Cephalosporins inhibit human metallo β-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo† †Electronic supplementary information (ESI) available: General experimental procedures and supplementary figures. See DOI: 10.1039/c6cc00529b Click here for additional data file.

The human metallo-β-lactamase fold DNA repair enzymes SNM1A and SNM1B can be inhibited by cephalosporin-family molecules, revealing a potential new strategy to chemosensitive tumours, as well as a novel evolutionary link.

MTT Assay: MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assays were carried out using clear 96-well microplates where 5000 cells/well of HeLa cells were seeded for at least 4 hours before they were treated with various concentraMons (0-50μM) of cephalosporin inhibitor. The inhibitors were lec on the cells for 72 hours before 100μl/well of 1mg/ml MTT (Sigma Aldrich, dissolved in PBS) was added. The cells were incubated with MTT for 2 hours at 37°C before it was removed and 100μl/well of DMSO was added. The plates were then shaken at room temperature for 20 minutes before the absorbance at 570nm of each well was measured using the SocMaxPro socware (Molecular Devices, Sunnyvale, CA, USA). To test for sensiMzaMon to the ICL inducer SJG-136 (Gregson et al. 2001;Hartley and Hochhauser 2012;Hopton and Thompson 2011), the MTT assay was used to determine if the IC 50 of HeLa cells in SJG-136 was affected by the presence of a fixed concentraMon of cephalosporin. IC 50 s were calculated using Prism socware (GraphPad Socware, Inc., La Jolla, CA, USA), fieng the data to a log(inhibitor) versus response curve. NMR Experiments: All NMR spectra were acquired at 298 K using a Bruker AVIII 700 spectrometer with 1 H/ 13 C/ 15 N TCI cryoprobe.
For 1 H NMR experiments, the data were recorded employing pulse sequence with water suppression (excitaMon sculpMng with gradients using perfect echo). Spectra were recorded with 11161 Hz sweep width, 2 s relaxaMon delay, 65536 data points and 8 scans. For data processing line broadening of 2 Hz was used. The NMR samples were prepared in 50 mM TRIS-d11 buffer pH 7.5, supplemented with 10% D 2 O.
Typical experimental parameters for Carr-Purcell-Meiboom-Gill (CPMG) NMR spectroscopy 20 were as follows: The PROJECT-CPMG sequence (90°x−[τ−180°y−τ−90°y−τ−180°y−τ]n−acq) was applied. Water suppression was achieved by pre-saturaMon. Data were collected with a sweep width of 11194 Hz and an acquisiMon Mme of 1.46 s. The filter width was 625000 s long and 64 scans were applied. Prior to Fourier transformaMon, the data were mulMplied with an exponenMal funcMon with a line broadening of 2 Hz.
In water-Ligand Observed Gradient SpectroscopY (wLOGSY) 18 , the typical experimental parameters were as follows: number of scans 512., the data were collected with a sweep width of 11160 Hz, an acquisiMon Mme of 2.9 s, and a relaxaMon delay of 2 s. Prior to Fourier transformaMon, the data were mulMplied with an exponenMonal funcMon with a line broadening of 2 Hz.
Thermal shic assay DifferenMal Scanning Fluorimetry assays were performed in white 8 x 6 PCR plates using a MiniOpMcon™ Real-Time PCR DetecMon System (BioRad). ReacMons were performed in 50 μl. Protein unfolding was monitored by measuring the fluorescence of the SYPRO Orange dye (Invitrogen). Temperature was increased from 25 to 85 °C, 1 °C increment every 30 seconds. The dye stock (5000x concentrate) was first diluted by adding 1 μl to 2.5 ml of 50 mM HEPES-NaOH pH 7.5, 200 mM NaCl metal free buffer to produce a 2x concentrated soluMon. Compounds were tested at 1 mM final concentraMon. The enzyme was diluted in SYPRO Orange buffer to obtain a final assay concentraMon of 6 μM. Measurements were performed in triplicate. For determinaMon of T M values, melMng curves for each triplicate data set were analyzed using GraphPad Prism (GraphPad Socware, Inc., La Jolla, CA, USA).