pf3644022  (Tocris)


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    Structured Review

    Tocris pf3644022

    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pf3644022/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pf3644022 - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy"

    Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

    Journal: iScience

    doi: 10.1016/j.isci.2024.109580


    Figure Legend Snippet:

    Techniques Used: Recombinant, Transfection, Electron Microscopy, Protease Inhibitor, Western Blot, Purification, SYBR Green Assay, Viability Assay, Sequencing, Software, Imaging


    Structured Review

    Carterra Inc chip carterra bio catalog 4279
    Chip Carterra Bio Catalog 4279, supplied by Carterra Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/chip carterra bio catalog 4279/product/Carterra Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    chip carterra bio catalog 4279 - by Bioz Stars, 2024-07
    86/100 stars

    Images


    Structured Review

    Ubiquigent ksq 4279 inhibition
    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Ksq 4279 Inhibition, supplied by Ubiquigent, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksq 4279 inhibition/product/Ubiquigent
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ksq 4279 inhibition - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279"

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    Journal: bioRxiv

    doi: 10.1101/2024.05.16.594330

    (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Figure Legend Snippet: (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Techniques Used: Activity Assay, Cryo-EM Sample Prep, Binding Assay

    (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.
    Figure Legend Snippet: (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Techniques Used:

    (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.
    Figure Legend Snippet: (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Techniques Used:

    (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.
    Figure Legend Snippet: (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Techniques Used: Solvent, Binding Assay, Negative Control

    (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.
    Figure Legend Snippet: (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Techniques Used: Binding Assay, Residue, Solvent, Generated


    Structured Review

    Ubiquigent ksq 4279
    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Ksq 4279, supplied by Ubiquigent, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksq 4279/product/Ubiquigent
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ksq 4279 - by Bioz Stars, 2024-07
    86/100 stars

    Images

    1) Product Images from "Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279"

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    Journal: bioRxiv

    doi: 10.1101/2024.05.16.594330

    (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Figure Legend Snippet: (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Techniques Used: Activity Assay, Cryo-EM Sample Prep, Binding Assay

    (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.
    Figure Legend Snippet: (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Techniques Used:

    (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.
    Figure Legend Snippet: (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Techniques Used:

    (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.
    Figure Legend Snippet: (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Techniques Used: Solvent, Binding Assay, Negative Control

    (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.
    Figure Legend Snippet: (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Techniques Used: Binding Assay, Residue, Solvent, Generated

    pf3644022  (Tocris)


    Bioz Verified Symbol Tocris is a verified supplier
    Bioz Manufacturer Symbol Tocris manufactures this product  
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  • 94

    Structured Review

    Tocris pf3644022

    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pf3644022/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pf3644022 - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy"

    Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

    Journal: iScience

    doi: 10.1016/j.isci.2024.109580


    Figure Legend Snippet:

    Techniques Used: Recombinant, Transfection, Electron Microscopy, Protease Inhibitor, Western Blot, Purification, SYBR Green Assay, Viability Assay, Sequencing, Software, Imaging

    ksq 4279  (MedChemExpress)


    Bioz Verified Symbol MedChemExpress is a verified supplier
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    Structured Review

    MedChemExpress ksq 4279
    Ksq 4279, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksq 4279/product/MedChemExpress
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ksq 4279 - by Bioz Stars, 2024-07
    86/100 stars

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    ksq 4279  (MedChemExpress)


    Bioz Verified Symbol MedChemExpress is a verified supplier
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  • 86

    Structured Review

    MedChemExpress ksq 4279
    Ksq 4279, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ksq 4279/product/MedChemExpress
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ksq 4279 - by Bioz Stars, 2024-07
    86/100 stars

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    pf  (Tocris)


    Bioz Verified Symbol Tocris is a verified supplier
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    Structured Review

    Tocris pf
    Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pf/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pf - by Bioz Stars, 2024-07
    94/100 stars

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    Structured Review

    SMAC Corp dna helicase inhibitor yk 4279
    Dna Helicase Inhibitor Yk 4279, supplied by SMAC Corp, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna helicase inhibitor yk 4279/product/SMAC Corp
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna helicase inhibitor yk 4279 - by Bioz Stars, 2024-07
    86/100 stars

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    Structured Review

    Agrisera atg13a product no as19 4279 antibodies
    Atg13a Product No As19 4279 Antibodies, supplied by Agrisera, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atg13a product no as19 4279 antibodies/product/Agrisera
    Average 86 stars, based on 1 article reviews
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    atg13a product no as19 4279 antibodies - by Bioz Stars, 2024-07
    86/100 stars

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    mk2 inhibitor pf3644022  (Tocris)


    Bioz Verified Symbol Tocris is a verified supplier
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    Tocris mk2 inhibitor pf3644022
    A. <t>MK2/3-deficient</t> MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.
    Mk2 Inhibitor Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mk2 inhibitor pf3644022/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mk2 inhibitor pf3644022 - by Bioz Stars, 2024-07
    94/100 stars

    Images

    1) Product Images from "5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling"

    Article Title: 5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

    Journal: bioRxiv

    doi: 10.1101/2023.03.03.530727

    A. MK2/3-deficient MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.
    Figure Legend Snippet: A. MK2/3-deficient MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.

    Techniques Used: Transduction, Expressing, Plasmid Preparation, Marker, Cytotoxicity Assay

    MK2-deficient MEFs transduced with MK2 expression or control vector were treated with TNF alone or in the presence of the inhibitor panel (at 10 μM concentration) for 6h and cell viability was quantified by CCK8 colorimetric assay. Each treatment was performed in triplicates. While TNF alone is not cytotoxic, several small molecules sensitize both MEF lines to TNF (comps. 15, 42, 133, 142). Compound 14/CAY10657 (IKK2 inhibitor), comp. 32/BIO (GSK3 inhibitor), comp. 54,55/Bisindolylmaleimide VIII/IX (PKC inhibitors), comp. 94/PIK-75 (PI3 kinase inhibitor) and comp. 130/5-Iodotubercidin (5-ITu) display specific sensitization effects dependent on MK2-deficiency. Average values of n = 3 independent wells are plotted ± SD.
    Figure Legend Snippet: MK2-deficient MEFs transduced with MK2 expression or control vector were treated with TNF alone or in the presence of the inhibitor panel (at 10 μM concentration) for 6h and cell viability was quantified by CCK8 colorimetric assay. Each treatment was performed in triplicates. While TNF alone is not cytotoxic, several small molecules sensitize both MEF lines to TNF (comps. 15, 42, 133, 142). Compound 14/CAY10657 (IKK2 inhibitor), comp. 32/BIO (GSK3 inhibitor), comp. 54,55/Bisindolylmaleimide VIII/IX (PKC inhibitors), comp. 94/PIK-75 (PI3 kinase inhibitor) and comp. 130/5-Iodotubercidin (5-ITu) display specific sensitization effects dependent on MK2-deficiency. Average values of n = 3 independent wells are plotted ± SD.

    Techniques Used: Transduction, Expressing, Plasmid Preparation, Concentration Assay, Colorimetric Assay

    A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/mL) for 6h. B. Cells of indicated genotype were treated with 10 μM 5-ITu for 6 and 24h and cell viability was quantified. C. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 μM ABT702, 100 nM gemcitabine, 5 μM etoposid, 5 μM doxorubicin and 5 μM staurosporine) in the presence or absence of TNF for 6h and cell viability was assessed. D. MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E, F. RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS - induced necroptosis (E) and SM-mediated autocrine TNF-dependent death (F). Average values of n = 3 independent wells are plotted ± s.d. (** denotes p-value ≤ 0.001, *** denotes p-value ≤ 0.0001, **** denotes p-value ≤ 0.0001).
    Figure Legend Snippet: A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/mL) for 6h. B. Cells of indicated genotype were treated with 10 μM 5-ITu for 6 and 24h and cell viability was quantified. C. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 μM ABT702, 100 nM gemcitabine, 5 μM etoposid, 5 μM doxorubicin and 5 μM staurosporine) in the presence or absence of TNF for 6h and cell viability was assessed. D. MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E, F. RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS - induced necroptosis (E) and SM-mediated autocrine TNF-dependent death (F). Average values of n = 3 independent wells are plotted ± s.d. (** denotes p-value ≤ 0.001, *** denotes p-value ≤ 0.0001, **** denotes p-value ≤ 0.0001).

    Techniques Used: Transduction, Expressing, Plasmid Preparation

    A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with IKK1/2 inhibitor BMS and 5-ITu in combination with TNF for 2h. MLKL and RIPK1 phosphorylation was monitored with additional control blots. RIPK1 band shift induced by MK2-mediated phosphorylation is absent in MK2-KO cells and EF2 is shown as loading control. B. MK2/3-deficient MEFs transduced with MK2 expression or control vector were treated with 5-ITu alone or in combination with TNF for 3h. Cells were pre-treated for 30 minutes with the MK2 inhibitor PF3604422, the RIPK1 inhibitor Nec-1 or the RIPK3 inhibitor GSK872, as indicated. C. Wild type (WT) - MEFs were treated with 5-ITu or TNF alone or in combination for a short-term kinetics (10-180 minutes). D. RAW264.7 cells were treated with 5-ITu or LPS alone or in combination for 10-180 minutes and signaling was monitored by immunoblotting as indicated. (A-D) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa.
    Figure Legend Snippet: A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with IKK1/2 inhibitor BMS and 5-ITu in combination with TNF for 2h. MLKL and RIPK1 phosphorylation was monitored with additional control blots. RIPK1 band shift induced by MK2-mediated phosphorylation is absent in MK2-KO cells and EF2 is shown as loading control. B. MK2/3-deficient MEFs transduced with MK2 expression or control vector were treated with 5-ITu alone or in combination with TNF for 3h. Cells were pre-treated for 30 minutes with the MK2 inhibitor PF3604422, the RIPK1 inhibitor Nec-1 or the RIPK3 inhibitor GSK872, as indicated. C. Wild type (WT) - MEFs were treated with 5-ITu or TNF alone or in combination for a short-term kinetics (10-180 minutes). D. RAW264.7 cells were treated with 5-ITu or LPS alone or in combination for 10-180 minutes and signaling was monitored by immunoblotting as indicated. (A-D) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa.

    Techniques Used: Transduction, Expressing, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Western Blot, Marker

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    Tocris pf3644022

    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ubiquigent ksq 4279 inhibition
    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
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    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
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    MedChemExpress ksq 4279
    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Ksq 4279, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pf  (Tocris)
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    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
    Pf, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
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    (A) Evaluation of the selectivity of ML323 and <t>KSQ-4279</t> inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.
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    Tocris mk2 inhibitor pf3644022
    A. <t>MK2/3-deficient</t> MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.
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    Image Search Results


    Journal: iScience

    Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

    doi: 10.1016/j.isci.2024.109580

    Figure Lengend Snippet:

    Article Snippet: PF3644022 , Tocris , Cat#4279.

    Techniques: Recombinant, Transfection, Electron Microscopy, Protease Inhibitor, Western Blot, Purification, SYBR Green Assay, Viability Assay, Sequencing, Software, Imaging

    (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Article Snippet: We first compared ML323 and KSQ-4279 inhibition in ubiquitin-rhodamine assays using the DUB profiler ™ assay (Ubiquigent).

    Techniques: Activity Assay, Cryo-EM Sample Prep, Binding Assay

    (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Article Snippet: We first compared ML323 and KSQ-4279 inhibition in ubiquitin-rhodamine assays using the DUB profiler ™ assay (Ubiquigent).

    Techniques:

    (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Article Snippet: We first compared ML323 and KSQ-4279 inhibition in ubiquitin-rhodamine assays using the DUB profiler ™ assay (Ubiquigent).

    Techniques:

    (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Article Snippet: We first compared ML323 and KSQ-4279 inhibition in ubiquitin-rhodamine assays using the DUB profiler ™ assay (Ubiquigent).

    Techniques: Solvent, Binding Assay, Negative Control

    (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Article Snippet: We first compared ML323 and KSQ-4279 inhibition in ubiquitin-rhodamine assays using the DUB profiler ™ assay (Ubiquigent).

    Techniques: Binding Assay, Residue, Solvent, Generated

    (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) Evaluation of the selectivity of ML323 and KSQ-4279 inhibitors across the DUBprofiler™ panel (Ubiquigent). a enzyme activated half-maximally with additional ubiquitin; b enzyme activated maximally with additional ubiquitin; c enzyme activated with proteasome-V5; d pS177; e with additional zinc added. (B) Gel-based assay to demonstrate USP1 activity against FANCI-FANCD2 Ub -dsDNA in the presence or absence of ML323 or KSQ-4279 (25 µM). USP1-UAF1 enzyme and FANCI-FANCD2 Ub substrate were used at 0.01 µM and 1 µM, respectively. Two technical replicates were performed. (C) Cryo-EM analysis of USP1 C90S -UAF1-FANCI-FANCD2 Ub -dsDNA with KSQ-4279. Density within 2.5 Å of KSQ-4279 is shown at 7.1σ (threshold of 0.085; sharpened map). (D) KSQ-4279 binding site. Sidechains of residues contacting KSQ-4279 and protein Cα atoms are shown; black dashed lines indicate hydrogen bonds.

    Article Snippet: The selectivity of KSQ-4279 and ML323 was then evaluated in the DUB profiler ™ assay (Ubiquigent) against 48 individual deubiquitinase enzymes, at concentrations of 0.01, 0.1 and 1, 10 and 100 µM of each compound, the latter being equivalent to at least 10,000-fold greater concentrations than the IC 50 against the primary target.

    Techniques: Activity Assay, Cryo-EM Sample Prep, Binding Assay

    (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) KSQ-4279 perturbs the β-turn, on which the catalytic aspartates D751 and D752 reside, to a lesser extent than ML323. (B) KSQ-4279 perturbs F101 whereas ML323 does not. Protein Cα atoms are shown.

    Article Snippet: The selectivity of KSQ-4279 and ML323 was then evaluated in the DUB profiler ™ assay (Ubiquigent) against 48 individual deubiquitinase enzymes, at concentrations of 0.01, 0.1 and 1, 10 and 100 µM of each compound, the latter being equivalent to at least 10,000-fold greater concentrations than the IC 50 against the primary target.

    Techniques:

    (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) KSQ-4279 disrupts β-strands of the inhibitor-free state leaving residues 163-176 disordered. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. KSQ-4279 is shown as space-filling spheres based on van der Waals radii. (B) A subset of ML323-bound particles have reordered residues 167-191 resulting in formation of a new helix and slipping of helix α4, in addition to bending of helix α3. The inhibitor-free structure (7ZH3 ) is shown in transparent grey. ML323 is shown as space-filling spheres based on van der Waals radii. (C) Additional interactions of the ML323 subset structure. Dashed lines indicate a potential hydrogen bond. Asterisk indicates where the isopropyl group of KSQ-4279 would clash with F163. ML323 is shown as sticks.

    Article Snippet: The selectivity of KSQ-4279 and ML323 was then evaluated in the DUB profiler ™ assay (Ubiquigent) against 48 individual deubiquitinase enzymes, at concentrations of 0.01, 0.1 and 1, 10 and 100 µM of each compound, the latter being equivalent to at least 10,000-fold greater concentrations than the IC 50 against the primary target.

    Techniques:

    (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) Solvent accessible surface of inhibitor-free USP1 (7ZH3 ) near to the cryptic binding site (top). KSQ-4279 and ML323 binding leaves only a small pocket remaining (middle and bottom, respectively). Solvent accessible pockets are shown as surfaces coloured by lipophilicity and inhibitors as space-filling spheres based on van der Waals radii. Superposition of each inhibitor-bound structures onto the inhibitor-free structure (7ZH3 ) was performed. (B) Thermal shift assays of USP1 (3.8 μM) in the presence or absence of ML323 or KSQ-4279 (50 μM) (solid lines). ML323, KSQ-4279, and DMSO without USP1 are shown as dashed lines. (C) Quantification of thermal shift assays using the inflection point to estimate the melting temperature. USP7 catalytic domain (USP7 CD ) was included as a negative control. Two technical replicates for each USP were performed and are shown as circles.

    Article Snippet: The selectivity of KSQ-4279 and ML323 was then evaluated in the DUB profiler ™ assay (Ubiquigent) against 48 individual deubiquitinase enzymes, at concentrations of 0.01, 0.1 and 1, 10 and 100 µM of each compound, the latter being equivalent to at least 10,000-fold greater concentrations than the IC 50 against the primary target.

    Techniques: Solvent, Binding Assay, Negative Control

    (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Journal: bioRxiv

    Article Title: Structural and biochemical insights on the mechanism of action of the clinical USP1 inhibitor, KSQ-4279

    doi: 10.1101/2024.05.16.594330

    Figure Lengend Snippet: (A) ML323 or KSQ-4279 binding disrupts hydrogen bonding of the catalytic aspartates and displaces the oxyanion stabilizing residue, N85. Hydrogen bonds of the inhibitor-free structure are shown as dashed lines. (B) Gel-based assay for reactions of USP1 (2 µM) in the presence or absence of ML323 or KSQ-4279 (25 µM) with Ub-Prg (6 μM) at room temperature. Fraction of the total signal corresponding to the reacted band is given below each reaction. Two technical replicates were performed. (C) Gel-based assay of reactions of 2 µM USP1 alone or with 25 µM inhibitor with excess Ub-Prg on ice using a single time-point. Fraction reacted was quantified using densitometric analysis of the bands. At least two technical replicates were performed. (D) Limited proteolysis reactions of USP1 Δ1Δ2 with different proteases. (E) AlphaFold model of USP1 Δ1Δ2 with residues of highly probable α-chymotrypsin cleavage sites and high solvent accessibility shown as spheres. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 1-3 are indicated. (F) AlphaFold model of USP1 Δ1Δ2 with residues of highly probably trypsin cleavage sites and high solvent accessibility shown as spheres. Protein cartoon is colored by Jones’ rainbow. Residues with high cleavage probability that are only accessible in inhibitor-bound structures are colored grey. The N-terminus and probable sites for cleavage products generated by cuts 2 and 3 are indicated.

    Article Snippet: The selectivity of KSQ-4279 and ML323 was then evaluated in the DUB profiler ™ assay (Ubiquigent) against 48 individual deubiquitinase enzymes, at concentrations of 0.01, 0.1 and 1, 10 and 100 µM of each compound, the latter being equivalent to at least 10,000-fold greater concentrations than the IC 50 against the primary target.

    Techniques: Binding Assay, Residue, Solvent, Generated

    A. MK2/3-deficient MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.

    Journal: bioRxiv

    Article Title: 5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

    doi: 10.1101/2023.03.03.530727

    Figure Lengend Snippet: A. MK2/3-deficient MEFs transduced with MK2 expression or control vector are treated with TNF in the presence of smac-mimetics (SM), a pro-apoptotic stimulus, for 60 and 90 minutes. B. Diverse apoptotic stimuli including combinations of TNF with the IKK1/2 inhibitor BMS345541 (BMS) and with BX795 (TBK1 inhibitor) induce predominant necroptotic response in MK2/3-deficient cells. C. MK2/3-deficient cells were pre-treated with RIPK3 inhibitor (GSK872) and Smac mimetics (SM) for 30 minutes followed by TNF-treatment for 2 hours. (A-C) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa. D. Sytox-green based cytotoxicity assay was performed with MK2/3-deficient and MK2-rescued cells treated in the presence and absence of caspase inhibitor (zVAD) as indicated.

    Article Snippet: The following reagents were used for cell treatments at given concentrations: Lipopolysaccharide ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100□ng□ml -1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10□ng/ml), Birinapant/Smac Mimetics (#HY-16591, MedChem Express, 1μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25□μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50μM), RIPK1 inhibitor Nec-1s (# 10-4544-5mg, Tebu-Bio, 50μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubericidin (#HY-15424, MedChem Express, 2.5-10μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5μM), Staurosporine (#81590, Cayman, 5μM), Etoposide (#E1383, Sigma, 5μM), Doxorubicin (#15007, Cayman, 5μM), Gemcitabine (#S1714, Selleckchem, 100nM)

    Techniques: Transduction, Expressing, Plasmid Preparation, Marker, Cytotoxicity Assay

    MK2-deficient MEFs transduced with MK2 expression or control vector were treated with TNF alone or in the presence of the inhibitor panel (at 10 μM concentration) for 6h and cell viability was quantified by CCK8 colorimetric assay. Each treatment was performed in triplicates. While TNF alone is not cytotoxic, several small molecules sensitize both MEF lines to TNF (comps. 15, 42, 133, 142). Compound 14/CAY10657 (IKK2 inhibitor), comp. 32/BIO (GSK3 inhibitor), comp. 54,55/Bisindolylmaleimide VIII/IX (PKC inhibitors), comp. 94/PIK-75 (PI3 kinase inhibitor) and comp. 130/5-Iodotubercidin (5-ITu) display specific sensitization effects dependent on MK2-deficiency. Average values of n = 3 independent wells are plotted ± SD.

    Journal: bioRxiv

    Article Title: 5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

    doi: 10.1101/2023.03.03.530727

    Figure Lengend Snippet: MK2-deficient MEFs transduced with MK2 expression or control vector were treated with TNF alone or in the presence of the inhibitor panel (at 10 μM concentration) for 6h and cell viability was quantified by CCK8 colorimetric assay. Each treatment was performed in triplicates. While TNF alone is not cytotoxic, several small molecules sensitize both MEF lines to TNF (comps. 15, 42, 133, 142). Compound 14/CAY10657 (IKK2 inhibitor), comp. 32/BIO (GSK3 inhibitor), comp. 54,55/Bisindolylmaleimide VIII/IX (PKC inhibitors), comp. 94/PIK-75 (PI3 kinase inhibitor) and comp. 130/5-Iodotubercidin (5-ITu) display specific sensitization effects dependent on MK2-deficiency. Average values of n = 3 independent wells are plotted ± SD.

    Article Snippet: The following reagents were used for cell treatments at given concentrations: Lipopolysaccharide ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100□ng□ml -1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10□ng/ml), Birinapant/Smac Mimetics (#HY-16591, MedChem Express, 1μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25□μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50μM), RIPK1 inhibitor Nec-1s (# 10-4544-5mg, Tebu-Bio, 50μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubericidin (#HY-15424, MedChem Express, 2.5-10μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5μM), Staurosporine (#81590, Cayman, 5μM), Etoposide (#E1383, Sigma, 5μM), Doxorubicin (#15007, Cayman, 5μM), Gemcitabine (#S1714, Selleckchem, 100nM)

    Techniques: Transduction, Expressing, Plasmid Preparation, Concentration Assay, Colorimetric Assay

    A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/mL) for 6h. B. Cells of indicated genotype were treated with 10 μM 5-ITu for 6 and 24h and cell viability was quantified. C. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 μM ABT702, 100 nM gemcitabine, 5 μM etoposid, 5 μM doxorubicin and 5 μM staurosporine) in the presence or absence of TNF for 6h and cell viability was assessed. D. MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E, F. RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS - induced necroptosis (E) and SM-mediated autocrine TNF-dependent death (F). Average values of n = 3 independent wells are plotted ± s.d. (** denotes p-value ≤ 0.001, *** denotes p-value ≤ 0.0001, **** denotes p-value ≤ 0.0001).

    Journal: bioRxiv

    Article Title: 5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

    doi: 10.1101/2023.03.03.530727

    Figure Lengend Snippet: A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/mL) for 6h. B. Cells of indicated genotype were treated with 10 μM 5-ITu for 6 and 24h and cell viability was quantified. C. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 μM ABT702, 100 nM gemcitabine, 5 μM etoposid, 5 μM doxorubicin and 5 μM staurosporine) in the presence or absence of TNF for 6h and cell viability was assessed. D. MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E, F. RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS - induced necroptosis (E) and SM-mediated autocrine TNF-dependent death (F). Average values of n = 3 independent wells are plotted ± s.d. (** denotes p-value ≤ 0.001, *** denotes p-value ≤ 0.0001, **** denotes p-value ≤ 0.0001).

    Article Snippet: The following reagents were used for cell treatments at given concentrations: Lipopolysaccharide ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100□ng□ml -1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10□ng/ml), Birinapant/Smac Mimetics (#HY-16591, MedChem Express, 1μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25□μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50μM), RIPK1 inhibitor Nec-1s (# 10-4544-5mg, Tebu-Bio, 50μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubericidin (#HY-15424, MedChem Express, 2.5-10μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5μM), Staurosporine (#81590, Cayman, 5μM), Etoposide (#E1383, Sigma, 5μM), Doxorubicin (#15007, Cayman, 5μM), Gemcitabine (#S1714, Selleckchem, 100nM)

    Techniques: Transduction, Expressing, Plasmid Preparation

    A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with IKK1/2 inhibitor BMS and 5-ITu in combination with TNF for 2h. MLKL and RIPK1 phosphorylation was monitored with additional control blots. RIPK1 band shift induced by MK2-mediated phosphorylation is absent in MK2-KO cells and EF2 is shown as loading control. B. MK2/3-deficient MEFs transduced with MK2 expression or control vector were treated with 5-ITu alone or in combination with TNF for 3h. Cells were pre-treated for 30 minutes with the MK2 inhibitor PF3604422, the RIPK1 inhibitor Nec-1 or the RIPK3 inhibitor GSK872, as indicated. C. Wild type (WT) - MEFs were treated with 5-ITu or TNF alone or in combination for a short-term kinetics (10-180 minutes). D. RAW264.7 cells were treated with 5-ITu or LPS alone or in combination for 10-180 minutes and signaling was monitored by immunoblotting as indicated. (A-D) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa.

    Journal: bioRxiv

    Article Title: 5-iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

    doi: 10.1101/2023.03.03.530727

    Figure Lengend Snippet: A. MK2-deficient MEFs transduced with MK2 expression or control vector were treated with IKK1/2 inhibitor BMS and 5-ITu in combination with TNF for 2h. MLKL and RIPK1 phosphorylation was monitored with additional control blots. RIPK1 band shift induced by MK2-mediated phosphorylation is absent in MK2-KO cells and EF2 is shown as loading control. B. MK2/3-deficient MEFs transduced with MK2 expression or control vector were treated with 5-ITu alone or in combination with TNF for 3h. Cells were pre-treated for 30 minutes with the MK2 inhibitor PF3604422, the RIPK1 inhibitor Nec-1 or the RIPK3 inhibitor GSK872, as indicated. C. Wild type (WT) - MEFs were treated with 5-ITu or TNF alone or in combination for a short-term kinetics (10-180 minutes). D. RAW264.7 cells were treated with 5-ITu or LPS alone or in combination for 10-180 minutes and signaling was monitored by immunoblotting as indicated. (A-D) Numbers at the right of the blot indicate molecular mass of the marker proteins in kDa.

    Article Snippet: The following reagents were used for cell treatments at given concentrations: Lipopolysaccharide ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100□ng□ml -1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10□ng/ml), Birinapant/Smac Mimetics (#HY-16591, MedChem Express, 1μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25□μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50μM), RIPK1 inhibitor Nec-1s (# 10-4544-5mg, Tebu-Bio, 50μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubericidin (#HY-15424, MedChem Express, 2.5-10μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5μM), Staurosporine (#81590, Cayman, 5μM), Etoposide (#E1383, Sigma, 5μM), Doxorubicin (#15007, Cayman, 5μM), Gemcitabine (#S1714, Selleckchem, 100nM)

    Techniques: Transduction, Expressing, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Western Blot, Marker