6 metimp  (Jena Bioscience)

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. Size exclusion chromatography (SEC) trace (left) and Coomassie-stained gel of PPAT-NUDT5-AMP complex (right) used for cryo-EM analysis. b. Motion-corrected and denoised micrograph of PPAT-NUDT5-AMP complex. c. Representative 2D-class averages for PPAT-NUDT5-AMP complex. Micrographs and 2D class averages from the 6-meTIMP and 6-benzylTIMP datasets were highly similar to that of the AMP-bound dataset. d, e. SEC and Coomassie-stained gels of PPAT-NUDT5 complexes formed in the presence of d. 6-meTIMP and e. 6-benzylTIMP. f. Cryo-EM data processing particle flow for the three nucleotide-bound PPAT-NUDT5 structures. All processing steps were performed in CryoSparc. g-i. Angular distribution of particles and FSC curves from refinements of PPAT-NUDT5-purine complexes with g. AMP, h. 6-meTIMP and i. 6-benzylTIMP.

Article Snippet: NUDT5 (75 μM) was incubated with PPAT (15 μM) directly after elution of PPAT from Streptactin resin for one hour in 2 mL of Streptactin elution buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 50 mM biotin) supplemented with 200 μM 6-meTIMP (Jena Biosciences, NU-1226) or 6-benzylTIMP (WuXi).

Techniques: Size-exclusion Chromatography, Staining, Cryo-EM Sample Prep

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. Left – Molecular structures of 6-meTIMP, 6-benzylthioinosine-5’-monophosphate (6-benzylTIMP), and 6-ethylthioinosine-5’-monophosphate (6-etTIMP). Right – PPAT activity assay measuring nucleotide-dependent inhibition in the presence and absence of NUDT5. Data points are the mean and error bars are SEM from n=3 independent experiments. b. Structural alignment of the region surrounding the molecular glue interface for the 6-benzylTIMP- and 6-meTIMP-bound PPAT-NUDT5 structures. Cryo-EM density for the 6-benzylTIMP nucleotide is shown as a transparent surface. c. Sharpened cryo-EM density of the I422-E436 loop in the 6-benzylTIMP and 6-meTIMP maps shown at the same contour. The dashed oval indicates a region of missing density in the 6-benzylTIMP map that is well-defined in the 6-meTIMP map. d. FACS-based growth competition experiment comparing ΔNUDT5 and endogenous NUDT5 L217A/K218A (LKAA) mutants to wildtype HEK293T treated with 6-ethylmercaptopurine riboside. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. e. Model of NUDT5-and purine-dependent molecular glue mechanism outlining effects on inhibition of de novo purine biosynthesis.

Article Snippet: NUDT5 (75 μM) was incubated with PPAT (15 μM) directly after elution of PPAT from Streptactin resin for one hour in 2 mL of Streptactin elution buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 50 mM biotin) supplemented with 200 μM 6-meTIMP (Jena Biosciences, NU-1226) or 6-benzylTIMP (WuXi).

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

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. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment with methotrexate (2 µM), 6-MP (50 µM), and MTX + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.

Article Snippet: NUDT5 (75 μM) was incubated with PPAT (15 μM) directly after elution of PPAT from Streptactin resin for one hour in 2 mL of Streptactin elution buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 50 mM biotin) supplemented with 200 μM 6-meTIMP (Jena Biosciences, NU-1226) or 6-benzylTIMP (WuXi).

Techniques: Transformation Assay, Cryo-EM Sample Prep, Activity Assay, Inhibition, Western Blot, Immunoprecipitation, Control, Labeling, Two Tailed Test, Mutagenesis

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: NUDT5 (75 μM) was incubated with PPAT (15 μM) directly after elution of PPAT from Streptactin resin for one hour in 2 mL of Streptactin elution buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT, 50 mM biotin) supplemented with 200 μM 6-meTIMP (Jena Biosciences, NU-1226) or 6-benzylTIMP (WuXi).

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