Transferrin Serves As a Mediator to Deliver Organometallic Ruthenium(II) Anticancer Complexes into Cells

We report herein a systematic study on interactions of organometallic ruthenium­(II) anticancer complex [(η⁶-arene)­Ru­(en)­Cl]⁺ (arene = <i>p</i>-cymene (<b>1</b>) or biphenyl (<b>2</b>), 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 <b>1</b>, <b>2</b>, or cisplatin, 1 mol of diferric hTf (holo-hTf) attached 0.62 mol of <b>1</b>, 1.01 mol of <b>2</b>, 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 <b>1</b> and <b>2</b> 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

High-Salt-Tolerance Matrix for Facile Detection of Glucose in Rat Brain Microdialysates by MALDI Mass Spectrometry

Due to its strong ultraviolet absorption, high salt tolerance, and little interference in the low molecular weight region, <i>N</i>-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) has been applied as a matrix to measure the level of glucose in rat brain microdialysates by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) in combination with in vivo microdialysis. By monitoring the ion signals of (glucose + Cl)⁻ in the mass spectra, we achieved a low detection limit of ∼10 μM for glucose in 126 mM NaCl, which is a typical component in artificial cerebrospinal fluid, without prior sample purification. It is concluded that NEDC-assisted laser desorption/ionization (LDI) MS is a fast and general method for sensitive detection of small molecules (such as glucose and amino acids) in high ionic strength solutions

2,3,4,5-Tetrakis(3′,4′-dihydroxylphenyl)thiophene: A New Matrix for the Selective Analysis of Low Molecular Weight Amines and Direct Determination of Creatinine in Urine by MALDI-TOF MS

Small organic matrixes are still the most commonly used ones in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) because of their advantages of high sensitivity, convenience, and cost-effectiveness. However, due to the matrix interference in the low mass region, the direct analysis of low molecular weight amines in complex surroundings with conventional organic matrixes remains a challenge. Here, a new Brønsted–Lowry acid compound 2,3,4,5-tetrakis­(3′,4′-dihydroxylphenyl)­thiophene (DHPT) was designed, synthesized, and applied as a matrix for analysis of low molecular weight amines by MALDI-TOF MS. DHPT displays good selectivity in the analysis of amines without matrix-related interference and the low picomole/femtomole limit-of-detection was obtained in positive ion mode. With DHPT, the metabolites including creatinine, glycine, alloxan, allantoin, and 3-hydroxyhippuric acid in human urine were directly analyzed by MALDI-TOF MS. The identity of these metabolites was confirmed by tandem mass spectrometry. Furthermore, the urine creatinine was quantitatively determined using isotope-labeled internal standard. This DHPT-assisted LDI MS method provides a general approach for both qualitative and quantitative analysis of low molecular weight amines

Mass Spectrometric Proteomics Reveals that Nuclear Protein Positive Cofactor PC4 Selectively Binds to Cross-Linked DNA by a <i>trans</i>-Platinum Anticancer Complex

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 <i>trans</i>-platinum anticancer complex, <i>trans</i>-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 <i>trans</i>-PtTz

Quantitative Mass Spectrometry Combined with Separation and Enrichment of Phosphopeptides by Titania Coated Magnetic Mesoporous Silica Microspheres for Screening of Protein Kinase Inhibitors

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 <i>r</i>² 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

Thymines in Single-Stranded Oligonucleotides and G‑Quadruplex DNA Are Competitive with Guanines for Binding to an Organoruthenium Anticancer Complex

Organometallic ruthenium­(II) complexes [(η⁶-arene)­Ru­(en)­Cl]⁺ (arene = e.g., biphenyl (<b>1</b>), 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 <b>1</b> 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 <b>1</b>. 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 <b>1</b> 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 <b>1</b>. 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