Journal: Nature

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

doi: 10.1038/s41586-026-10790-3

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 Strep-Tactin resin for 1 h in 2 ml Strep-Tactin elution buffer (50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM DTT and 50 mM biotin) supplemented with 200 μM 6-meTIMP (Jena Biosciences, NU-1226) or 6-benzylTIMP (WuXi).

Techniques: Size-exclusion Chromatography, Staining, Cryo-Electron Microscopy, Analysis, SDS Page, Single Particle

Journal: Nature

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

doi: 10.1038/s41586-026-10790-3

Figure Lengend Snippet: a , Metabolism of 6-MP and 6-TG. Orange asterisk indicates 6-TGMP conversion to 6-meTGMP, which is a PPAT–NUDT5 glue. b , AMP and 6-meTIMP structures. c , Left, alignment of the AMP and 6-meTIMP glue interface with cryo-EM density. Right, PPAT residue rearrangement in 6-meTIMP-bound (dark) versus AMP-bound (light) structures. d , PPAT hydrophobic pocket engaged by 6-meTIMP. e , 6-meTIMP glue interface. f , PPAT activity assay with wild-type NUDT5 and nucleotide (0.25 mM PRPP). Data are mean ± s.e.m. from n = 3 independent experiments ( n = 2 for 6-TIMP). g , PRPP-induced PPAT–NUDT5 dissociation (AlphaLisa) with AMP and 6-meTIMP. Data are mean ± s.e.m. from n = 3 independent experiments. Data are also shown in Fig. . h , NUDT5–3×Flag immunoprecipitation (left) and quantification (right) after treatment with MTX (2 µM) and/or 6-MP (50 µM). Data are mean ± s.e.m. with individual values from n = 3 independent biological replicate experiments. i , AMP (M+2) and GMP (M+3) fractional enrichment in [ 15 N-amide]-glutamine labelling of cells treated with 6-MP. Data are mean ± s.e.m. with individual values from n = 6 biological replicates from 2 independent experiments. j , FACS growth competition: ∆NUDT5 and NUDT5 L217A/L218A (LKAA) mutants versus wild-type HEK293T cells treated with 6-MP or 6-TG. Data are mean with individual values from n = 3 biological replicates. k , Viability of wild-type, ∆NUDT5 and NUDT5 L217A/L218A ALL cells treated with 6-meTIMP. Data are mean ± s.e.m. of n = 6 biological replicates from 3 independent experiments. FC (fold change) is the ratio of mutant to wild-type IC 50 . K i and IC 50 values in f , g , k were compared by global nonlinear regression using an extra sum-of-squares F-test. Significance was determined by two-tailed one-sample t -test relative to DMSO control in h and by one-way ANOVA followed by Dunnett’s multiple-comparisons test against wild type in i . NS, not significant. * P < 0.05 and *** P < 0.001. Exact P values are included as source data.

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

Techniques: Cryo-Electron Microscopy, Residue, Activity Assay, Immunoprecipitation, Mutagenesis, Two Tailed Test, Control, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay, Enzymatic Assay

Journal: Nature

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

doi: 10.1038/s41586-026-10790-3

Figure Lengend Snippet: a . FACS-based growth competition comparing Δ NUDT5 and rescue mutants with wild-type HEK293T cells treated with 6-TG. Data represent the mean with individual values from n = 3 biological replicates. b . Cryo-EM density of the PPAT–NUDT5 6-meTIMP molecular-glue interface with model fit. c , d . PPAT activity assay measuring inhibitory effects of 6-meTIMP in the absence and presence of wild-type NUDT5 and indicated mutants ( c ) and compared to AMP only ( d ) with 0.25 mM PRPP. Data represent the mean ± s.e.m. from n = 3 independent experiments. e . AlphaLisa binding assay quantifying the stabilization of PPAT–NUDT5 binding by AMP and 6-meTIMP in the presence of PRPP (1 mM). Data represent the mean ± s.e.m. from n = 3 independent experiments. Data for the AMP condition are also shown in Extended Data Fig. . f . Western blot of endogenous PPAT–3×Flag immunoprecipitations after 16-h treatment with MTX (2 µM), 6-MP (50 µM) and MTX + 6-MP. Blot is representative of two independent experiments g . Time-resolved microscopy (incucyte) growth assays of wild-type and mutant HEK293T cells treated with the indicated drugs. Data represent the mean ± s.e.m. of n = 6 biological replicates. h . Levels of intracellular 6-TIMP and 6-meTIMP metabolites after 16-h treatment with 6-MP (20 µM). Data represent the mean ± s.e.m. with individual values from n = 3 biological replicates. i . PPAT activity assay measuring inhibitory effects of 6-meTGMP in the absence and presence of wild-type NUDT5 and mutants. Data represent the mean ± s.e.m. from n = 3 independent experiments. K i values in c , d , i as well as EC 50 in e were compared by global nonlinear regression using an extra sum-of-squares F-test. Statistical significance in h was determined by one-way ANOVA followed by Dunnett’s multiple-comparisons test against wild type. Significance is indicated as ns (not significant), * P < 0.05, ** P < 0.01 and *** P < 0.001. Exact P values are included as source data.

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

Techniques: Cryo-Electron Microscopy, Activity Assay, Binding Assay, Western Blot, Microscopy, Mutagenesis

Journal: Nature

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

doi: 10.1038/s41586-026-10790-3

Figure Lengend Snippet: a , Chemical structures of 6-meTIMP, 6-benzylthioinosine-5′-monophosphate (6-benzylTIMP) and 6-ethylthioinosine-5′-monophosphate (6-etTIMP). b , PPAT activity assay measuring nucleotide-dependent inhibition in the absence and presence of NUDT5. Data are mean ± s.e.m. from n = 3 independent experiments. The same data for 6-meTIMP are shown in both panels for clarity. c , 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 is shown as a transparent surface. d , 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. e , Viability of wild-type, ∆NUDT5 and endogenous NUDT5 L217/K218A mutant cells treated with 6-etTIMP for 72 h. Data are mean ± s.e.m. of n = 6 biological replicates from 3 independent experiments. FC (fold change) is the ratio of mutant to wild-type IC 50 , representing fold change in 6-etTIMP resistance. Selectivity index (SI) is the ratio of 6-etTIMP to 6-meTIMP fold change relative to wild type, quantifying relative on-target cytotoxicity. An SI greater than 1 is indicative of increased on-target cytotoxicity. f , Model of the NUDT5- and purine- or thiopurine-dependent metabolite-glue mechanism. The double asterisks represent the PPAT hydrophobic pocket. K i and IC 50 values in b , e were compared by global nonlinear regression using an extra sum-of-squares F-test. NS, not significant. *** P < 0.001. Exact P values are included as source data.

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

Techniques: Activity Assay, Inhibition, Cryo-Electron Microscopy, Mutagenesis

Journal: JCI Insight

Article Title: Purine metabolism enhances neovascularization of type H vessels in the induced membrane technique

doi: 10.1172/jci.insight.200820

Figure Lengend Snippet: ( A – D ) Representative immunohistochemical images showing expression of ADSS1 ( A ) and IMPDH2 ( B ) in the induced membrane. Quantification of ADSS1 ( C ) and IMPDH2 ( D ) staining using ImageJ. Each dot represents one animal (IM, n = 6; BD, n = 6). Data are presented as area fraction and normalized to DAPI areas. Box plots represent the median (line), interquartile range (box), and minimum to maximum values (whiskers). P < 0.05 was considered statistically significant. ( E ) Summary of showing alterations in serum and induced membrane at week 6 in IMT rats. Scale bars: 50 μm. ADSS1, adenylosuccinate synthase 1; IMPDH2, inosine monophosphate dehydrogenase 2; BD, bone defect; IM, induced membrane.

Article Snippet: The primary antibodies used included adenylosuccinate synthase 1 (ADSS1; A6516, rabbit polyclonal antibody, ABclonal), inosine monophosphate dehydrogenase 2 (IMPDH2; A15626, rabbit polyclonal antibody, ABclonal), CD31 conjugated with Alexa Flour 488 (#42777, clone 89C2, Cell Signaling), and endomucin (EMCN) conjugated with Alexa Flour 555 (BS-4808R-A555, rabbit polyclonal antibody, Bioss).

Techniques: Immunohistochemical staining, Expressing, Membrane, Staining