Journal: Antimicrobial Agents and Chemotherapy

Article Title: Proof of concept: targeted protein degradation of the stress granules component G3BP1 as an antiviral strategy against norovirus infection

doi: 10.1128/aac.01118-25

Figure Lengend Snippet: HaloPROTAC3 efficiently binds Halotag and HaloG3BP1 fusion proteins. ( A ) Chemical structure of HaloPROTAC3 and the enantiomeric form ent -HaloPROTAC3; the scheme indicates the warhead-binding VHL E3 ubiquitin ligase (VH285) and the chloroalkene moiety-binding Halotag (generated using Marvin JS 22.11.1, web-based chemical sketch tool by Chemaxon). ( B ) BV2 ΔG3BP1 + HaloG3 BP1 cells were treated with the indicated concentrations of HaloPROTAC3 for 48 h, and metabolic cell death was measured using CellTiter-Blue. Cell death was calculated as relative to the DMSO control, and CC 50 is indicated by a dotted line. ( C–E ) BV2 + Halotag, BV2 ΔG3BP1 + Halotag, or BV2 ΔG3BP1 + HaloG3 BP1 cells treated with the indicated concentrations of HaloPROTAC3 for 48 h were subjected to flow cytometry analysis after incubation for 2 h with the fluorescent ligand Halotag Janelia 647. ( C ) Gating strategy of Halotag + cells in the live cell population. ( D ) Representative histograms and ( E ) dose-response curves of the quantified percentage of Halotag + cells. The corresponding curve fitting parameters for each cell line are shown.

Article Snippet: HaloPROTAC3 (VH285-PEG4-C4-Cl, HY-111997) and VH032 (HY-120217) were purchased from MedChemExpress. ent -HaloPROTAC3 was obtained from Promega.

Techniques: Binding Assay, Ubiquitin Proteomics, Generated, Control, Flow Cytometry, Incubation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Proof of concept: targeted protein degradation of the stress granules component G3BP1 as an antiviral strategy against norovirus infection

doi: 10.1128/aac.01118-25

Figure Lengend Snippet: HaloPROTA3 degrades Halotag and HaloG3BP1 fusion proteins. ( A–E ) Protein samples from BV2 + Halotag, ΔG3BP1 + Halotag, or ΔG3BP1 + HaloG3 BP1 cells treated for either 24 h or 48 h with HaloPROTAC3 or ent -HaloPROTAC3 were subjected to Western blot analysis using antibodies against Halotag, G3BP1, or GAPDH. ( A ) Representative Western blots and ( B ) quantification of the Halotag protein expression in cells treated with HaloPROTAC3 normalized to GAPDH. ( C ) BV2 ΔG3BP1 + HaloG3 BP1 cells treated for 48 h with either 1.2 µM of HaloPROTAC3 or the indicated concentrations of ent -HaloPROTAC3 were subjected to flow cytometry analysis, as stated in . Data is expressed as mean ± standard error of the mean with ∗∗∗∗ indicating P ≤ 0.0001. ( D ) Representative Western blots and ( E ) quantification of the Halotag protein expression in cells treated with ent- HaloPROTAC3 normalized to GAPDH.

Article Snippet: HaloPROTAC3 (VH285-PEG4-C4-Cl, HY-111997) and VH032 (HY-120217) were purchased from MedChemExpress. ent -HaloPROTAC3 was obtained from Promega.

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Proof of concept: targeted protein degradation of the stress granules component G3BP1 as an antiviral strategy against norovirus infection

doi: 10.1128/aac.01118-25

Figure Lengend Snippet: Degradation of G3BP1 using HaloPROTAC3 shows antiviral activity against norovirus. BV2 ΔG3BP1 + HaloG3 BP1 cells were treated for 48 h with the shown concentrations of HaloPROTA3 and infected with MNV1. After 1 h of virus adsorption, the virus inoculum was removed, and HaloPROTAC3 was re-added to the cell culture medium for the remaining infection time. ( A ) Scheme of the experimental setup of the antiviral assays. ( B–D ) Cells infected with MOI 1 TCID 50 /cell for 16 h were incubated with fluorescent ligand Halotag Janelia 647 for 2 h, harvested, and stained with anti-dsRNA antibody to assess the percentage of infection. Cells were then subjected to flow cytometry analysis. ( B ) Gating strategy showing first the duplet exclusion for selection of single cells, followed by dead cell exclusion within the single-cell population. Halotag + cells and dsRNA + cells were gated exclusively from the live cell population. ( C ) Quantification of the percentage of Halotag + and ( D ) dsRNA + (infected) cells across HaloPROTAC3 concentrations. ( E ) Total RNA was isolated from cells infected with MOI 1 TCID 50 /cell for 10 h and used to quantify the levels of viral RNA (expressed as vRNA copies/µg RNA, normalized to the DMSO control), by RT-qPCR. ( F ) Extracellular infectious virus yield in supernatants from cells infected with MOI 1 TCID 50 /cell for 16 h and treated with the indicated concentrations of HaloPROTAC3 was quantified by TCID 50 in BV2 cells. ( G, H ) Infection-induced cell death was detected using the NIR dye measured in the live-cell imaging IncuCyte system. ( G ) Representative pictures after 24 h.p.i.; NIR + cells are shown in blue. ( H ) Quantification of the total number of NIR + cells for the shown HaloPROTAC3 concentrations. Data is expressed as mean ± standard error of the mean with ∗ , ∗∗ , ∗∗∗ , and ∗∗∗∗ indicating P ≤ 0.05, P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001 respectively.

Article Snippet: HaloPROTAC3 (VH285-PEG4-C4-Cl, HY-111997) and VH032 (HY-120217) were purchased from MedChemExpress. ent -HaloPROTAC3 was obtained from Promega.

Techniques: Activity Assay, Infection, Virus, Adsorption, Cell Culture, Incubation, Staining, Flow Cytometry, Selection, Single Cell, Isolation, Control, Quantitative RT-PCR, Live Cell Imaging

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Proof of concept: targeted protein degradation of the stress granules component G3BP1 as an antiviral strategy against norovirus infection

doi: 10.1128/aac.01118-25

Figure Lengend Snippet: Degradation of G3BP1 is required for HaloPROTAC3 antiviral activity. ( A, B ) BV2 ΔG3BP1 + HaloG3 BP1 cells were treated for 48 h with 1.2 µM of HaloPROTAC3 or ent -HaloPROTAC3, as indicated, and infected with MNV1. After 1 h of virus adsorption, the virus inoculum was removed and PROTACs re-added to the cell culture medium. ( A ) Extracellular infectious virus yield in supernatants from cells infected with MOI 1 TCID 50 /cell for 16 h and treated with either HaloPROTAC3 or ent -HALOPROTAC3 was quantified by TCID 50 in BV2 cells. ( B ) Infection-induced cell death was detected using the NIR dye measured in the live-cell imaging IncuCyte system. Shown is the quantification of the total number of NIR + cells for cells treated with either HaloPROTAC3 or ent -HaloPROTACs, measured every 3 h and followed for a total of 72 h.p.i. ( C–F ) BV2 ΔG3BP1 + HaloG3 BP1 cells were concomitantly treated for 48 h with 1.2 µM of HaloPROTAC3 and VH032 in the indicated concentrations. ( C ) Metabolic cell death was measured using CellTiter-Blue. Cell death was calculated as relative to the DMSO control. A concentration of 75 µM of VH032 is indicated by a dotted line. ( D ) Protein samples from cells treated for either 24 or 48 h with the indicated concentrations of HaloPROTAC3 and VH032 were subjected to Western blot analysis using antibodies directed Halotag or GAPDH. Bar plot shows the quantification of the Halotag protein bands from three independent experiments for all the experimental conditions. ( E ) Cells were infected with MOI 1 TCID 50 /cell of MNV1. After 1 h of virus adsorption, the virus inoculum was removed and drugs re-added to the cell culture medium. Infection-induced cell death was detected using the NIR dye measured in the live-cell imaging IncuCyte system, as stated in B . Shown is the quantification of the total number of NIR + cells measured after 24 h.p.i. ( F ) Extracellular infectious virus yield quantified by TCID 50 in BV2 cells from supernatants of cells infected with MOI 1 TCID 50 /cell for 16 h and treated with 1.2 µM of HaloPROTAC3 and 30 µM of VH032. Data is expressed as mean ± standard error of the mean with ∗∗ , ∗∗∗ , and ∗∗∗∗ indicating P ≤ 0.01, P ≤ 0.001, and P ≤ 0.0001 respectively. ‘ns’ denotes non-significant.

Article Snippet: HaloPROTAC3 (VH285-PEG4-C4-Cl, HY-111997) and VH032 (HY-120217) were purchased from MedChemExpress. ent -HaloPROTAC3 was obtained from Promega.

Techniques: Activity Assay, Infection, Virus, Adsorption, Cell Culture, Live Cell Imaging, Control, Concentration Assay, Western Blot

Journal: Current Protocols in Pharmacology

Article Title: Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3

doi: 10.1002/cpph.81

Figure Lengend Snippet: Schematic of HaloPROTAC3 degradation of HaloTag protein fusions in live cells. First, CRISPR/Cas9 technology is used to insert HaloTag or HiBiT‐HaloTag into the genomic locus of the target protein using a dsDNA donor plasmid and Cas9‐crRNA complex (1). After the HaloTag fusion is expressed, cells are treated with HaloPROTAC3(2). HaloPROTAC3 induces a ternary complex between the VHL E3 ligase component and HaloTag protein fusion, resulting degradation of the HaloTag target protein via the ubiquitin‐proteasomal pathway (3).

Article Snippet: HaloTag or HiBiT‐HaloTag CRIPSR/Cas9‐edited cells DPBS (Gibco, cat. no. 14190‐144) 0.05% trypsin/EDTA (Invitrogen, cat. no. 25300‐054) or 0.25% trypsin/EDTA (Invitrogen, cat. no 25200‐056), depending on cell line Complete growth medium for cell type of choice HaloPROTAC3 ligand (Promega, cat. no. GA3110) ent ‐HaloPROTAC3 ligand (Promega, cat. no. GA4110) Dimethyl sulfoxide (DMSO; Sigma, cat. no. 02660‐100 ml) Optional materials for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: LgBiT Expression Vector (Promega, cat. no. N2681) Nano‐Glo Endurazine Live Cell Substrate (Promega, cat. no. N2570/1/2) CO 2 ‐independent medium (Gibco, cat. no. 18045‐088) FBS (VWR, cat. no. 89510‐194) Six‐well plates Appropriate incubator for cell line Dilution reservoirs (Dilux, cat. no. D‐1002) White 96‐well plates (Corning, cat. no. 3917) Optional material for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader (Promega, cat. no. GM3000) or CLARIOstar Plus (BMG Labtech)

Techniques: CRISPR, Plasmid Preparation

Journal: Current Protocols in Pharmacology

Article Title: Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3

doi: 10.1002/cpph.81

Figure Lengend Snippet: Endpoint analysis of a homozygous β‐catenin‐HaloTag‐HiBiT endogenous protein fusion in HEK293 cells that stably express LgBiT after degradation with HaloPROTAC3. Cells contained the endogenous β‐catenin‐HaloTag‐HiBiT protein fusion were treated with HaloPROTAC3 for 3 or 24 hr, and degradation was detected with an endpoint HiBiT lytic assay ( A ). HaloPROTAC3 caused rapid reduction in the amount of tagged protein in the cell, which was visualized through luminescence imaging on an Olympus LV200 microscope ( B ). Phenotypic characterization was performed on cells that were treated with HaloPROTAC3. Cells containing the endogenous β‐catenin‐HaloTag‐HiBiT protein fusion were transfected with a TCF firefly luciferase reporter before being treating with mWnt3a. HaloPROTAC3 was used to degrade the beta‐catenin‐HaloTag‐HiBiT protein fusion, and after 24 hr a lytic dual luciferase assay was performed to measure firefly luciferase expressed from the TCF reporter ( C ) and β‐catenin HiBiT luminescence ( D ). TCF expression was not induced in cells that were treated with HaloPROTAC3 because β‐catenin‐HaloTag‐HiBiT protein fusions were degraded and could not enter the nucleus upon treatment with mWnt3a.

Article Snippet: HaloTag or HiBiT‐HaloTag CRIPSR/Cas9‐edited cells DPBS (Gibco, cat. no. 14190‐144) 0.05% trypsin/EDTA (Invitrogen, cat. no. 25300‐054) or 0.25% trypsin/EDTA (Invitrogen, cat. no 25200‐056), depending on cell line Complete growth medium for cell type of choice HaloPROTAC3 ligand (Promega, cat. no. GA3110) ent ‐HaloPROTAC3 ligand (Promega, cat. no. GA4110) Dimethyl sulfoxide (DMSO; Sigma, cat. no. 02660‐100 ml) Optional materials for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: LgBiT Expression Vector (Promega, cat. no. N2681) Nano‐Glo Endurazine Live Cell Substrate (Promega, cat. no. N2570/1/2) CO 2 ‐independent medium (Gibco, cat. no. 18045‐088) FBS (VWR, cat. no. 89510‐194) Six‐well plates Appropriate incubator for cell line Dilution reservoirs (Dilux, cat. no. D‐1002) White 96‐well plates (Corning, cat. no. 3917) Optional material for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader (Promega, cat. no. GM3000) or CLARIOstar Plus (BMG Labtech)

Techniques: Stable Transfection, Imaging, Microscopy, Transfection, Luciferase, Expressing

Journal: Current Protocols in Pharmacology

Article Title: Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3

doi: 10.1002/cpph.81

Figure Lengend Snippet: Kinetic degradation profiles of endogenous nuclear, mitochondrial membrane, and cytoplasmic HiBiT‐HaloTag protein fusions after treatment with HaloPROTAC3. Cells were treated with a series of dilutions of HaloPROTAC3 ( A , C , E ) or ent ‐HaloPROTAC3 ( B , D , F ), and degradation was followed by luminescence detection in live cells through a kinetic degradation assay for 24 hr. An N‐terminal HiBiT‐HaloTag‐nuclear endogenous protein fusion in HEK293 cells that stably express LgBiT, a C‐terminal HaloTag‐HiBiT mitochondrial membrane endogenous protein fusion in parent HEK293 cells with LgBiT introduced by transient transfection, and a C‐terminal HaloTag‐HiBiT‐cytoplasmic protein fusion in HEK293 cells that stably express LgBiT were used, respectively. No degradation was detected in the ent ‐HaloPROTAC3‐treated samples, confirming that the protein loss is due to a PROTAC‐mediated mechanism. ( G ) Rapid degradation rates were observed in all samples, with >50% degradation occurring in the first 3 hr. Around 80% degradation was achieved with each HiBiT‐HaloTag protein fusion regardless of cellular location ( H ). DC 50 values were 8.1 nM for the mitochondrial membrane and nuclear protein and 18.6 nM for the cytoplasmic protein.

Article Snippet: HaloTag or HiBiT‐HaloTag CRIPSR/Cas9‐edited cells DPBS (Gibco, cat. no. 14190‐144) 0.05% trypsin/EDTA (Invitrogen, cat. no. 25300‐054) or 0.25% trypsin/EDTA (Invitrogen, cat. no 25200‐056), depending on cell line Complete growth medium for cell type of choice HaloPROTAC3 ligand (Promega, cat. no. GA3110) ent ‐HaloPROTAC3 ligand (Promega, cat. no. GA4110) Dimethyl sulfoxide (DMSO; Sigma, cat. no. 02660‐100 ml) Optional materials for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: LgBiT Expression Vector (Promega, cat. no. N2681) Nano‐Glo Endurazine Live Cell Substrate (Promega, cat. no. N2570/1/2) CO 2 ‐independent medium (Gibco, cat. no. 18045‐088) FBS (VWR, cat. no. 89510‐194) Six‐well plates Appropriate incubator for cell line Dilution reservoirs (Dilux, cat. no. D‐1002) White 96‐well plates (Corning, cat. no. 3917) Optional material for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader (Promega, cat. no. GM3000) or CLARIOstar Plus (BMG Labtech)

Techniques: Degradation Assay, Stable Transfection, Transfection

Journal: Current Protocols in Pharmacology

Article Title: Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3

doi: 10.1002/cpph.81

Figure Lengend Snippet: Comparison between a HaloTag insertion with HaloPROTAC3 degradation and an FKBP12 F36V insertion with dTag‐13 degradation. Endogenous EPOP was tagged at the C terminus with either HaloTag‐HiBiT or FKBP12 F36V ‐HiBiT in HEK293 cells. HiBiT was used in combination with both tags to allow the detection of luminescent live cells once LgBiT was introduced by transient transfection. Both HaloPROTAC3 ( A ) and dTag‐13 ( B ) produced rapid and robust degradation of the protein fusion with the ∼80% degradation after 24 hr and similar levels of compound ( C ). However, with the dTag‐13 compound ( B ), a hook effect was detected in which at higher concentrations, the rate slows and degradation decreases.

Article Snippet: HaloTag or HiBiT‐HaloTag CRIPSR/Cas9‐edited cells DPBS (Gibco, cat. no. 14190‐144) 0.05% trypsin/EDTA (Invitrogen, cat. no. 25300‐054) or 0.25% trypsin/EDTA (Invitrogen, cat. no 25200‐056), depending on cell line Complete growth medium for cell type of choice HaloPROTAC3 ligand (Promega, cat. no. GA3110) ent ‐HaloPROTAC3 ligand (Promega, cat. no. GA4110) Dimethyl sulfoxide (DMSO; Sigma, cat. no. 02660‐100 ml) Optional materials for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: LgBiT Expression Vector (Promega, cat. no. N2681) Nano‐Glo Endurazine Live Cell Substrate (Promega, cat. no. N2570/1/2) CO 2 ‐independent medium (Gibco, cat. no. 18045‐088) FBS (VWR, cat. no. 89510‐194) Six‐well plates Appropriate incubator for cell line Dilution reservoirs (Dilux, cat. no. D‐1002) White 96‐well plates (Corning, cat. no. 3917) Optional material for live‐cell kinetic degradation of HiBiT‐HaloTag fusions: Luminometer such as the GloMax Discover Microplate Reader (Promega, cat. no. GM3000) or CLARIOstar Plus (BMG Labtech)

Techniques: Transfection, Produced