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6 timp  (Jena Bioscience)


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    Structured Review

    Jena Bioscience 6 timp
    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of <t>intracellular</t> <t>6-TIMP</t> and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.
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    Images

    1) Product Images from "Metabolic glues as a means of purine sensing and chemotherapeutic response"

    Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

    Journal: bioRxiv

    doi: 10.64898/2026.05.05.723063

    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.
    Figure Legend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

    Techniques Used: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis



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    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of <t>intracellular</t> <t>6-TIMP</t> and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.
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    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of <t>intracellular</t> <t>6-TIMP</t> and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.
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    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of <t>intracellular</t> <t>6-TIMP</t> and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.
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    a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

    Journal: bioRxiv

    Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

    doi: 10.64898/2026.05.05.723063

    Figure Lengend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

    Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

    Techniques: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis