Structural studies of the effect that dimethyl sulfoxide (DMSO) has on cisplatin and carboplatin binding to histidine in a protein

Abstract

The anticancer complexes cisplatin and carboplatin target the DNA major groove, forming intrastrand and interstrand cross-links between guanine bases through their N7 atoms, causing distortion of the DNA helix and apoptotic cell death. A major side effect of these drugs is toxicity, which is caused via binding to many proteins in the body. A range of crystallographic studies have been carried out involving the cocrystallization of hen egg-white lysozyme (HEWL) as a test protein with cisplatin and carboplatin in aqueous and dimethyl sulfoxide (DMSO) conditions. Different cryoprotectants, glycerol and Paratone, were used for each of the cisplatin and carboplatin cocrystallization cases, while silicone oil was used for studies involving N-acetylglucosamine (NAG). Both cisplatin and carboplatin do not bind to HEWL in aqueous media on the timescales of the conditions used here, but upon addition of DMSO two molecules of cisplatin or carboplatin bind either side of His15, which is the only His residue in lysozyme and is assumed to be an imidazolyl anion or a chemical resonance moiety, i.e. both imidazole N atoms are chemically reactive. To identify the platinum-peak positions in the 'with DMSO conditions', anomalous scattering maps were calculated as a cross-check with the F(o) - F(c) OMIT maps. Platinum-occupancy σ values were established using three different software programs in each case. The use of EVAL15 to process all of the diffraction data sets provided a consistent platform for a large ensemble of data sets for the various protein and platinum-compound model refinements with REFMAC5 and then SHELXTL. Overall, this extensive set of crystallization and cryoprotectant conditions allowed a systematic evaluation of cisplatin and carboplatin binding to lysozyme as a test protein via detailed X-ray crystal structure characterizations. DMSO is used as a super-solvent for drug delivery as it is deemed to cause no effect upon drug binding. However, these results show that addition of DMSO causes the platinum anticancer drugs to bind to HEWL. This effect should be considered in toxicity assessments of these drugs and perhaps more widely.

Say No to DMSO: Dimethylsulfoxide Inactivates Cisplatin, Carboplatin, and Other Platinum Complexes

“…For example, a crystallographic study of cisplatin and carboplatin binding to histidines of hen egg-white lysozyme found one platinum bound to His15 when cisplatin was dissolved in aqueous medium. When dissolved in DMSO, two platinum atoms bound to His15, leading to the conclusion that DMSO could facilitate platinum-protein binding (50). Uribe et al reported increased sensory hair cell death in zebrafish co-treated with cisplatin and DMSO but not other solvents, which was ascribed to the cell permeabilizing effects of DMSO, and warned that DMSO could produce false-positive effects in other drug screens due to DMSO's biological activity (51).…”

A site-selective, irreversible inhibitor of the DNA replication auxiliary factor proliferating cell nuclear antigen (PCNA)

“…T2Pt and N3-T2Pt were insoluble in either aqueous buffers or ethanol, yet readily dissolved in DMSO. The co-ordination of cisplatin by DMSO has previously been described and may reduce its DNA reactivity 16 while increasing its reactivity with the histidine residues of protein 17 . Correspondingly, the solvation of T2Pt in DMSO was expected to increase the selectivity of T2Pt for PCNA H44 over DNA.…”

Cisplatin coordination chemistry determination at hen egg white lysozyme His15 with ligand distances and angles, and their standard uncertainties, and also reporting a split occupancy effect

“…We have extended our previously published crystal structure refinements of cisplatin binding to hen egg lysozyme using the existing diffraction data for our PDB entries 4dd4 and 4dd6 (Tanley et al 2012). This follows the interest of Messori and Merlino 2016 in the platinum ions' precise coordination geometry.…”

“…Overall approach As an extension of our original model refinement (Tanley et al 2012) we have now been able to use the 'validation within model refinement computational environment' of Phenix_Refine (Afonine et al 2010) that is now available within the Windows computer environment, and which we have found very advantageous e.g. for discerning solute molecule assignments and avoiding clashes with the protein.…”

“…We plan then an extensive reanalysis of all the existing datasets of cisplatin bound to histidine with a detailed look for such split occupancy effects, which we will now undertake and report later in the rather new 'data review' style of article. All these extended analyses, reported here and that we plan in our data review, are outside the scope of our original paper (Tanley et al 2012) and whose conclusions are obviously not affected, namely that DMSO is important for cisplatin binding to histidine versus under aqueous conditions. Such new and extended analyses, based on improved crystal structure analysis tools and/or more recent structural insights, are likely to be increasingly important in macromolecular crystallography and are within the vision of the 'Living PDB and Living Publication' (espoused by Helliwell, Terwilliger and McMahon (2012).…”