Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: X-ray crystal structure of the PDZ2 domain of ZO-1 bound to the SARS-CoV-2 E protein PBM . ( A ) Ribbon diagram of the final crystal structure model of the ZO-1 PDZ2 domain in complex with the SARS-CoV-2 E protein PBM peptide. The structure forms a swapped dimer, with one monomer colored light blue and the other dark blue. The bound peptides are shown in orange. Secondary structure elements are labeled according to the canonical PDZ domain scheme. ( B ) Close-up view of the SARS-CoV-2 E protein PBM bound within the PDZ domain. Key interacting residues are shown as sticks and labeled. Intermolecular hydrogen bonds and polar interactions are indicated by cyan dashed lines.

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Labeling

Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: HTRF-based screening of compounds targeting the interaction between ZO-1 PDZ2 and the SARS-CoV-2 E protein PBM. The assay was conducted in 96-well, white, low-volume HTRF plates using 5 µL of purified GST-ZO-1-PDZ2 at a final concentration of 3 nM, 5 µL of purified His 6 -Ecyto at a final concentration of 60 nM, and 1 µL of each of the 1000 compounds from a focused-PPI chemical library. The histogram shows the % residual HTRF ratio. Among the 1,000 compounds screened, only the 36 compounds with % residual HTRF ratio below 30% were classified as primary hits (C1 to C36). Horizontal lines indicate the mean with SD ( n = 2).

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Purification, Concentration Assay

Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: Characterization of C19, a promising antiviral compound. ( A ) Chemical structure of C19, generated using ChemDraw software. ( B,C ) Docking poses of C19 targeting the ZO-1 PDZ2 swapped dimer. Panels show the best predicted binding poses of C19 when docked to the ZO-1 PDZ2 dimer either in its free form ( B ) or in complex with the E protein PBM ( C ), using DOCK6 simulations. The PDZ domains are shown in cartoon representation. The compound is positioned within key binding regions of the ZO-1 PDZ2 dimer, preferentially occupying the PBM-binding site. ( D ) HTRF inhibition dose-response curve of C19. The assay was performed in 96-well, white, low-volume HTRF plates with 5 µL of purified GST-ZO-1-PDZ2 (final concentration: 3 nM), 5 µL of purified His₆-Ecyto (final concentration: 60 nM), and 1 µL of C19, tested in a 10-point, twofold serial dilution (100 µM to 195 nM). The resulting curve represents the percentage of residual interaction (%R) based on the normalized FRET signal. A sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized response ( n = 1). ( E,F ) Antiviral activity of C19. The assay was conducted in 96-well culture plates using Vero-E6 cells (40,000 cells/well) infected with a NanoLuciferase-expressing recombinant SARS-CoV-2 virus at an MOI of 0.001. Cells were incubated for 72 h in the presence of C19, applied as a 9-point, twofold serial dilution ranging from 20 µM to 78 nM. Viral replication was assessed by measuring luminescence in the cell culture supernatant using the Nano-Glo assay kit ( E ). Data were normalized to both infected untreated and uninfected untreated control conditions. Sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized luminescence signal ( n =3). Viral quantification was performed using a plaque assay on Vero-E6 cells ( F ). Plaques were observed at 3 days post-infection (dpi) following infection with serial dilutions of supernatants collected from infected non-treated (INT) or infected treated (IT) cells, treated for 72 h with 20 µM of compound C19. Viral titers are reported as plaque-forming units per milliliter (PFU/mL). Horizontal lines represent the mean ± SEM, and dashed lines indicate the limit of detection ( n =3; nd=not detected). Statistical significance was determined by a t -test ( p < 0.005).

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Generated, Software, Binding Assay, Inhibition, Purification, Concentration Assay, Serial Dilution, Activity Assay, Infection, Expressing, Recombinant, Virus, Incubation, Cell Culture, Glo Assay, Control, Plaque Assay

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Schematic of IQGAP1 domains. Abbreviations: CHD, calponin homology domain; WW, WW domain; GRD, GAP-related domain; LBR, ligand binding region. (B) Images from TIRF assays containing 1 µM actin monomers (20% Oregon Green(OG)-label) and concentrations of IQGAP1. Scale bars, 25 µm. (C) Actin filament nucleation 200 s after initiation from reactions in B (n = 3 fields of view). Error bars indicate SE. Statistics, ANOVA: a, significantly different from control (No IQGAP1); ns, not different from control. (D) Actin filament elongation rates from TIRF reactions in B (n = 75 filaments per condition; pooled from 3 different trials). Statistics as in C. (E) Example actin filament that exhibits a pause in growth (red arrows). Scale bar, 3 µm. (F) Example length over time plots from reactions containing actin (grey) or actin and 75 nM IQGAP1 (teal). Red shading indicates the duration of filament pausing events. (G) Frequency distribution plots of the duration of IQGAP1 capping events from reactions measured in D (n = 75 filaments, and 31–70 pause events per condition, 331 pauses total measured). The mean duration of individual capping events was 20.6 s in the presence of IQGAP1 (regardless of concentration).

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Ligand Binding Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) IQGAP1 constructs that cap (+) and fail to cap (–) actin filaments. Specific functional regions and formin (mDia1) binding area are highlighted. DD, dimerization domain. Purple dots, two residues necessary for capping activity. (B) TIRF images from assays containing 1 µM actin (20% OG-label) or actin and 75 nM IQGAP1 proteins. CD, capping deficient. Scale bars, 25 µm. (C) Actin filament elongation rates from B (n = 75–324 filaments per condition; pooled from at least 3 different experiments). Error bars indicate SE. Statistics, ANOVA: a, significantly different from actin; b, significantly different from actin and 75 nM FL-IQGAP1. IQGAP1(CD) does not cap actin filaments shown by (D) filament length traces and (E) frequency plots of the duration of IQGAP1-mediated pauses (n = 75 filaments per condition; n = 159 pauses for IQGAP1; n = 12 for IQGAP1(CD)).

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Construct, Functional Assay, Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Single-molecules of labeled IQGAP1 or IQGAP1(CD) subjected to step-photobleaching. (B) Fluorescence intensity profiles of representative step photobleaching events for 5 nM SNAP-IQGAP1 proteins as imaged in A. Red lines emphasize photobleaching steps. (C) Fluorescence intensity predictions and analysis of SNAP-IQGAP1 molecules (n = 300 molecules per protein, pooled from 3 experiments) as in D-E. (D) Two-color TIRF images showing the localization of 649-IQGAP1 or 488-IQGAP1(CD). Reactions contain: 1 µM actin (10% 488- or 647-Alexa label) and 5 nM SNAP-IQGAP1 construct. Arrows depict filament end- or side-binding events. Scale bars, 5 µm. (E) Actin filament elongation rates comparing actin alone control with 5 nM untagged or 5 nM SNAP-tagged versions of IQGAP1. Conditions as in A (n = 33–75 filaments (dots) per condition pooled from 3 independent experiments). Error bars indicate SE. Statistics, ANOVA: a, significantly different from no IQGAP1 control; b, significantly different from actin and untagged IQGAP1; ns, not different from control. (F) Representative length traces of filaments from reactions in B. Red shading indicates the duration of filament capping events. (G) Percent of all actin filaments with labeled-IQGAP1 molecules present on the plus-end (n = 73–144 filaments per field of view, 335–372 measured total). (H) Percent of actin filaments from G with side bound IQGAP1 molecules. (I) Actin filament bundling was quantified at 1200 s from TIRF fields described in B, with skewness parameter (n = 3 fields of view per condition).

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Labeling, Fluorescence, Construct, Binding Assay, Control

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Schematic of formin (mDia1) constructs that do not bind (FH1-C) or bind (∆DAD) to IQGAP1. Abbreviations: GBD, GTPase-binding domain; DID, Diaphanous inhibitory domain; FH1, formin homology region 1; FH2, formin homology 2 domain; DAD, Diaphanous autoregulatory domain. Structural features of mDia1 are labeled with green lines. DD, dimerization domain. (B) Single molecule colocalization of 10 nM 549-mDia1 constructs with 10 nM 649-IQGAP1 or 10 nM 488-IQGAP1(CD) by TIRF. Arrows highlight examples of SNAP-IQGAP1 (pink), SNAP-mDia1 (green) or colocalized molecules (white). Scale bars, 5 µm. (C) Colocalization of formin-IQGAP1 complexes as in B. Error bars indicate SE. Dots are individual values for n = 3–4 replicates. Statistics, ANOVA: a, significantly more association compared to mDia1(FH1-C). (D) Triple-color TIRF of actin filaments (blue; 10% 488- or 647-Alexa label) polymerizing in the presence of 5 µM profilin, 10 nM 549-mDia1(∆DAD) (green), and 10 nM 649-IQGAP1 or 488-IQGAP1(CD) (pink). Arrows as in B. Scale bars, 3 µm. (E) Elongation rates correlate with arrival and dissociation of 649-IQGAP1 or mDia1(∆DAD) at the barbed end. (F) Effects of IQGAP1 on the rate of mDia1-mediated actin filament elongation. Reactions as in D with unlabeled proteins. (G) Effects of IQGAP1(CD) on the rate of mDia1-mediated elongation. Reactions as described in D. Error bars in F-G indicate SE. Dots in F-G represent individual filaments measured (n = 24–105 per condition, pooled from at least 3 independent trials). Statistics as in C: a, different from actin alone and formin controls lacking profilin; b, different from reactions containing formin and profilin.

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Construct, Binding Assay, Labeling

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Schematic of approach. Pink filament seeds with available ends will become bicolor filaments upon addition of blue actin monomers (free ends) or remain pink (blocked/capped ends). (B) Two-color actin filament assay visualized by TIRF. Reactions contain biotinylated 647-actin filament “seeds” polymerized for 2–3 mins before the reaction volume is replaced with 0.5 µM free-actin monomers (20% OG (top row only) or 10% 488-Alexa label) and buffer (control), 250 nM IQGAP1, 250 nM IQGAP1(CD), or 10 nM CP. (C) Quantification of blocked ends from reactions in B. Statistics, ANOVA: a, significantly different from control; b, significantly different from reactions containing IQGAP1. (D) Reactions performed as in B with the following conditions: buffer or 10 nM mDia1 protein, or 250 nM IQGAP1. (E) Quantification and statistics as in C for reactions in D. (F) Reactions performed as in B and D in the following combinations: buffer, CP, CP and mDia1(∆DAD), CP and IQGAP1, or CP, mDia1(∆DAD), and IQGAP1. All scale bars, 10 µm. (G) Quantification and statistics as in C for reactions in F.

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Control

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Four-color TIRF microscopy images of plus-end complexes. Reactions contain: 1 µM actin (30% 405-Alexa label), 10 nM 488-mDia1(∆DAD), 10 nM 549-CP, and 10 nM 649-IQGAP1. Scale bars, 2 µm. (B) Fluorescence intensity for examples in (A) showing the formation or dissolution of plus-end complexes. (C) Dissociation of single molecules and complexes from actin filament plus-ends. (D) The plus-end half-life of each complex determined in C.

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Microscopy, Fluorescence, Dissolution

Journal: bioRxiv

Article Title: Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

doi: 10.1101/2023.05.04.539490

Figure Lengend Snippet: (A) Representative cell morphology of 3T3 cells expressing endogenous IQGAP1 (green), IQGAP1 (−/−) (blue), IQGAP1 (−/−) transfected with Halo-IQGAP1 (pink), or IQGAP1 (−/−) transfected with Halo-IQGAP1(CD), plated on micropatterns. Scale bars, 10 µm. (B) Quantification of mean fluorescence (pixel count) from cells in A. Dots represent values from individual cells (n = 20–41 cells). Error bars, SE. Statistics, ANOVA: a, significantly different from endogenous; b, significantly different from IQGAP1 (−/−) . Cells expressing IQGAP1(CD) plasmid were not significantly different than cells expressing IQGAP1 plasmid (p = 0.99). (C) Representative images of phalloidin-stained actin filaments from cells as in A. (D) Quantification and statistics of actin filament morphology as in B (n = 20–41 cells). Cells expressing IQGAP1(CD) plasmid were not significantly different than cells expressing IQGAP1 plasmid (p = 0.11). (E) Representative images from cells as in A 12 h post-wounding event. Scale bars, 200 µm. (F) Quantification of wound healing assays in E. Histograms represent means from n = 3–4 independent assays (dots). (G) Summary of IQGAP1 actin filament plus-end activities highlighting differences in on-rates and average dwell time. IQGAP1 displaces plus-end factors >18-fold more than the mDia1-CP decision complex. In cells, IQGAP1-filament capping activity may promote more turnover of molecules on filament plus-ends.

Article Snippet: GST-tagged IQGAP1 in pGEX-2T (AddGene: 30107) was expressed (as above) and induced for 18 h at 18 °C.

Techniques: Expressing, Transfection, Fluorescence, Plasmid Preparation, Staining, Activity Assay

Journal: Cells

Article Title: Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

doi: 10.3390/cells11010119

Figure Lengend Snippet: Binding preferences of mTOR FRB domain to different PAs vary in terms of acyl chain structure. Experiments were performed using POPC liposomes containing 10 mol% POPA, SAPA, or DPPA. ( A ) Percentage of FRB domain of mTOR in the fractions collected after flotation and analyzed by dot-blot/densitometry. For POPA and SAPA, no or very weak binding occurs, but for DPPA strong interaction is observed. Error bars are standard deviations of three independent experiments. ( B ) Distribution of median intensity per pixel of individual GUVs (of variable diameter) incubated with FRB-GFP in green fluorescent channel. GUVs were captured after addition of FRB GFP protein and Rh-PE as membrane marker (data from three independent sets of experiments). Each dot represents a single GUV. The number ( n ) of analyzed GUVs is indicated in the legend. ( C ) Representative images of GUVs (with Rh-PE as membrane marker in red) and FRB-GFP (green channel). The scale bar corresponds to 10 μm.

Article Snippet: The pGEX-2T plasmid containing the sequence for the FRB domain of human mTOR protein was bought from Addgene (Cambridge, MA, USA) (cat. no. 26607).

Techniques: Binding Assay, Liposomes, Dot Blot, Incubation, Membrane, Marker

Journal: Cells

Article Title: Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

doi: 10.3390/cells11010119

Figure Lengend Snippet: Effect of cholesterol on binding preferences of mTOR FRB domain towards different PA species. Experiments were performed using POPC/CH/PA (55/35/10 molar ratio) liposomes with POPA, SAPA, or DPPA, and results were directly compared to the data already presented in . ( A ) Percentage of FRB domain of mTOR protein in fractions collected after flotation and analyzed by dot-blot/densitometry. Error bars are standard deviations of three independent experiments. ( B ) Distribution of median intensity per pixel of individual GUVs incubated with FRB-GFP in green fluorescent channel. Each dot represents a single GUV (with CH represented by blue dots, and without CH by green). Data collected in three independent experiments. The number ( n ) of analyzed GUVs is indicated in the legend. ( C) Representative images of GUVs (with Rh-PE as membrane marker in red channel) and FRB GFP (green channel). The scale bar corresponds to 10 μm. ( D ) Boxplot of data presented in panel B. Each dot represents a single GUV. The mean value for each GUV lipid composition tested is indicated as a line and standard deviation as whiskers.

Article Snippet: The pGEX-2T plasmid containing the sequence for the FRB domain of human mTOR protein was bought from Addgene (Cambridge, MA, USA) (cat. no. 26607).

Techniques: Binding Assay, Liposomes, Dot Blot, Incubation, Membrane, Marker, Standard Deviation

Journal: Cells

Article Title: Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

doi: 10.3390/cells11010119

Figure Lengend Snippet: Association and dissociation curves of PA-containing liposomes. mTOR FRB domain was immobilized on Ni-NTA sensors tips prior association. Curves represent the mean values of triplicate measurements of each concentration. Dashed lines represent raw data, continuous lines fit. ( A ) POPC/CH/PA liposomes, ( B ) POPC/PA liposomes, ( C ) normalized curves of all studied liposome mixtures in 24 µM PA concentration.

Article Snippet: The pGEX-2T plasmid containing the sequence for the FRB domain of human mTOR protein was bought from Addgene (Cambridge, MA, USA) (cat. no. 26607).

Techniques: Liposomes, Concentration Assay

Journal: Cells

Article Title: Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol

doi: 10.3390/cells11010119

Figure Lengend Snippet: Kinetic rate constants and affinities determined for the liposome/mTOR FRB domain interactions.

Article Snippet: The pGEX-2T plasmid containing the sequence for the FRB domain of human mTOR protein was bought from Addgene (Cambridge, MA, USA) (cat. no. 26607).

Techniques: