The in situ molecular characterization of reaction intermediates and products at electrode–electrolyte interfaces is central to mechanistic studies of complex electrochemical processes, yet a great challenge. The coupling of electrochemistry (EC) and mass spectrometry (MS) has seen rapid development and found broad applicability in tackling challenges in analytical and bioanalytical chemistry. However, few truly in situ and real-time EC-MS studies have been reported at electrode–electrolyte interfaces. An innovative EC-MS coupling method named in situ liquid secondary ion mass spectrometry (SIMS) was recently developed by combining SIMS with a vacuum compatible microfluidic electrochemical device. Using this novel capability, we report the first in situ elucidation of the electro-oxidation mechanism of a biologically significant organic compound, ascorbic acid (AA), at the electrode–electrolyte interface. The short-lived radical intermediate was successfully captured, which had not been detected directly before. Moreover, we demonstrated the power of this new technique in real-time monitoring of the formation and dynamic evolution of electrical double layers at the electrode–electrolyte interface. This work suggests further promising applications of in situ liquid SIMS in studying more complex chemical and biological events at the electrode–electrolyte interface.

An addressable single cell imaging strategy combining ToF-SIMS and confocal fluorescence microscopy imaging has been developed, and sucessfully applied to visualize the subcellular distribution of an organoruthenium anticancer complex, [(η6-benzene)Ru(N,N-L)Cl]+ (1; L: 4-anilinoquinazoline ligand), showing its accumulation in both cell membrane and nuclei, and verifying its dual-targeting feature.

A high mobility group box 1 (HMGB1) protein has been reported to recognize both 1,2-intrastrand crosslinked DNA by cisplatin (1,2-cis-Pt-DNA) and monofunctional platinated DNA using trans-[PtCl2(NH3)(thiazole)] (1-trans-PtTz-DNA). However, the molecular basis of recognition between the trans-PtTz-DNA and HMGB1 remains unclear. In the present work, we described a hydrogen/deuterium exchange mass spectrometry (HDX-MS) method in combination with docking simulation to decipher the interactions of platinated DNA with domain A of HMGB1. The global deuterium uptake results indicated that 1-trans-PtTz-DNA bound to HMGB1a slightly tighter than the 1,2-cis-Pt-DNA. The local deuterium uptake at the peptide level revealed that the helices I and II, and loop 1 of HMGB1a were involved in the interactions with both platinated DNA adducts. However, docking simulation disclosed different H-bonding networks and distinct DNA-backbone orientations in the two Pt-DNA-HMGB1a complexes. Moreover, the Phe37 residue of HMGB1a was shown to play a key role in the recognition between HMGB1a and the platinated DNAs. In the cis-Pt-DNA-HMGB1a complex, the phenyl ring of Phe37 intercalates into a hydrophobic notch created by the two platinated guanines, while in the trans-PtTz-DNA-HMGB1a complex the phenyl ring appears to intercalate into a hydrophobic crevice formed by the platinated guanine and the opposite adenine in the complementary strand, forming a penta-layer π–π stacking associated with the adjacent thymine and the thiazole ligand. This work demonstrates that HDX-MS associated with docking simulation is a powerful tool to elucidate the interactions between platinated DNAs and proteins.

Gastric cancer (GC) is the fourth most commonly diagnosed cancer and the third leading cause of cancer-related deaths. Most GC deaths can be prevented by early diagnosis. However, biomarkers with high sensitivity and specificity are rare. Comprehensive serum phosphopeptides analysis may lead to the discovery of novel biomarkers for GC. In this work, we report a mass spectrometric strategy for the evaluation of serum phosphopeptides separated and enriched by ZrAs-Fe3O4@SiO2 nanoparticles. Four endogenous phosphopeptides in sera generated by degradation of fibrinogen were synthesized with light- and heavy-glycine residues, respectively, as external/internal standards, and used to gain multi-point standard calibration curves for absolute quantification of phosphopeptides by LC-ESI-MS following ZrAs-Fe3O4@SiO2 enrichment. The ESI-MS signal ratios of the four pairs of light-/heavy-phosphopeptide standards captured by ZrAs-Fe3O4@SiO2 from aqueous solutions are linearly correlated with the molar ratios of the “light” to “heavy” phosphopeptides over the range of 0.05–5 μM with an r2 of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 94.7–107.5% with RSDs in the range of 0.6–8.2%. The validated method was utilized to measure the phosphopeptide levels of serum samples from 30 healthy persons and 60 GC patients. Receiver operating characteristic (ROC) analysis shows that one phosphopeptide (F3: DpSGEGDFLAEGGGVR) yields high sensitivity and specificity of 90.0% and 96.7%, respectively, in the validation set for discriminating GC patients from healthy controls. Overall, 53 of 60 GC cases and 29 of 30 controls were correctly classified, including eight of nine GC patients at stage I. These results suggest that F3 in sera may be a potential biomarker for GC diagnosis, particularly for early stage cases.

Secondary ion mass spectrometry, including nanoscale secondary ion mass spectrometry (NanoSIMS) and time-of-flight secondary ion mass spectrometry (ToF–SIMS), has emerged as a powerful tool for biological imaging, especially for single cell imaging. SIMS imaging can provide information on subcellular distribution of endogenous and exogenous chemicals, including metallodrugs, from membrane through to cytoplasm and nucleus without labeling, and with high spatial resolution and chemical specificity. In this mini-review, we summarize recent progress in the field of SIMS imaging, particularly in the characterization of the subcellular distribution of metallodrugs. We anticipate that the SIMS imaging method will be widely applied to visualize subcellular distributions of drugs and drug candidates in single cells, exerting significant influence on early drug evaluation and metabolism in medicinal and pharmaceutical chemistry.

Ruthenium-based anticancer complexes are promising antitumor agents for their low system toxicity and versatile chemical structures. Epidermal growth factor receptor (EGFR) has been found to be overexpressed in a broad range of tumor cells and is regarded as a drug target in developing novel antitumor drugs. In this work, five ruthenium(II) polypyridyl complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were synthesized and characterized. These complexes showed both high EGFR-inhibiting activity and strong DNA minor groove-binding activity. In vitro antiproliferation screening demonstrated that the prepared ruthenium complexes are highly cytotoxic against a series of cancer cell lines, in particular non-small-cell lung A549 and human epidermoid carcinoma A431. Fluorescence-activated cell sorting analysis and fluorescence microscopy revealed that the most active complex, K4, induced much more late-stage cell apoptosis and necrosis than gefitinib, the first EGFR-targeting antitumor drug in clinical use. These results indicate that the ruthenium(II) polypyridyl complexes bearing EGFR-inhibiting 4-anilinoquinazolines possess highly active dual-targeting anticancer activity and are promising in developing new anticancer agents.

•Study on interactions of anticancer Ru arene complexes with human copper chaperone Cox17.•Ru arene complexes are much less reactive than cisplatin to Cox17.•Both Ru and Pt can bind to Cys27 which is the Cu(I) binding site on Cox17.•Cox17 may not be involved in the action of anticancer Ru arene complexes.•First report about interactions of Ru arene complexes with Cox17.Herein we report investigation of the interactions between anticancer organoruthenium complexes, [(η6-arene)Ru(en)(Cl)]PF6 (en = ethylenediamine, arene = p-cymene (1) or biphenyl (2)), and the human copper chaperone protein Cox17 by mass spectrometry with cisplatin as a reference. The electrospray ionization mass spectrometry (ESI-MS) results indicate much weaker binding of the ruthenium complexes than that of cisplatin to apo-Cox172s-s, the functional state of Cox17. Up to tetra-platinated Cox17 adducts were identified while only mono-ruthenated and a little amount of di-ruthenated Cox17 adducts were detected even for the reactions with 10-fold excess of the Ru complexes. However, ESI-MS analysis coupled with liquid chromatography of tryptic digests of metalated proteins identified only three platination sites as Met4, Cys27 and His47 residues, possibly due to the lower abundance or facile dissociation of Pt bindings at other sites. Complexes 1 and 2 were found to bind to the same three residues with Met4 as the major site. Inductively coupled plasma mass spectrometry results revealed that ~ 7 mol Pt binding to 1 mol apo-Cox172s-s molecules, compared to only 0.17 (1) and 0.10 (2) mol Ru to 1 mol apo-Cox172s-s. This is in line with the circular dichroism results that much larger unfolding extent of α-helix of apo-Cox172s-s was observed upon cisplatin binding than that upon organoruthenium bindings. These results collectively indicate that Cox17 might not participate in the action of these anticancer organoruthenium complexes, and further verify the distinct anticancer mechanism of the organoruthenium(II) complexes from cisplatin.The interactions of anticancer organoruthenium(II) complexes with human copper chaperone Cox17 were investigated by mass spectrometry compared with cisplatin, revealing that they share the same binding sites as cisplatin but are much less reactive towards Cox17. This implies that Cox17 may not be involved in action of organoruthenium(II) complexes.

Platinum-based anticancer drugs, including cisplatin and its analogues, have played important roles in the clinical treatment of solid tumors over the past 38 years. However, poor selectivity, high toxicity and intrinsic or acquired drug resistance profoundly limit their application, which encourages the development of novel transition metal-based anticancer agents with different mechanisms of action. To this end, transition metal complexes that can simultaneously act on more than one target, termed as single-molecule multi-targeting complexes, have attracted increasing attention because of their enhanced efficacy and diminished chance of drug resistance. In this review, we systematically discuss the recent progress in the development of platinum- and ruthenium-based anticancer agents, in particular the rational design of platinum and ruthenium complexes with multi-targeting features.

We have recently demonstrated that complexation with (η6-arene)RuII fragments confers 4-anilinoquinazoline pharmacophores a higher potential for inducing cellular apoptosis while preserving the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine (Chem. Commun., 2013, 49, 10224–10226). Reported herein are the synthesis, characterisation and evaluation of the biological activity of a new series of ruthenium(II) complexes of the type [(η6-arene)Ru(N,N-L)Cl]PF6 (arene = p-cymene, benzene, 2-phenylethanol or indane, L = 4-anilinoquinazolines). These organometallic ruthenium complexes undergo fast hydrolysis in aqueous solution. Intriguingly, the ligation of (arene)RuII fragments with 4-anilinoquinazolines not only makes the target complexes excellent EGFR inhibitors, but also confers the complexes high affinity to bind to DNA minor grooves while maintaining their reactivity towards DNA bases, characterising them with dual-targeting properties. Molecular modelling studies reveal that the hydrolysis of these complexes is a favourable process which increases the affinity of the target complexes to bind to EGFR and DNA. In vitro biological activity assays show that most of this group of ruthenium complexes are selectively active inhibiting the EGF-stimulated growth of the HeLa cervical cancer cell line, and the most active complex [(η6-arene)Ru(N,N-L13)Cl]PF6 (4, IC50 = 1.36 μM, L13 = 4-(3′-chloro-4′-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline) is 29-fold more active than its analogue, [(η6-arene)Ru(N,N-ethylenediamine)Cl]PF6, and 21-fold more active than gefitinib, a well-known EGFR inhibitor in use clinically. These results highlight the strong promise to develop highly active ruthenium anticancer complexes by ligation of cytotoxic ruthenium pharmacophores with bioactive organic molecules.

Ruthenium based complexes are promising antitumour candidates due to their lower toxicity and better water-solubility compared to the platinum antitumour complexes. An epidermal growth factor receptor (EGFR) has been found to be overexpressed in a large set of tumour cells. In this work, a series of organoruthenium complexes containing EGFR-inhibiting 4-anilinoquinazoline pharmacophores were synthesised and characterised. These complexes exhibited excellent inhibitory activity against EGFR and high affinity to interact with DNA via minor groove binding, featuring dual-targeting properties. In vitro screening demonstrated that the as-prepared ruthenium complexes are anti-proliferating towards a series of cancer cell lines, in particular the non-small-cell lung cancer cell line A549. Fluorescence-activated cell sorting analysis and fluorescence microscopy revealed that the most active complex 3 induced much more early-stage cell apoptosis than its cytotoxic arene ruthenium analogue and the EGFR-inhibiting 4-anilinoquinazolines, verifying the synergetic effect of the two mono-functional pharmacophores.

•An MS method was developed to quantify bindings of Ru arene complexes to GSTπ.•Binding affinity to GSTπ and inhibition potency of the complexes was correlated.•Ru-bindings to Cys15/Cys48 predominate the inhibition of the complexes on GSTπ.•Ru-bindings to Met92 and Cys102 contribute little to the inhibition activity.Electrospray ionization mass spectrometry (ESI-MS) has been widely used to identify binding sites of metal complexes to proteins. However, the MS quantification of the metal–protein coordination remains a challenge. We have recently demonstrated by ESI-MS analysis that organometallic ruthenium complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine) bound to human glutathione-S-transferase π (GSTπ) at Cys15 and Cys48 within the G-site, and Cys102 and Met92 on the interface of the GSTπ dimer, showing inhibitory potency against the enzyme (J. Inorg. Biochem., 128 (2013) 77–84). Herein, we developed a mass spectrometric method to quantify the binding stoichiometry of the three complexes to GSTπ. The differences in signal intensities of the heavy-labelled peptides produced by tryptic digestion of the ruthenated GSTπ complexes and the respective light-labelled peptides in the tryptic digest of equimolar GSTπ were used to calculate the binding stoichiometry at specific residues. The results indicated that the pre-complexation of GSTπ with its substrate GSH significantly reduced the bindings of the ruthenium complexes at Met92 and Cys102, but had little impact on the bindings at Cys15 and Cys48. As the inhibitory activities of the ruthenium complexes against GSTπ are similar to those against GSTπ in complexation with GSH, these results suggest that the inhibition of the ruthenium complexes on GSTπ is attributed to the ruthenation at Cys15 and Cys48. The present work provides not only insights into the understanding on the inhibitory mechanism of ruthenium complexes GSTπ, but also a general method for quantitative characterization of metal–protein interactions.An ESI-MS method was developed to quantify the bindings of organoruthenium anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene, biphenyl or dihydrophenanthrene; en = ethylenediamine) to GSTπ, demonstrating that the coordination of {(η6-arene)Ru(en)} units to Cys15 and Cys48 of GSTπ is determinant to their inhibitory potency against GSTπ.

The use of an argon cluster ion sputtering source has been demonstrated to perform superiorly relative to traditional oxygen and cesium ion sputtering sources for ToF-SIMS depth profiling of insulating materials. The superior performance has been attributed to effective alleviation of surface charging. A simulated nuclear waste glass (SON68) and layered hole-perovskite oxide thin films were selected as model systems because of their fundamental and practical significance. Our results show that high sputter rates and accurate interfacial information can be achieved simultaneously for argon cluster sputtering, whereas this is not the case for cesium and oxygen sputtering. Therefore, the implementation of an argon cluster sputtering source can significantly improve the analysis efficiency of insulating materials and, thus, can expand its applications to the study of glass corrosion, perovskite oxide thin film characterization, and many other systems of interest.

An MS-based proteomic strategy combined with chemically functionalized gold nanoparticles as affinity probes was developed and validated by successful identification and quantification of HMGB1, which is well characterized to interact selectively with 1,2-cross-linked DNA by cisplatin, from whole cell lysates. The subsequent application of this method to identify proteins responding to 1,3-cross-linked DNA by a trans-platinum anticancer complex, trans-PtTz (Tz = thiazole), revealed that the human nuclear protein positive cofactor PC4 selectively binds to the damaged DNA, implying that PC4 may play a role in cellular response to DNA damage by trans-PtTz.

We here report the identification of the binding sites of an organometallic ruthenium anticancer complex [(η6-biphenyl)Ru(en)Cl]+ (1) to single-stranded oligodeoxynucleotides (ODNs) 5′-CCCA4G5C6CC-3′ (I) and 5′-CCC3G4A5CCC-3′ (II) by mass spectrometry. The MS analysis of exonuclease ladders demonstrated that the 5′-exonuclease bovine spleen phosphodiesterase digestion of I and II mono-ruthenated by complex 1 was arrested solely at A4 and partially at C3 and G4, respectively, and that the 3′-exonuclease snake venom phosphodiesterase digestion of the ruthenated ODNs was arrested solely at G5 and G4, respectively, due to the ruthenation. These results did not allow unambiguous identification of ruthenation sites on the metallated ODNs. In contrast, tandem mass spectrometry analysis with CID fragmentation of the mono-ruthenated ODNs provided sequential and complementary [ai − B]/wi fragments, leading to unambiguous identification of G5 in I and G4 in II as the ruthenation sites on the ODN adducts, which is in line with the high selectivity of the complex towards guanine base as reported previously. These findings suggest that caution should be raised with regard to the identification of the binding sites of metal complexes, in particular complexes with bulky ligands, like biphenyl in complex 1, to DNA by MS analysis of exonuclease ladders of the metallated adducts, because the bulky ligands may adopt such an orientation that they block the exonuclease cleavage of the 5′- or 3′-side phosphodiester bonds adjacent to the binding sites, leading to digestion stalling at the nucleotides before the binding sites.

•Five Ru complexes bearing 4-anilinoquinazolines were synthesised and characterised.•A RuII complex was characterised crystallographically.•Three Ru complexes exhibit highly inhibitory potential towards EGFR.•A RuIII complex exhibits EGF-dependent antiproliferative activity against MCF-7.•The RuIII complex is more active to induce early-stage apoptosis than gefitinib.The ruthenium DMSO complexes cis-RuIIC12(DMSO)4 and [(DMSO)2H][trans-RuIIICl4(DMSO)2] reacted with 4-(3′-chloro-4′-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline (L1), 4-(3′-chloro-4′-fluoroanilino)-6-(2-(1H-imidazol-1-yl)ethoxy)-7-methoxy quinazoline (L2), N-(benzo[d]imidazol-4-yl)-6,7-dimethoxyquinazolin-4-amine hydrochloride (L3), 5-(6,7-dimethoxyquinazolin-4-ylamino)quinolin-8-ol hydrochloride (L4), respectively, to afford [RuIICl2(DMSO)2(L1)] (1), [RuIIICl3(DMSO)(L1)] (2), [RuIIICl4(DMSO)(H-L2)] (3), [RuIIICl4(DMSO)(H-L3)] (4), and [RuIIICl3(DMSO)(H-L4)] (5), which were characterised by mass spectrometry, NMR, elementary analysis and single crystal X-ray diffraction (complex 1). Experimental screening (ELISA) showed that complexes 1, 2 and 3 are remarkably inhibitory towards epidermal growth factor receptor (EGFR) with IC50 values at submicromolar or nanomolar level. Docking studies indicated that complexation with ruthenium has little interference with the formation of the two essential H-bonds between the N3 of the quinazoline ring in L1 and L2 and O–H of Thr766 through a water molecule, and the N1 of the quinazoline ring and N–H of Met769 in EGFR. Moreover, complex 2 was shown to be more active against the EGF-stimulated proliferation of human breast cancer cell line MCF-7 than the better EGFR inhibitor 4-(3′-chloro-4′-fluoroanilino)-6,7-dimethoxyquinazoline, being more potential to induce early-stage apoptosis than gefitinib. These imply that apart from inhibiting EGFR, complex 2 may involve in regulating other biological events related to the proliferation of MCF-7, implicating a novel type of multi-targeting metal-based anticancer agents.A ruthenium complex (see picture) bearing an EGFR inhibiting 4-anilinoquinazoline ligand exhibits selectively anti-proliferative activity on EGF-stimulated growth of MCF-7, being more active to induce early-stage apoptosis than gefitinib.

•A MS-based absolute quantification method was developed for serum phosphopeptides.•Titania Coated Silica Microspheres were used to enrich phsophopeptides.•Isolated phosphopeptides were isobarically labeled for absolute quantification.•An endogenous phosphopeptide is a potential discriminator of gastric cancer.Mass spectrometric quantification of phosphopeptides is a challenge due to the ion suppression effect of highly abundant non-phosphorylated peptides in complex samples such as serum. Several strategies for relative quantification of serum phosphopeptides based on MS have been developed, but the power of relative quantities was limited when making direct comparisons between two groups of samples or acting as a clinical examination index. Herein, we describe an MS absolute quantification strategy combined with Titania Coated Magnetic Hollow Mesoporous Silica Microspheres (TiO2/MHMSM) enrichment and stable isotopic acetyl labeling for phosphopeptides in human serum. Four endogenous serum phosphopeptides generated by degradation of fibrinogen were identified by LC–ESI–MS/MS following TiO2/MHMSM enrichment. The ESI–MS signal intensity ratios of the four phosphopeptide standards labeled with N-acetoxy-H3-succinimide (H3-NAS) and N-acetoxy-D3-succinimide (D3-NAS), following TiO2/MHMSM capture are linearly correlated with the molar ratios of the “light” to “heavy” phosphopeptides over the range of 0.1–4 with an r2 of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 98.4–111.9% with RSDs ranging 2.0–10.1%. The absolute quantification of the phosphopeptides in serum samples of 20 healthy persons and 20 gastric cancer patients by the developed method demonstrated that 3 out of the 4 phosphopeptides showed remarkable variation in serum level between healthy and cancer groups, and the phosphopeptide DpSGEGDFLAEGGGVR is significantly down-regulated in the serum of patients, being a potential biomarker for gastric cancer diagnosis.

The complexation with organoruthenium fragments confers 4-anilinoquinazoline pharmacophores with higher potential for inducing cellular apoptosis while the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine are well preserved.

Cu(I) binding promotes the platination of Atox1, although cisplatin binds to the copper coordination sites. In addition, Cu(I) binding enhances the competition of Atox1 with DTT in the reaction of cisplatin. These results indicate that cuprous ions could regulate the cellular trafficking of cisplatin.

Organometallic ruthenium(II) complexes [(η6-arene)Ru(en)Cl]+ (arene = e.g., biphenyl (1), dihydrophenanthrene, tetrahydroanthracene) show promising anticancer activity both in vitro and in vivo and are cytotoxic to cisplatin-resistant cancer cells, implying that these monofunctional complexes have a different mechanism of action from that of bifunctional cisplatin. We demonstrate here that complex 1 binds selectively to the guanine base in the 15-mer single-stranded oligodeoxynucleotides (ODNs) 5′-CTCTCTX7G8Y9CTTCTC-3′ [X = Y = T; X = C, Y = A; X = A, Y = T; X = T, Y = A] to form thermodynamically stable adducts, but thymine bases (T7/T11 or T6/T11) compete kinetically with guanine for binding to 1. The T-bound monoruthenated species eventually convert to diruthenated products via a second step of binding at G or/and to G-bound monoruthenated species through dissociation of the diruthenated adducts. Complex 1 was further shown to bind preferentially to the middle T in a sequence rather than to a T near the terminus and favor coordination to a 5′-T compared to a 3′-T. Interestingly, the T bases in the human telomeric G-quadruplex sequence (5′-AGGGTTAGGGTTAGGGTTAGGG-3′) were found to be more competitive both kinetically and thermodynamically with G bases for binding to 1. These results suggest that thymine bases play a unique role in the pathways of ruthenation of DNA by organoruthenium anticancer complexes and illustrate that kinetic studies can provide new insight into the mechanism of action of metallodrugs in addition to study of the structures and functions of the thermodynamically stable end products.

We report herein a systematic study on interactions of organometallic ruthenium(II) anticancer complex [(η6-arene)Ru(en)Cl]+ (arene = p-cymene (1) or biphenyl (2), en = ethylenediamine) with human transferrin (hTf) and the effects of the hTf-ligation on the bioavailability of these complexes with cisplatin as a reference. Incubated with a 5-fold excess of complex 1, 2, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of 1, 1.01 mol of 2, or 2.14 mol of cisplatin. Mass spectrometry revealed that both ruthenium complexes coordinated to N-donors His242, His273, His578, and His606, whereas cisplatin bound to O donors Tyr136 and Tyr317 and S-donor Met256 in addition to His273 and His578 on the surface of both apo- and holo-hTf. Moreover, cisplatin could bind to Thr457 within the C-lobe iron binding cleft of apo-hTf. Neither ruthenium nor platinum binding interfered with the recognition of holo-hTf by the transferrin receptor (TfR). The ruthenated/platinated holo-hTf complexes could be internalized via TfR-mediated endocytosis at a similar rate to that of holo-hTf itself. Moreover, the binding to holo-hTf well preserved the bioavailability of the ruthenium complexes, and the hTf-bound 1 and 2 showed a similar cytotoxicity toward the human breast cancer cell line MCF-7 to those of the complexes themselves. However, the conjugation with holo-hTf significantly reduced the cellular uptake of cisplatin and the amount of platinated DNA adducts formed intracellularly, leading to dramatic reduction of cisplatin cytotoxicity toward MCF-7. These findings suggest that hTf can serve as a mediator for the targeting delivery of Ru(arene) anticancer complexes while deactivating cisplatin.

•Three ruthenium arene anticancer complexes inhibit human GSTπ.•Inhibitory potencies of Ru complexes towards GSTπ are comparable to their cytotoxicity.•Ru complexes bind to Cys15, Cys48, Met92 and/or Cys102 of GSTπ.•Ru binding sites are either within the G-site or at the dimer interface of GSTπ.•Ru binding induces irreversible oxidation of targeted cysteinyl residues.The organometallic ruthenium(II) anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4 ± 1.3, 63.2 ± 0.4 and 37.2 ± 1.1 μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}2 + fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ.Organoruthenium anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene, biphenyl or dihydrophenanthrene; en = ethylenediamine) bind to thiol and thioetherer sulfurs in the G-site and in the dimer interface of human GSTπ and subsequently induce oxidation of the thiolate ligands, showing different inhibitory potency towards GSTπ.

We report identification of the binding sites for an organometallic ruthenium anticancer complex [(η6-biphenyl)Ru(en)Cl][PF6] (1; en = ethylenediamine) on the 15-mer single-stranded oligodeoxynucleotides (ODNs), 5′-CTCTCTX7G8Y9CTTCTC-3′ [X = Y = T (I); X = C and Y = A (II); X = A and Y = T (III); X = T and Y = A (IV)] by electrospray ionization mass spectrometry (ESI-MS) in conjunction with enzymatic digestion or tandem mass spectrometry (top-down MS). ESI-MS combined with enzymatic digestion (termed MS-based ladder-sequencing), is effective for identification of the thermodynamically-favored G-binding sites, but not applicable to determine the thermodynamically unstable T-binding sites because the T-bound adducts dissociate during enzymatic digestion. In contrast, top-down MS is efficient for localization of the T binding sites, but not suitable for mapping ruthenated G bases, due to the facile fragmentation of G bases from ODN backbones prior to the dissociation of the phosphodiester bonds. The combination of the two MS approaches reveals that G8 in each ODN is the preferred binding site for 1, and that the T binding sites of 1 are either T7 or T11 on I and IV, and either T6 or T11 on II and III, respectively. These findings not only demonstrate for the first time that T-bases in single-stranded oligonucleotides are kinetically competitive with guanine for such organoruthenium complexes, but also illustrate the relative merits of the combination of ladder-sequencing and top-down MS approaches to elucidate the interactions of metal anticancer complexes with DNA.

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO2/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r2 being 0.99. The IC50 values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.

Cisplatin, cis-[Pt(Cl2(NH3)2], can crosslink residues His67 of domain I and His247 of domain II in human albumin, occupying the major binding site for the essential metal zinc on the protein.

We present direct ESI Q-TOF MS and X-ray evidence for remarkable structural and metal ion size effects on the formation of thymidine-based T-tetramers. The conventional H-bond acceptors on the ribose and deoxyribose may disfavor the formation of T-tetramers, and in the series of alkali metal ions, lithium did not induce T-tetramer due to its small ion size. Sodium, potassium, rubidium and caesium could produce thymidine-based T-tetramers. Furthermore, rubidium and caesium could induce T-pentamers and dimeric T-pentamers probably due to their larger ion sizes.

The organometallic anticancer complex [(η6-p-cymene)Ru(en)Cl]PF6 (1, en = ethylenediamine) readily reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological activity. Protein tyrosine phosphatase 1B (PTP1B), a therapeutic target, contains a catalytic cysteinyl thiol and is involved in the regulation of insulin signaling and the balance of protein tyrosine kinase activity. On oxidation, the catalytic Cys215 can form an unusual sulfenyl-amide intermediate which can subsequently be reduced by glutathione. Here we study reactions of 1 with 2-mercaptobenzanilide, 2, a recognized model for the active site of PTP1B. We have characterized crystallographically compound 2 and its oxidized sulfenyl-amide derivative 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), which shows a close structural similarity to the sulfenyl-amide in oxidized PTP1B. At pH 7.4 and 5.3, 1 reacted with 2, affording a mono-ruthenium thiolato complex [(η6-cym)Ru(en)(S-RS)]+ (7+, R = (C6H4)CONH(C6H5)) and a triply-S-bridged thiolato complex [((η6-cym)Ru)2(μ-S-RS)3]+ (8+), respectively. Coordination of Ru to the S atom in 7 allows formation of a strong H-bond (2.02 Å) between the en-NH and the carbonyl oxygen. To assess the possible effect of ruthenium coordination on the redox regulation of PTP1B, reactions of these thiolato products with H2O2 and/or GSH were then investigated, demonstrating that coordination to Ru largely retards both the oxidation (deactivation) of the thiol in compound 2 by H2O2 and the subsequent reduction (reactivation) of the sulfenyl-amide by GSH, implying that the inhibition of complex 1 on PTP1B (IC50 of 19 μM) may be attributed to coordination to its catalytic cysteine.

Six analogs of imatinib, an Abl kinase inhibitor clinically used as a first-line therapeutic agent for chronic myeloid leukaemia (CML), have been synthesized and characterized. And their potency as Abl kinase inhibitors have been screened by a robust virtual screening method developed based on the crystal structure (PDB code 2hyy) of Abl-imatinib complex using Surflex-Docking. The docking results are consistent with the inhibitory potency of the compounds characterized by MS method. And the H-bonds between imatinib analogs and Thr315 and Met318 residues in Abl kinase are shown to be crucial for achieving accurate poses and high binding affinities for the ATP-competitive kinase inhibitors.A robust virtual screening method has been developed based on the crystal structure (PDB code 2hyy) of Abl-imatinib complex using Surflex-Docking to screen six analogs of imatinib newly synthesized as potential Abl kinase inhibitors.

Metallothioneins (MTs) are a group of low molecular weight (6–7 KDa) proteins featured by high cysteine content which allows the proteins to bind to a diverse range of metals. MTs, of which the gene transcription can be induced by a variety of stimuli including hormones, cytokines and metal ions, have important biological functions such as storage, trafficking and homeostasis of metal ions, detoxification of heavy metals, resistance to ionizing radiation, scavenging hydroxyl radical, etc. Importantly, it has been reported that MTs play a role in oncogenesis and cancer prognosis, and are implicated in resistance of cancer cells to anticancer metallodrug cisplatin. In this work, we present a comparative study on interactions between MTs and cisplatin and ruthenium arene anticancer complexes using MALDI-TOF-MS. The results show that cisplatin coordinates to MT-I and MT-II at either pH 3.0 or 7.4, and exhibits a higher affinity to MT-II than to MT-I. The MT–Pt complexes increase significantly in content with decrease in pH values of solutions, indicative that cisplatin competes with zinc for coordination to cysteine residues on MTs and interferes with the binding of zinc to the proteins. While the ruthenium arene anticancer complexes [(η6-arene)Ru(en)Cl]PF6 (arene = benzene or biphenyl, en = ethylenediamine) hardly bind to MTs at acidic pH and coordinate to MTs at a much lower level than cisplatin at neutral pH, which may account for the less toxicity and lack of cross-resistance to cisplatin for this class of ruthenium anticancer complexes. With the MT–Pt complexes as model protein complexes, DDT, the widely used unfolding thiol-containing agent in proteomic research was shown to reduce the coordination of platinum to MTs significantly, implying that DDT used as unfolding reagent at high concentration may cause dissociation of bound metallodrug and should be applied with care.Graphical abstractResearch highlights▶ MALDI-TOF-MS studies reactivity of metal-based anticancer complexes to MTs. ▶ Ruthenium arene complexes bind to MTs at a much lower level than cisplatin. ▶ The low reactivity to MTs may account for less toxicity and non-resistance.

A MALDI-TOF mass spectrometric method for rapid screening of protein tyrosine kinase (PTK) inhibitors has been developed. To circumvent the ion suppression of phosphorylated substrate peptides caused by the presence of high abundant non-phosphorylated peptides in the enzymatic reaction mixtures, a separation and enrichment process of the phosphorylated peptides from complex mixtures was carried out by using an in-house fabricated TiO2 nanoparticle-coated capillary column prior to the MS analysis. With a synthetic phosphopeptide (DAIpYAAPFAKKK), of which the sequence is similar to that of the substrate (EAIYAAPFAKKK) of the Abelson tyrosine kinase (Abl), as the internal standard, the signal ratio of the phosphorylated substrate to the standard detected by MALDI-TOF MS is linearly correlated with the molar ratio of the two phosphopeptides over the range of 0.3 to 3 with r2 = 0.99. We validated the MS method by determining the IC50 value of imatinib, an Abl inhibitor for clinical treatment of chronic myelogenous leukaemia (CML). The obtained IC50 value (234 nM) is consistent with that determined by ELISA (291 nM). Then, six analogues of imatinib synthesized in our laboratory were screened using the method, giving rise to inhibitory potential results which are in good agreement with the docking analysis data. The developed method is sensitive, operationally simple, does not require isotope-labelling and is cost/time effective, providing an alterative method for rapid screening of PTK inhibitors as therapeutic agents for tumours.

The organoruthenium complex [(η6-hmb)Ru(en)(Cl)][PF6] (hmb is hexamethylbenzene, en is ethylenediamine) undergoes facile aquation and then reacts with KSCN in unbuffered solution to give the S-coordinated thiocyanato product [(η6-hmb)Ru(en)(S-SCN)]+ which slowly converts to the thermodynamically favored N-bound complex [(η6-hmb)Ru(en)(N-NCS)]+ (1+). Complex 1 was synthesized and characterized by X-ray crystallography and mass spectrometry. Despite its lack of hydrolysis over 24 h, complex 1 exhibits moderate cytotoxicity (IC50 24 μM) towards the human ovarian cancer cell line A2780, comparable with that of the chlorido analogue which is thought to be activated (towards potential target DNA) via a rapid aquation (Wang et. al. in Proc Natl Acad Sci USA 102:18269–18274, 2005). Detailed kinetic studies suggest that complex 1 binds to guanosine 5′-monophosphate (GMP) through direct N7 substitution of the N-bound SCN ligand. In the presence of a high concentration of chloride (104 mM), however, complex 1 may bind partly to GMP via Cl substitution.

•Three ruthenium arene anticancer complexes inhibit human GSTπ.•Inhibitory potencies of Ru complexes towards GSTπ are comparable to their cytotoxicity.•Ru complexes bind to Cys15, Cys48, Met92 and/or Cys102 of GSTπ.•Ru binding sites are either within the G-site or at the dimer interface of GSTπ.•Ru binding induces irreversible oxidation of targeted cysteinyl residues.The organometallic ruthenium(II) anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4 ± 1.3, 63.2 ± 0.4 and 37.2 ± 1.1 μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}2 + fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ.Organoruthenium anticancer complexes [(η6-arene)Ru(en)Cl]+ (arene = p-cymene, biphenyl or dihydrophenanthrene; en = ethylenediamine) bind to thiol and thioetherer sulfurs in the G-site and in the dimer interface of human GSTπ and subsequently induce oxidation of the thiolate ligands, showing different inhibitory potency towards GSTπ.Download full-size image

Boc5, the first nonpeptidic agonist of Glucagon-like peptide-1 receptor, has been recognized as a potential candidate for treatment of diabetes. However, the metabolic behaviors of this novel molecule in both human and experimental animals remain unclear. This study aimed to explore the metabolic behaviors of Boc5 in biological preparations from human, pig and rat. Boc5 was found to be very stable in liver microsomes of human, pig and rat, but it can be degraded to two metabolites in plasma from all three species, via the successive hydrolysis of the C-22 esters. Chemical inhibition studies using selective esterase inhibitors and assays with purified enzymes suggested that Boc5 hydrolysis in human was totally mediated by human serum albumin (HSA) rather than esterases. ESI-TOF-MS/MS analysis revealed that Lys525 of HSA could be modified by treatment with Boc5, strongly suggesting the pseudo-esterase activity of albumin. Studies on species differences in this albumin-mediated metabolism showed large species differences in degradation rate of Boc5, the half lives of Boc5 in plasma from three various species varied from 23.5 h to 83.1 h, but they were much closer to the half lives of Boc5 in corresponding serum albumins, implying the predominant role of serum albumin in plasma metabolism of Boc5. Additionally, the effects of various ligands including fatty acids and several drugs with unambiguous binding sites on HSA, on the pseudo-esterase activity of HSA, were also investigated using both experimental and molecular modelling studies. These results showed that the binding of various ligands to HSA could significantly affect the pseudo-esterase activity of HSA towards Boc5, due to the ligand-induced conformation changes of HSA.Download high-res image (73KB)Download full-size image

The complexation with organoruthenium fragments confers 4-anilinoquinazoline pharmacophores with higher potential for inducing cellular apoptosis while the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine are well preserved.

Cisplatin, cis-[Pt(Cl2(NH3)2], can crosslink residues His67 of domain I and His247 of domain II in human albumin, occupying the major binding site for the essential metal zinc on the protein.

Cu(I) binding promotes the platination of Atox1, although cisplatin binds to the copper coordination sites. In addition, Cu(I) binding enhances the competition of Atox1 with DTT in the reaction of cisplatin. These results indicate that cuprous ions could regulate the cellular trafficking of cisplatin.

We have recently demonstrated that complexation with (η6-arene)RuII fragments confers 4-anilinoquinazoline pharmacophores a higher potential for inducing cellular apoptosis while preserving the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine (Chem. Commun., 2013, 49, 10224–10226). Reported herein are the synthesis, characterisation and evaluation of the biological activity of a new series of ruthenium(II) complexes of the type [(η6-arene)Ru(N,N-L)Cl]PF6 (arene = p-cymene, benzene, 2-phenylethanol or indane, L = 4-anilinoquinazolines). These organometallic ruthenium complexes undergo fast hydrolysis in aqueous solution. Intriguingly, the ligation of (arene)RuII fragments with 4-anilinoquinazolines not only makes the target complexes excellent EGFR inhibitors, but also confers the complexes high affinity to bind to DNA minor grooves while maintaining their reactivity towards DNA bases, characterising them with dual-targeting properties. Molecular modelling studies reveal that the hydrolysis of these complexes is a favourable process which increases the affinity of the target complexes to bind to EGFR and DNA. In vitro biological activity assays show that most of this group of ruthenium complexes are selectively active inhibiting the EGF-stimulated growth of the HeLa cervical cancer cell line, and the most active complex [(η6-arene)Ru(N,N-L13)Cl]PF6 (4, IC50 = 1.36 μM, L13 = 4-(3′-chloro-4′-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline) is 29-fold more active than its analogue, [(η6-arene)Ru(N,N-ethylenediamine)Cl]PF6, and 21-fold more active than gefitinib, a well-known EGFR inhibitor in use clinically. These results highlight the strong promise to develop highly active ruthenium anticancer complexes by ligation of cytotoxic ruthenium pharmacophores with bioactive organic molecules.

The organometallic anticancer complex [(η6-p-cymene)Ru(en)Cl]PF6 (1, en = ethylenediamine) readily reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological activity. Protein tyrosine phosphatase 1B (PTP1B), a therapeutic target, contains a catalytic cysteinyl thiol and is involved in the regulation of insulin signaling and the balance of protein tyrosine kinase activity. On oxidation, the catalytic Cys215 can form an unusual sulfenyl-amide intermediate which can subsequently be reduced by glutathione. Here we study reactions of 1 with 2-mercaptobenzanilide, 2, a recognized model for the active site of PTP1B. We have characterized crystallographically compound 2 and its oxidized sulfenyl-amide derivative 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), which shows a close structural similarity to the sulfenyl-amide in oxidized PTP1B. At pH 7.4 and 5.3, 1 reacted with 2, affording a mono-ruthenium thiolato complex [(η6-cym)Ru(en)(S-RS)]+ (7+, R = (C6H4)CONH(C6H5)) and a triply-S-bridged thiolato complex [((η6-cym)Ru)2(μ-S-RS)3]+ (8+), respectively. Coordination of Ru to the S atom in 7 allows formation of a strong H-bond (2.02 Å) between the en-NH and the carbonyl oxygen. To assess the possible effect of ruthenium coordination on the redox regulation of PTP1B, reactions of these thiolato products with H2O2 and/or GSH were then investigated, demonstrating that coordination to Ru largely retards both the oxidation (deactivation) of the thiol in compound 2 by H2O2 and the subsequent reduction (reactivation) of the sulfenyl-amide by GSH, implying that the inhibition of complex 1 on PTP1B (IC50 of 19 μM) may be attributed to coordination to its catalytic cysteine.

We present direct ESI Q-TOF MS and X-ray evidence for remarkable structural and metal ion size effects on the formation of thymidine-based T-tetramers. The conventional H-bond acceptors on the ribose and deoxyribose may disfavor the formation of T-tetramers, and in the series of alkali metal ions, lithium did not induce T-tetramer due to its small ion size. Sodium, potassium, rubidium and caesium could produce thymidine-based T-tetramers. Furthermore, rubidium and caesium could induce T-pentamers and dimeric T-pentamers probably due to their larger ion sizes.

A high mobility group box 1 (HMGB1) protein has been reported to recognize both 1,2-intrastrand crosslinked DNA by cisplatin (1,2-cis-Pt-DNA) and monofunctional platinated DNA using trans-[PtCl2(NH3)(thiazole)] (1-trans-PtTz-DNA). However, the molecular basis of recognition between the trans-PtTz-DNA and HMGB1 remains unclear. In the present work, we described a hydrogen/deuterium exchange mass spectrometry (HDX-MS) method in combination with docking simulation to decipher the interactions of platinated DNA with domain A of HMGB1. The global deuterium uptake results indicated that 1-trans-PtTz-DNA bound to HMGB1a slightly tighter than the 1,2-cis-Pt-DNA. The local deuterium uptake at the peptide level revealed that the helices I and II, and loop 1 of HMGB1a were involved in the interactions with both platinated DNA adducts. However, docking simulation disclosed different H-bonding networks and distinct DNA-backbone orientations in the two Pt-DNA-HMGB1a complexes. Moreover, the Phe37 residue of HMGB1a was shown to play a key role in the recognition between HMGB1a and the platinated DNAs. In the cis-Pt-DNA-HMGB1a complex, the phenyl ring of Phe37 intercalates into a hydrophobic notch created by the two platinated guanines, while in the trans-PtTz-DNA-HMGB1a complex the phenyl ring appears to intercalate into a hydrophobic crevice formed by the platinated guanine and the opposite adenine in the complementary strand, forming a penta-layer π–π stacking associated with the adjacent thymine and the thiazole ligand. This work demonstrates that HDX-MS associated with docking simulation is a powerful tool to elucidate the interactions between platinated DNAs and proteins.

An addressable single cell imaging strategy combining ToF-SIMS and confocal fluorescence microscopy imaging has been developed, and sucessfully applied to visualize the subcellular distribution of an organoruthenium anticancer complex, [(η6-benzene)Ru(N,N-L)Cl]+ (1; L: 4-anilinoquinazoline ligand), showing its accumulation in both cell membrane and nuclei, and verifying its dual-targeting feature.