Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Cascade assay for target engagement confirms the mechanisms of action for reference compounds. (A) Schematic of MK2/3 signaling. (B) Isolated reactions tested in the biochemical cascade assays. (C) Concentration-response profiles for CC-99677 and ATI-450 against active kinases on their own (open symbols) or for cascade assays using active p38α to activate unactive MK2 or MK3 (solid symbols). Phosphorylation of the respective substrates for the individual enzymes, or in the cascade assays for the unactive enzyme (MK2 or MK3), were measured by HTRF.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Drug discovery, Isolation, Concentration Assay, Phospho-proteomics

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: HTRF Assay, Bioprocessing, Assay Development, Recombinant, Positive Control, Drug discovery, Quantitative Proteomics, Knock-Out, Concentration Assay

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: NanoBRET assay development. (A) Emission spectrum of NanoLuciferase measured live in dimethyl sulfoxide-treated HeLa cells over-expressing either amino- or carboxy-terminal MK2 fusions, with or without 0.17 mM (0.004%) Triton X-100 permeabilization (dashed or solid lines, respectively). The filter band passes used to measure NanoBRET signal are highlighted (centered at 452 nm for NanoLuciferase and 600 nm for BODIPY585). Magnified portion of the spectrum (right) shows the fraction of light from the NanoLuciferase donor that overlaps with –and should be subtracted from– the BODIPY acceptor emission signal. (B) Characterization of BODIPY|rev and BODIPY585|cov tracers, which compete for the same binding pocket as CC-99677. Structures of tracers (left) are shown alongside magnified emission spectra when added to HeLa cells transfected with plasmids encoding either amino- or carboxy-terminal MK2 fusions (middle) as well as the ratiometric NanoBRET signal following a titration of each tracer (right). Signal was measured 1 hour after tracer addition to the live intact (solid) or permeabilized (open/dashed line) cells using a CLARIOstar Plus (average of 5 replicates with standard deviation is shown). Line weight and style follow the legend in panel (A). (C) and (D) NanoBRET signal kinetics in the presence of BODIPY585|cov tracer, using well-by-well acquisitions (C) on the CLARIOstar Plus (CLARIOstar) or full-plate imaging (D) with a GNF Systems luminescence plate reader (LPR). NanoLuciferase signal (blue profiles in shaded background) decayed over time whereas the NanoBRET ratio (red profiles in white background) increased with exposure time to tracer. Continuous measurements for longer than 1 hour were achieved by supplementing with Endurazine substrate. (E) and (F) Measurement of k -on (E) and k -off (F) for CC-99677 using NanoBRET with BODIPY585|cov on a luminescence plate reader. The time points denote when CC-99677-containing medium was exchanged for medium containing excess BODIPY585|cov, which irreversibly quenched the system by saturating free binding sites. The profile for a reversible compound that occupies the same pocket as the covalent CC-99677 (gray, open circles) is included in the k -off plots (F).

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Assay Development, Expressing, Binding Assay, Transfection, Titration, Standard Deviation, Imaging, Microplate Reader Luminescence Measurement

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Comparison of compound target occupancy assays. (A) to (C) Measurement of MK2 target occupancy using dual HTRF, for endogenous protein targeting, in human HCC1428 cells (A) or murine RAW264.7 cells (B) versus NanoBRET in HeLa cells transfected with NanoLuciferase amino-terminally fused to human MK2 (C). Cells were treated with compound for 3 hours before washout. Curve fits are shown for the “free” signal (the ratio of “tracer”/“total”) for CC-99677 treatment, with regression ( R 2 ) values of 0.96 (RAW264.7 in HTRF); 0.92 (HCC1428 in HTRF), and 0.99 (HeLa in NanoBRET). (D) and (E) NanoBRET target occupancy data for 3-hour CC-99677 exposure in the presence of increasing concentrations of fetal bovine or human serum (D) to model in vivo compound availability using the NanoBRET assay. The right-shifted phenotype of CC-99677 is independent of human serum lot (E), suggesting that the free-fraction of CC-99677 compound will be reduced in circulation.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Comparison, Transfection, In Vivo

Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: A. Comparison of cell toxicity versus viral-GFP intensity (relative fluorescence units) at 7 days post-infection with RSVA or hPIV3 (both at MOI 0.02) with the epithelial cell lines VeroE6, Calu-3 or LLC-MK2 (n=3). Dashed box indicates cell lines chosen for subsequent antiviral assays. B-C. Representative images of the antiviral infection assay in 96-well format demonstrating dynamic range, here showing RSV infection of VeroE6 cells with and without single or dual-drug antiviral treatments. Cytopathology is visualised by endpoint crystal violet staining of adherent cells (B), whilst the fluorescence micrograph of same representative assay plate (C) demonstrates visualisation of GFP-tagged RSVA infection. D. Relationship between cell viability (quantified by crystal violet absorbance) and viral GFP fluorescence, as normalised against virus-only and mock controls. Representative dose response curves are modelled on data for remdesivir against RSVA. E-L. Dose response curves for monotherapy concentrations of remdesivir (E&I), favipiravir (F&J), EIDD-1931 (G&K), and ribavirin (H&L) tested against RSVA (E-H) or hPIV3 (I-L) infection. The viral inhibition % model is shown as the blue line, while cytotoxicity (as a % reduction of adherent cells) is indicated by the red line. The shaded box indicates drug concentrations with cytotoxicity exceeding 20%. (n=5 for RSVA, n=3-4 for hPIV3)

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Comparison, Fluorescence, Infection, Staining, Virus, Inhibition

Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: Dual-combinations of antivirals were assayed for viral inhibitory dose response and cytotoxicity for hPIV3 infection of LLC-MK2 cells. For each panel (A-F), the dose response grid represents % relative viral inhibition and the cytotoxicity response grid indicates % cell viability. Also shown are 3D contour plots of synergy scores as calculated by the HSA model, alongside cytotoxicity at the corresponding antiviral concentrations. (n=3 per combination). Favi = favipiravir, Rem = remdesivir, Mol = EIDD-1931, Riba = ribavirin.

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Infection, Inhibition

Journal: bioRxiv

Article Title: Antiviral drug synergy and mutational signatures in different epithelial cell models of RSV and hPIV infection

doi: 10.64898/2026.01.13.699296

Figure Lengend Snippet: A. Graphical representation of methodology for culture of primary airway epithelial cell model at air-liquid interface (ALI) and infection with RSVA or hPIV3. B-C. Representative confocal micrographs of primary airway epithelial cells in ALI culture. Cultures were infected with RSVA, hPIV3, or a mock control and fixed for immunofluorescent staining and imaging at 7 days post infection (dpi). Channels represent signal for DAPI staining of cell nuclei (blue), anti-GFP for the GFP-tagged virus (green), phalloidin for tight junctions (white), alpha-tubulin for ciliated epithelial cells (violet), and MUC5AC to indicate mucus-secretory cells (yellow). Orthogonal views (C) are also shown for representative cultures, with the same immunofluorescent staining. D-G. Cilia-beat frequency (CBF) was analysed using high speed video microscopy at 7 dpi with RSVA (D&F) or hPIV3 (E&G) versus mock. Distribution of measured CBF is shown by histogram and box plot (D-E) for each virus. Representative regions of interest (ROI) from video analysis indicate the relative abundance of detectable cilia movement in mocks versus RSVA (F) and hPIV3 (G) infected cultures. H. Representative well scans showing brightfield and GFP signal at 7 dpi for mock and infection conditions with each virus using cell lines (VeroE6 or LLC-MK2 in 96-well format) compared to primary ALI cultures (24-well format). I. The Ct result from qRT-PCR performed on supernatant (for cell lines) or apical wash (for ALI) samples used for viral genomic sequencing, indicating respective viral load at 7 dpi. J-L. Sequencing results for total mutations (J), intrahost single nucleotide variants (iSNV) and single nucleotide polymorphisms (SNP) (K) for each virus from cell line of ALI culture infection model, and the proportion of mutations which were observed in both models or unique (L). Representative images and functional analyses are shown from triplicate ALI cultures per experimental condition.

Article Snippet: Human lung adenocarcinoma cell line Calu-3 (HTB-55, ATCC) and rhesus monkey kidney cell line LLC-MK2 (CCL-7, Caltag-Medsystems Ltd) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1× penicillin/streptomycin.

Techniques: Infection, Control, Staining, Imaging, Virus, Microscopy, Quantitative RT-PCR, Genomic Sequencing, Sequencing, Functional Assay