Journal: International journal of molecular sciences

Article Title: Chloroquine-Induced DNA Damage Synergizes with Nonhomologous End Joining Inhibition to Cause Ovarian Cancer Cell Cytotoxicity.

doi: 10.3390/ijms23147518

Figure Lengend Snippet: Figure 1. Panobinostat does not affect DSB repair by NHEJ. (A) Left panel: time-response of NHEJ- related proteins (Ku70, Ku80) after LBH treatment (50 nM) in SK-OV-3 cell line. Right panel: Ku70 and Ku80 expression after 24 h of LBH treatment (20 nM) in OCCLs. β-actin was used as a loading control. (B) Map of pEGFP-Pem1-Ad2. An Ad2 exon is present in the middle of the Pem1 intron, and efficient splicing inactivates the GFP activity and makes the starting substrate GFP-negative. However, both sides of the Ad2 exon present HindII/I-SceI restriction sites. Cleavage with either of these endonucleases removes the Ad exon, and upon successful intracellular plasmid circularization, GFP expression is restored and can be quantified by flow cytometry [47]. (C) Percentage of NHEJ using HindIII- or I-SceI-digested plasmid in IGROV-1 and SK-OV-3 cell lines. Cells were pre-treated or not with the indicated doses of LBH or NU-7026, transfected with the linearized pEGFP-Pem1-Ad2 or supercoiled pEGFP-Pem1 together with the pDSRed plasmid, and incubated again with LBH or NU-7026 for 72 h. The percentage of NHEJ was calculated as described in the Materials and Methods. (D) Top panel: Map of NHEJ-C reporter construct [48]. Bottom panel: Dot plots of nontransfected SK-OV-3 and IGROV-1 cells carrying the NHEJ reporter cassette, and the same cell lines co-transfected with 5 µg of an I-SceI endonuclease-expressing plasmid and 0.5 µg of pDsRed2-N1. The latter were incubated in the presence or absence (C-) of LBH or NU-7026 for an additional 72 h. Correct NHEJ repair restored the GFP gene, which was detected as GFP+ cells. NHEJ efficiency was calculated as the ratio of GFP+ to DsRed+ cells and then normalized to the untreated control. C-: negative control (untreated cells). Data are the mean of three independent experiments. Error bars represent the SD (** p < 0.01, * p < 0.05 compared to controls).

Article Snippet: Nonhomologous end joining inhibitors (NHEJi) KU-57788 (KU), NU-7026 (NU), and SCR7 pyrazine (SCR7), a stable form of SCR7, were obtained from MedChemExpress, South Brunswick Township, NJ, USA.

Techniques: Expressing, Control, Activity Assay, Plasmid Preparation, Cytometry, Transfection, Incubation, Construct, Negative Control

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Chemical structure of ′(3′)-O-(N-methyl-anthraniloyl) ATPγS (mant-ATPγS). (B) ATPγS is not hydrolyzed by nsP2. Under the conditions tested, 50 nM nsP2 converted ∼80% of 1 mM ATP to ADP within 30 minutes, whereas no detectable hydrolysis of 1 mM ATPγS was observed after 120 minutes. No luminescence signal was detected in reactions containing ATP or ATPγS in the absence of enzyme (data not shown). (C) Representative tryptophan to mant FRET emission spectra collected using excitation at 80 nm. nsP2 alone (1 μM) exhibits an emission peak at 350 nm, whereas mant-ATPγS alone (10 μM) shows weak emission at 445 nm under 280-nm excitation. Addition of mant-ATPγS to nsP2 products an increase in 445-nm emission, consistent with FRET arising from formation of the nsP2·mant-ATPγS complex. Data in panels D-G were generated by subtracting mant-ATPγS-only emission at 445 nm from spectra collected in the presence of nsP2. (D) Direct binding of mant-ATPγS to nsP2. ns2P (0. 25 μM) was titrated with 0.002 5 μM mant-ATPγS. Data represent mean ± SD ( n = 3). (E-G) Competitive binding experiments. nsP2 (0. 5 μM) was incubated with 0.1 μM mant-ATPγS and increasing concentrations of unlabeled competitor. ATPγS (E; 0-10 μM), ADP (F; 0-9 mM), or inorganic phosphate (Pi) and tripolyphosphate (TPP) (G; 0-40 mM) were added as indicated. Fluorescence data in panels E and F were normalized to percent relative fluorescence, with the signal in the absence of competitor defined as 100%. Data were fit by nonlinear regression, and IC₅₀ values were converted to inhibition constants ( K i ) using the Cheng-Prusoff equation.

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Generated, Binding Assay, Incubation, Fluorescence, Inhibition

Journal: bioRxiv

Article Title: Linking the kinetic mechanism to structural dynamics required for nucleotide hydrolysis by an alphavirus nsP2 RNA helicase

doi: 10.64898/2026.05.08.723793

Figure Lengend Snippet: (A) Exp rimental design for ATPγS association kinetics. nsP2 was rapidly mixed with mant-ATPγS under stopped-flow conditions, and binding was monitored by tryptophan-to-mant FRET. (B) ATPγS association kinetics. Representative fluorescence time courses following rapid mixing of mant-ATPγS (0.1 μM) with increasing concentrations of nsP2 (0.5-3 μM). (C) Observed rate constants ( k obs ) extracted from single-phase fits to the association traces in panel B were replotted as a function of nsP2 concentration (n = 3 independent experiments). Linear regression was used to determine the second-order association rate constant ( k ₒₙ). (D) Experimental design for ATPγS dissociation kinetics. Pre-formed nsP2·mant-ATPγS complexes were rapidly mixed with excess unlabeled ATPγS to initiatw ligand displacement. (E) ATPγS dissociation kinetics. Time-dependent loss of sensitized Mant fluorescence following competition with unlabeled ATPγS. Traces were fit to a two-phase exponential decay, revealing fast and slow dissociation components ( k off,fast and k off,slow ). (F) Experimental design for ATP dissociation in the presence of inhibitor. Pre-formed nsP2·mant-ATP complexes were rapidly mixed with excess unlabeled ATP in the presence of the nsP2 inhibitor RA-NSP2- (5 μM). (G) ATP dissociation kinetics in the presence of inhibitor. Representative fluorescence decay trace fit to a single-phase exponential model, yielding the apparent ATP dissociation rate constant ( k off ).

Article Snippet: Mant-ATP and mant-ATPγS were from Jena Bioscience.

Techniques: Binding Assay, Fluorescence, Concentration Assay

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: 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: 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: 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