Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) Engineered U2OS cell expressing mTurquoise2-NDC80 and β-tubulin-TC-FlAsH. NDC80 (gray), mTurquoise2 (blue) and TC-FlAsH (green). ( B ) Two-photon microscopy images of the engineered U2OS cells not exposed to FlAsH (top) and exposed to FlAsH (bottom). 3 μm scale bar. mTurquoise2 (blue) and FlAsH (green). ( C ) Example fluorescence decay curves of mTurquoise2-NDC80 in the engineered U2OS cells not exposed to FlAsH (left, green circle) and exposed to FlAsH (right, orange triangle), plotted with the best-fit single-exponential decay models (black and blue dotted lines). Corresponding weighted residuals (the deviation of data from model, divided by the square root of the number of photons) are plotted below after being smoothened to display systematic deviations. ( D ) The fluorescence decay curve of mTurquoise2-NDC80 in the presence of FlAsH labeling (orange triangle, same as ( C )), plotted with the best-fit two-exponential model (blue solid line). The single-exponential model fit to the fluorescence decay curve in the absence of FlAsH labeling (black dotted line) plotted together for comparison. Corresponding smoothened weighted residual (described above) for the two-exponential model is plotted below. Long- and short-lifetime exponentials correspond to the mTurquoise2-NDC80 populations in non-FRET state and FRET state, respectively, and their relative amplitudes give the fraction of each population. To facilitate the comparison, the fluorescence decay curves in the absence and presence of FlAsH labeling were normalized such that they asymptotically overlap. Data points and source FLIM data are available in Figure 1-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Expressing, Microscopy, Fluorescence, Labeling, Comparison

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) to ( D ) Schematic descriptions, example cell images, and example mTurquoise2 fluorescence decay curves from three different FRET-negative control experiments and a nocodazole treatment experiment. mTurquoise2 fluorescence decay curves (blue circles) are plotted with best-fit single- (black dotted line) or two-exponential decay model (black solid line), and the associated weighted residuals are plotted below (blue curve). 3 μm scale bar. ( A ) Negative control 1. Nuf2 N-terminally labeled with mTurquoise2, and no FlAsH labeling. ( B ) Negative control 2. Nuf2 C-terminally labeled with mTurquoise2 (far from kMT), and no FlAsH labeling. ( C ) Negative control 3. Nuf2 C-terminally labeled with mTurquoise2 (far from kMT), and β-tubulin C-terminally labeled with FlAsH. ( D ) Nocodazole treatment experiment. Nuf2 N-terminally labeled with mTurquoise2, and β-tubulin C-terminally labeled with FlAsH. Cell was incubated with 5 µM nocodazole for >10 min to depolymerize microtubules. ( E ) Boxplot of fluorescence lifetimes estimated from single-exponential models fit to the negative control fluorescence decays. n = 32, 11, and 6 cells for Neg Ctrl 1, 2, and 3, respectively.

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Fluorescence, Negative Control, Labeling, Incubation

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) Fluorescence decay curves of cells expressing mTurquoise2-TC in the absence (green circle) and the presence (orange triangle) of FlAsH. A single-exponential model (black solid line) was fit to the fluorescence decay curve in the absence of FlAsH. For easier comparison, the fluorescence decay curves were normalized such that they asymptotically overlap. ( B ) The conformational ensemble of the flexible tether between mTurquiose2 and TC were modeled by Monte Carlo protein simulations, and the distance, r , between mTurquiose2 (blue cartoon) and TC-FlAsH (green ball) was estimated. This distribution is denoted by p ( r ). ( C ) The measured fluorescence decay of mTurquoise2-TC-FlAsH (orange triangles, same as ( A ) but not normalized) plotted with the best-fit decay model (black dotted line, model described in the box and derived in Materials and methods). Associated weighted residual (deviation of model from data, divided by the square root of the number of photons) plotted below. Fitting the decay model to the data estimated the Förster radius to be 5.90 ± 0.10 nm (SE).

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Fluorescence, Expressing, Comparison, Derivative Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) The conformational ensemble of the flexible tether between mTurquoise2 and Nuf2 (red) and the disordered C-terminal tails of beta-tubulins around the NDC80 (green) were modeled by large-scale Monte Carlo protein simulations, which were then used to calculate the distances, r i , between the mTurquoise2 and the TC motifs. FlAsH labeling was assigned to the TC motifs with 26.1% probability (which is the measured labeling fraction of beta-tubulin). Fluorescence lifetimes of the mTurquoise2 were calculated for randomly sampled sets of distances, r → = ( r i ) , based on which fluorescence decay curves were simulated. ( B ) (top) Fluorescence decay curves for various distances between NDC80 and MT were simulated and then were fit using single- and two-exponential decay models. Difference in Bayesian information criteria (BIC) between single- and double-exponential models is plotted against the NDC80-MT distance. Data points are mean and SD. (bottom) Example simulated fluorescence decay curves (green dots) for 0, 4, and 9 nm NDC80-MT distances are plotted with the best-fit single- (blue line) and two-exponential (red line) models. Corresponding smoothened weighted residuals plotted below. ( C ) Fluorescence decay curves for various NDC80 binding fractions ( f b ) were simulated and fit by using two-exponential decay model to estimate FRET fraction ( f FRET ). (left) Three example simulated fluorescence decay curves (green dots) for 0, 30, and 60% binding fractions with the best-fit two-exponential models (red line), and the corresponding smoothened weighted residuals plotted below. (right) NDC80 FRET fractions ( f FRET ) plotted against NDC80 binding fractions ( f b ) (blue dots), and the linear fit (black line). Gray-shaded area represents the uncertainty in the slope, which was determined from the uncertainties in the measured beta-tubulin labeling fraction and Förster radius (see Supplemental experiments in Materials and methods).

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Labeling, Fluorescence, Binding Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) (left) kMTs predominantly depolymerize at leading kinetochores and polymerize at trailing kinetochores. (right) K-K distance is a proxy for centromere tension. Measuring NDC80-kMT binding along with the kinetochore movement and K-K distance therefore reveals how NDC80-kMT binding is related to the kMT dynamics and centromere tension. ( B ) Image of a metaphase cell with mTurquoise2-NDC80 (blue) and β-tubulin-TC-FlAsH (green), and kinetochore tracking (yellow circles) and pairing (red lines) results. 3 μm scale bar. ( C ) NDC80 FRET fraction vs. kinetochore speed for leading (green circle) and trailing (orange triangle) kinetochores (n = 17 cells, 681 kinetochores/data point). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar velocities. ( D ) NDC80 FRET fraction vs. K-K distance for untreated cells (green circle, n = 17 cells, 984 kinetochores/data point), cells treated with 10 μM taxol (orange triangle, n = 7 cells, 525 kinetochores/data point), and cells treated with 5 μM STLC (purple square, n = 16 cells, 493 kinetochores/data point). For STLC data, only poleward-facing kinetochores are plotted (see for comparison between poleward and anti-poleward kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( E ) Histograms of K-K distances for the untreated (top, green), taxol-treated (middle, orange), and STLC-treated (bottom, purple) cells. 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tublin-TC-FlAsH (green). ***p-value (Welch’s t-test) less than 10 −30 . Data points and source FLIM data are available in Figure 3-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Binding Assay, Comparison

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) (top) Cell images showing mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). (bottom) Time course of NDC80 FRET fraction in response to Aurora B inhibition by 3 μM ZM447439 (n = 15 cells). ( B ) Images of cells with mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green) after depleting endogenous Hec1 by siRNA and expressing siRNA-insensitive WT or three different phosphomimetic mutants of Hec1: 9A-, 2D-, and 9D-Hec1 (see Materials and methods). ( C ) NDC80 FRET fraction of cells whose endogenous Hec1 are replaced with WT or phosphomimetic Hec1 (see Materials and methods). Black dots are from individual cells and red error bars are mean ± SEM. n = 19, 22, 12, and 17 cells for WT-, 9A-, 2D-, and 9D-Hec1. *p<0.1; **p<0.01; ***p<0.001; ****p<0.0001. ( D ) Time course of NDC80 FRET fraction of 2D-Hec1-expressing cells in response to Aurora B inhibition by 3 μM ZM447439 (n = 12 cells). ( E ) (top) Cell images color-coded with Aurora B sensor non-FRET fraction. (bottom) Time course of the non-FRET fraction of the cytoplasmic Aurora B FRET sensor in response to 3 μM ZM447439 (n = 10 cells). In (A), (D) and (E), black squares and error bars are the weighted mean and SEM of the data points (green circles) in equally spaced time intervals of 1 min. Red solid and dashed lines are the best-fit exponential decay models and their 95% confidence intervals, respectively. 5 μm scale bar for all images. (F)NDC80 FRET fraction (from (A)) and NDC80 binding fraction (converted from the FRET fraction) plotted against the fraction of phosphorylated Aurora B phosphorylation sites in NDC80 (converted from Aurora B FRET sensor non-FRET fraction in (E)). Red solid and dashed lines are the best-fit NDC80-kMT binding model (derived in Mathematical modeling in Materials and methods) and its 95% confidence interval. Data points and source FLIM data are available in Figure 4-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Inhibition, Expressing, Binding Assay, Phospho-proteomics, Derivative Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet: ( A ) NDC80 FRET fraction vs. K-K distance for 9A-Hec1-expressing cells with no drug treatment (green circle, n = 12 cells, 803 kinetochores/data point), with 10 μM taxol treatment (orange triangle, n = 9 cells, 1113 kinetochores/data point), or with 5 μM STLC treatment (purple square, n = 10 cells, 855 kinetochores/data point). For STLC data, only poleward-facing kinetochores are included (see for comparison between poleward-facing and anti-poleward-facing kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( B ) Histograms of K-K distances for the untreated 9A-Hec1 cells (top, green), untreated cells with endogenous Hec1 (top, black line), 9A-Hec1 cells treated with taxol (middle, orange), and 9A-Hec1 cells treated with STLC (bottom, purple). 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). ***p<10 −6 (Welch’s t-test). ( C ) Haspin kinase phosphorylates histone H3 at Thr3 (H3T3), which recruits the chromosome passenger complex (CPC, red) to centromeres. 5-Iodotubercidin (5-ITu) inhibits haspin kinase, thereby displacing Aurora B from centromeres. ( D ) Spinning-disk confocal microscopy images of cells expressing mNeonGreen-Nuf2 (green) and INCENP-mCherry (red) before (top) and after (bottom) haspin inhibition by 10 μM 5-ITu treatment. 3 μm scale bar. ( E ) NDC80 FRET fraction vs. K-K distance for cells treated with 10 μM 5-ITu (green circle, n = 15 cells, 1170 kinetochores/data point), for cells treated with both 10 μM 5-ITu and 10 μM taxol (orange triangle, n = 3 cells, 359 kinetochores/data point), and for cells treated with 10 μM 5-ITu and 5 μM STLC (purple square, n = 12 cells, 564 kinetochores/data point). For 5-ITu + STLC data, only poleward-facing kinetochores are included (see for comparison between poleward-facing and anti-poleward-facing kinetochores). Data points are the mean, y-error bars the SEM, and the x-error bars the interquartile ranges within groups of kinetochores with similar K-K distances. Gray area is the 95% confidence interval for the linear fit to the combined data. ( F ) Histograms of K-K distances for the 5-ITu-treated (top, green), untreated (top, black line), 5-ITu + taxol treated (middle, orange), and 5-ITu + STLC treated cells (bottom, purple). 3 μm scale bar in the cell images of mTurquoise2-NDC80 (blue) and beta-tubulin-TC-FlAsH (green). ***p<10 −6 (Welch’s t-test). ( G ) NDC80 FRET fraction and ( H ) the non-FRET fraction of Nuf2-targeted Aurora B FRET sensor (proxy for Aurora B activity at NDC80) for different drug treatments. Each data point (gray circle) corresponds to an individual cell, and the error bar (red) shows the mean and SEM. P-values from two-sided Welch’s t-test. Data points and source FLIM data are available in Figure 5-Data .

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Expressing, Comparison, Confocal Microscopy, Inhibition, Activity Assay

Journal: eLife

Article Title: Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

doi: 10.7554/eLife.36392

Figure Lengend Snippet:

Article Snippet: An Aurora B FRET sensor was constructed by replacing CyPet in a previous construct (Addgene plasmid # 45215) ( ) with mTurquoise2.

Techniques: Transfection, Construct, Labeling, Retroviral, Plasmid Preparation, Selection, Marker, Modification, Recombinant, Sequencing, Synthesized, Electroporation, Software, Concentration Assay

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Top, experimental setup. Following 4-h treatment with 100 ng/mL nocodazole (NOC), mitotic cells were collected via shake off and treated for 1 h with PLK1i (1 μM BI-2536), Chk2i (10 μM BML-277), or DMSO, then collected and immunoblotted. Left, representative blot of A549 cells. Right, quantification of N = 3 (A549) or N = 4 (RPE-1) biological replicates. * p < 0.05, two-tailed t -test. B Following 4-hour arrest in 10 μM S-trityl- l -cysteine (STLC), inhibitors or DMSO were added for 1 h. Cells were then fixed and stained for p-TCTP. Scale bar, 5 μm. Points are individual cells ( n = 172 DMSO, 115 PLK1i, 168, Chk2i) combined from 3 experiments. * p < 0.05, two-tailed t -test of normalized replicate means. C A549 wild-type (WT) or Chk2 −/ − cells treated as in ( A ). Right, quantification of N = 3 (DMSO, Chk2i) or N = 2 (PLK1i) experimental replicates * p < 0.05, unpaired one-way ANOVA with Bonferroni multiple comparison correction. ns, not significant. D Quantification of normalized CFP/FRET change for each condition in RPE-1 and U2OS cells. Error bars SD of individual cell values combined from N = 3 (U2OS) or N = 5 (RPE-1) experimental replicates. Individual cells n = 24, RPE-1 DMSO; n = 18, RPE-1 PLK1i; n = 16, RPE-1 Chk2i; n = 56, U2OS DMSO; n = 28 U2OS PLK1i; n = 28 U2OS Chk2i. * p < 0.05, two-tailed t -test of individual cell values. DMSO values are the same as in Fig. . E Top, protein sequence alignment of kinase ATP-binding pocket residues. Putative gatekeeper residues are red. Bottom, cartoon of analog-sensitive (as)Chk2 allele. F ADP-Glo quantification of kinase activity with DMSO or 10 μM 3-MB-PP1. Points denote an experimental replicate. Error bars SD ( N = 6 WT and asChk2 DMSO; N = 3 WT and asChk2 3-MB-PP1). ns not significant, * p < 0.05, two-tailed t -test. G Quantification of relative change in CFP/FRET ratio 1 h after addition of inhibitors or DMSO in cells co-expressing PLK1 FRET and asChk2-mCherry. Each point represents one cell normalized to the average value in the DMSO condition for each replicate. Error bars SD of individual cell values combined from ≥3 experimental replicates, n = 26 DMSO, n = 16 PLK1i, n = 19 Chk2i. * p < 0.05, two-tailed t -test of individual cell values. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Two Tailed Test, Staining, Comparison, Sequencing, Binding Assay, Activity Assay, Expressing

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Representative western blot (left) and quantification (right) of U2OS cells treated with MG132 (20 μM) in combination with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), CDK1i (5 μM RO-3306), CDK1i Chk2i combination, or a DMSO vehicle control. Each point represents one experimental replicate * p < 0.05, paired 1-way ANOVA with Bonferroni multiple comparisons correction. ns not significant ( p > 0.05). Error bars SD. B Representative western blot (left) and quantification (right) of U2OS cells treated with PLK1i (1 μM BI2536), Chk2i (10 μM BML-277), AurAi (50 nM MLN-8237), AurAi Chk2i combination, or a DMSO vehicle control. * p < 0.05, one-way ANOVA with Bonferroni multiple comparisons correction. ns, not significant ( p > 0.05). Error bars SD. C Left, immunoblot of mitotic HeLa cells treated with a vehicle control or Chk2 inhibitor for 1 h. Right, immunoblot of the same samples on a PhosTag gel. The putative phosphorylated specifies of PLK1 is indicated. *, uncharacterized bands recognized by PLK1 antibody. Right, quantification of phosphorylated PLK1 (slower-migrating PLK1 band on PhosTag gel) normalized to the total PLK1 protein abundance (68 kDa band on SDS-Page gel). Each point represents one biological replicate. * p < 0.05, two-tailed t -test. Error bars SD. D Experimental setup and representative western blot of PLK1 immunoprecipitated from mitotic HeLa cells treated with DMSO or Chk2i. E Quantification of phospho-antibody signal normalized to total PLK1 abundance from ( D ). Each point represents the value from one experimental replicate. * p < 0.05, two-tailed t -test. Error bars SD. F In vitro kinase assay with recombinant HIS-PLK1, MBP-Chk2 incubated with DMSO, PLK1i, or Chk2i as marked. Recombinant proteins were incubated in kinase assay buffer in a 1:1 molar ratio with 500 μM ATP for 30 minutes at 30 °C with gentle agitation. The reaction was quenched with 5 mM EDTA before being combined with denaturing sample buffer and analyzed via western blotting. p-PLK1 T210 antibody shown is Cell Signaling Technology D5H7. *, nonspecific recognition of MBP-Chk2. This experiment has been replicated 5 times with similar results. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Western Blot, Control, Quantitative Proteomics, SDS Page, Two Tailed Test, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Incubation, Gentle

Journal: Nature Communications

Article Title: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation

doi: 10.1038/s41467-024-54922-7

Figure Lengend Snippet: A Top, experimental setup. Bottom, relative cell viability (DMSO = 1) measured by crystal violet following 3-day treatment with PLK1 inhibitor BI-2536. Points represent average of N = 4 experimental replicate values averaged from technical triplicates. Error bars SEM of replicate averages. B Representative blots of cells treated for 3 days with either a nontargeting siRNA (control) or siRNA against Chk2 in Hela, U2OS, and A549 cells. C Quantification of cell viability following siRNA treatment and 2.27 nM BI-2536. Each point marks the mean of N = 3 HeLa, N = 4 U2OS and A549 experimental replicates, each averages from technical triplicates and normalized to DMSO-treated cells. Error bars SD of experimental replicates. p = 0.049 HeLa, p = 0.065 U2OS, p = 0.0176 A549 ratio paired two-tailed t -test of replicate mean values. D Map of Chk2 protein with indicated domains including S/T-Q cluster, FHA2, kinase, and nuclear localization sequence (NLS). Top, trace file of Chek2 locus from HCT-116 cells. Gray shading indicates the heterozygous L355P mutation. E Top, experimental setup. Bottom, representative western blot of HCT-116 cells with or without Chk2-mCherry challenged with etoposide (Etop, 10 μM) or in combination with Chk2i (10 μM BML-277) for 6 h. Numbers are quantification of p-Y15 signal normalized to GAPDH loading control and to DMSO-treated condition. This experiment has been repeated 1 other time with similar results. F Left, quantification of p-TCTP abundance in STLC-arrested HCT-116 cells transduced with a doxycycline (DOX)-inducible 3xFLAG-Chk2 construct. Cells were either treated with 0.1 μM DOX or untreated for 48 h prior to STLC addition. Each point represents one cell normalized to the average value in the untreated condition for each replicate. Error bars SD of individual cell values combined from N = 3 experimental replicates, n = 131 -DOX, n = 141 + DOX. * p < 0.05, two-tailed t -test of replicate mean values. Right, representative immunofluorescence image. Scale bar, 5 μm. G Representative western blot of HCT-116 cells with or without expression of the Chk2-mCherry rescue construct. H Quantification of relative cell viability in HCT-116 cells following 3-day treatment with BI-2536 titration. Points represent average of 3–4 experimental replicate values averaged from technical triplicates. Error bars SEM of replicate averages. Source data are provided as a Source Data file.

Article Snippet: Plk1 FRET sensor c-jun substrate was a gift from Michael Lampson (Addgene plasmid #45203; http://n2t.net/addgene:45203 ; RRID:Addgene 45203).

Techniques: Control, Two Tailed Test, Sequencing, Mutagenesis, Western Blot, Transduction, Construct, Immunofluorescence, Expressing, Titration

Journal: Virology

Article Title: A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors

doi: 10.1016/j.virol.2022.07.006

Figure Lengend Snippet: The inhibitory activity and mechanism of a natural product anacardic acid (AA) against SARS-CoV-2 PLpro in vitro . (A) The 4-step procedure of the FP screening assay for rapid screening of PLpro inhibitors. Step 1: 29 μL PLpro solution (1 μM) was incubated with 1 μL sample of a natural product (2 mg/mL) for 30 min at RT in a black 96-well microplate. Step 2: 20 μL FP probe solution (30 nM) was added and incubated for 30 min at RT. Step 3: The reaction was quenched by adding 10 μL avidin solution (240 nM) followed by 5 min incubation. Step 4: The mP value was measured by a microplate reader (BioTek). (B) The natural product layout in a black 96-well microplate for a large-scale screening. The positive (GRL0617), negative, and background wells were highlighted. (C) The illustration for the primary screening cycle of a natural product library using the FP screening assay. The red dotted line indicates a baseline (equivalent to 50% inhibition) in the primary screening cycle, and 5 candidate compounds were identified. (D) The chemical structure of anacardic acid. (E) Inhibitory effect of anacardic acid on the PLpro activity in the FP screening assay. (F) Inhibitory effect of anacardic acid on the PLpro activity in the FRET screening assay. GRL0617 was used as a positive control. (G) Inhibitory effect of anacardic acid on the Mpro activity in the FRET screening assay. PF-07321332 served as a positive control. (H) Inhibitory effect of anacardic acid on the PLpro isopeptidase activity. The fluorogenic peptide Z -RLRGG-AMC was used as the substrate for inhibition of PLpro isopeptidase activity by anacardic acid in vitro . GRL0617 served as a positive control. (I) The Lineweaver-Burk double-reciprocal plots for inhibition of PLpro activity by anacardic acid in vitro for the FRET substrate. (J) The secondary plots for a K i value.

Article Snippet: The FRET substrate (Dabcyl-FTLKGGAPTKVTE-Edans; λ ex /λ em = 340/500 nm), FP probe (FITC-FTLKGGAPTKVTK-Biotin; λ ex /λ em = 485/535 nm), FITC-FTLKGG peptide, and the fluorogenic peptide Z -RLRGG-AMC (λ ex /λ em = 340/460 nm) were purchased from GL Biochem (Shanghai, China), respectively.

Techniques: Activity Assay, In Vitro, Screening Assay, Incubation, Avidin-Biotin Assay, Inhibition, Positive Control