metalloproteinase inhibitor marimastat and superior to the metal chelator dimercaprol, doubling the current global portfolio of SVMP inhibitors. Following analysis of their chemical structure and ADME properties, two hit-to-lead compounds were identified. These clear starting points for the initiation of medicinal chemistry campaigns provide the basis for the first ever designer snakebite specific small molecules.</p