Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

Indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and its Na+ analogue (KP1339) are two of the most prominent non-platinum antitumor metal complexes currently undergoing clinical trials. After intravenous administration, they are known to bind to human serum albumin (HSA) in a noncovalent manner. To elucidate their HSA binding sites, displacement reactions with the established site markers warfarin and dansylglycine as well as bilirubin were monitored by spectrofluorimetry, ultrafiltration-UV-vis spectrophotometry, and/or capillary zone electrophoresis. Conditional stability constants for the binding of KP1019 and KP1339 to sites I and II of HSA were determined, indicating that both Ru(III) compounds bind to both sites with moderately strong affinity (log K (1)' = 5.3-5.8). No preference for either binding site was found, and similar results were obtained for both metal complexes, demonstrating low influence of the counter ion on the binding event

Monitoring of the metal displacement from the recombinant mouse liver metallothionein Zn7-complex by capillary zone electrophoresis with electrospray MS detection

International audienceA solution of a Zn-complex of recombinant mouse MT-1 isoform (Zn7-MT-1) was prepared and titrated with Cd2+ ions. A method based on the coupling of capillary zone electrophoresis (CZE) with electrospray MS detection was developed for the analysis of the stoichiometry of the species formed during the titration. The method offered the possibility of the on-line removal of up to 100 mM Tris or phosphate buffer solutions that would otherwise suppress the electrospray signal. By allowing the determination of the metal stoichiometry of the complex species present in solution the method was shown to be complementary to circular dichroism and UV-VIS spectrophotometry conventionally used for similar studies. The titration of the ZN7-MT complex with Cd(II) showed the sequential displacement of the Zn by Cd. The unusually high stability of the Cd6Zn1-MT species was observed which suggests a structural role of the remaining Zn(II) ion. © 2002 Elsevier Science B.V. All rights reserved

Is Ag(I) an adequate probe for Cu(I) in structural copper-metallothionein studies?: The binding features of Ag(I) to mammalian metallothionein 1

International audienceThe binding abilities of silver(I) silver(l) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408-417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu-MT studies. The Zn/Ag replacement in recombinant mouse Zn7-MT 1 and corresponding Zn4-? MT 1 and Zn3-?MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV-vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn7-MT 1, Zn4-? MT 1 and Zn3-?MT 1 with AgClO4 at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn7-MT 1 at pH 7.5 has revealed the subsequent formation of Ag4Zn5-MT, Ag7Zn3-MT, Ag8Zn3-MT, Ag10Zn2-MT, Ag 12Zn1-MT, Agx-MT, x = 14-19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Agx-MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the M xZny-MT (0 ? y < 7) species found, only M 4IZn5-MT [(Zn4)?(M 4Zn1)?] and M7IZn3-MT [(M3Zn2)? (M4Zn1)?], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I)

Capillary electrophoresis hyphenated to inductively coupled plasma-mass spectrometry: A novel approach for the analysis of anticancer metallodrugs in human serum and plasma

The development of metal-based chemotherapeutics lacks methods which are capable of providing early indication on the potential of new metal complexes as future anticancer drugs. Since most of these compounds are administered intravenously, serum proteins are the first available biological binding partners in the bloodstream. For platinum-based anticancer drugs the interaction with serum proteins is regarded as an important contribution to the side effects accompanying chemotherapy. In contrast, newly developed ruthenium compounds are thought to be transported into the tumor in a protein-bound form. In here, the application of CE hyphenated to inductively coupled plasma (ICP)-MS, applying Polybrene-coated capillaries, is demonstrated for studying the interaction of indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) with HSA and transferrin, which are important transport proteins. Furthermore, the applicability of the method to human serum and plasma and, more importantly, to real-world patient samples was proven. KP1019 was found to bind to a high degree to HSA both in serum, plasma and the patient samples. Only minor fractions of ruthenium were found attached to other proteins

Elucidation of the interactions of an anticancer ruthenium complex in clinical trials with biomolecules utilizing capillary electrophoresis hyphenated to inductively coupled plasma-mass spectrometry

The application of capillary electrophoresis (CE) combined with highly sensitive inductively-coupled-plasma mass spectrometric (ICP-MS) detection allows the interactions of metal complexes with biomols. to be characterized. This technique has been used to provide new insights into the mode of action of the ruthenium-based anticancer drug candidate indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019). While the compd. binds rapidly and efficiently to serum proteins, esp. albumin, its reactivity towards the model DNA compd. 2'-deoxyguanosine 5'-monophosphate (5'-dGMP) is moderate.LCO