Deborah J. Kerwood

Pt(IV) complexes as prodrugs for cisplatin

The antitumor effects of platinum(IV) complexes, considered prodrugs for cisplatin, are believed to be due to biological reduction of Pt(IV) to Pt(II), with the reduction products binding to DNA and other cellular targets. In this work we used pBR322 DNA to capture the products of reduction of oxoplatin, c,t,c-[PtCl(2)(OH)(2)(NH(3))(2)], 3, and a carboxylate-modified analog, c,t,c-[PtCl(2)(OH)(O(2)CCH(2)CH(2)CO(2)H)(NH(3))(2)], 4, by ascorbic acid (AsA) or glutathione (GSH). Since carbonate plays a significant role in the speciation of platinum complexes in solution, we also investigated the effects of carbonate on the reduction/DNA-binding process. In pH 7.4 buffer in the absence of carbonate, both 3 and 4 are reduced by AsA to cisplatin (confirmed using ((195))Pt NMR), which binds to and unwinds closed circular DNA in a manner consistent with the formation of the well-known 1, 2 intrastrand DNA crosslink. However, when GSH is used as the reducing agent for 3 and 4, ((195))Pt NMR shows that cisplatin is not produced in the reaction medium. Although the Pt(II) products bind to closed circular DNA, their effect on the mobility of Form I DNA is different from that produced by cisplatin. When physiological carbonate is present in the reduction medium, ((13))C NMR shows that Pt(II) carbonato complexes form which block or impede platinum binding to DNA. The results of the study vis-à-vis the ability of the Pt(IV) complexes to act as prodrugs for cisplatin are discussed.

Modification and Uptake of a Cisplatin Carbonato Complex by Jurkat Cells

The interactions of Jurkat cells with cisplatin, cis-[Pt(15NH3)2Cl2](1), are studied using 1H-15N heteronuclear single quantum coherence (HSQC) NMR and inductively coupled plasma mass spectrometry. We show that Jurkat cells in culture rapidly modify the monocarbonato complex cis-[Pt(15NH3)2(CO3)Cl]- (4), a cisplatin species that forms in culture media and probably also in blood. Analysis of the HSQC NMR peak intensity for 4 in the presence of different numbers of Jurkat cells reveals that each cell is capable of modifying 0.0028 pmol of 4 within ∼0.6 h. The amounts of platinum taken up by the cell, weakly bound to the cell surface, remaining in the culture medium, and bound to genomic DNA were measured as functions of time of exposure to different concentrations of drug. The results show that most of the 4 that has been modified by the cells remains in the culture medium as a substance of molecular mass <3 kDa, which is HSQC NMR silent, and is not taken up by the cell. These results are consistent with a hitherto undocumented extracellular detoxification mechanism in which the cells rapidly modify 4, which is present in the culture medium, so it cannot bind to the cell. Because there is only a slow decrease in the amount of unmodified 4 remaining in the culture medium after 1 h, -1.1 ± 0.4 μM h-1, the cells subsequently lose their ability to modify 4. These observations have important implications for the mechanism of action of cisplatin.

Adsorption of the PtII Anticancer Drug Carboplatin by Mesoporous Silica

MCM‐41, a mesoporous silica nanomaterial with a high surface area for adsorption of small molecules, is a potential new type of delivery vehicle for therapeutic and diagnostic agents. In this report, we show that MCM‐41 adsorbs the front‐line anticancer drug carboplatin, [Pt(CBDCA‐O,O′)(NH3)2] (CBDCA=cyclobutane‐1,1‐dicarboxylate; 1), which is used to treat ovarian, lung, and other types of cancer. UV/Visible difference absorption spectroscopy shows that MCM‐41 adsorbs 1.8±0.2% of its own weight of carboplatin after a 24 h exposure to 26.9 mM drug in H2O. The pseudo‐first‐order rate constant for adsorption of carboplatin by MCM‐41, measured using [1H,15N] heteronuclear single quantum coherence (HSQC) NMR, and 15N‐labeled carboplatin is k1=2.92±2.17×10−6 s−1 at ca. 25°.

Formation of Carbonato and Hydroxo Complexes in the Reaction of Platinum Anticancer Drugs with Carbonate

The second-generation Pt(II) anticancer drug carboplatin is here shown to react with carbonate, which is present in blood, interstitial fluid, cytosol, and culture medium, to produce platinum-carbonato and -hydroxo complexes. Using [(1)H-(15)N] HSQC NMR and (15)N-labeled carboplatin, we observe that cis-[Pt(CBDCA-O)(OH)(NH(3))(2)](-), cis-[Pt(OH)(2)(NH(3))(2)], cis-[Pt(CO(3))(OH)(NH(3))(2)](-), and what may be cis-[Pt(CO(3))(NH(3))(2)] are produced when 1 is allowed to react in 23.8 mM carbonate buffer. When (15)N-labeled carboplatin is allowed to react in 0.5 M carbonate buffer, these platinum species, as well as other hydroxo and carbonato species, some of which may be dinuclear complexes, are produced. Furthermore, we show that the carbonato species cis-[Pt(CO(3))(OH)(NH(3))(2)](-) is also produced when cisplatin is allowed to react in carbonate buffer. The study outlines the conditions under which carboplatin and cisplatin form carbonato and aqua/hydroxo species in carbonate media.

Stability of carboplatin and oxaliplatin in their infusion solutions is due to self-association

Carboplatin and oxaliplatin are commonly used platinum anticancer agents that are sold as ready-to-use aqueous infusion solutions with shelf lives of 2 and 3 years, respectively. The observed rate constants for the hydrolysis of these drugs, however, are too large to account for their long shelf lives. We here use electrospray-trap mass spectrometry to show that carboplatin and oxaliplatin are self-associated at concentrations in their ready-to-use infusion solutions (~27 mM and 13 mM, respectively) and, as expected, when the drug concentration is reduced to more physiologically relevant concentrations (100 μM and 5 μM, respectively) the association equilibrium is shifted in favor of the monomeric forms of these drugs. Using (1)H NMR we measure the intensity of the NH resonance of the two symmetry-equivalent NH(3) molecules of carboplatin, relative to the intensity of the γ-methylene CH resonance, as a function of total drug concentration. Then, by fitting the data to models of different molecularity, we show that the association complex is a dimer with a monomer-dimer association constant of K (M(-1)) = 391 ± 127. The work presented here shows that carboplatin and oxaliplatin mainly exist as association complexes in concentrated aqueous solution, a property that accounts for the long term stability of their ready-to-use infusion solutions, and that these association complexes may exist, to some extent, in the blood after injection.

New extracellular resistance mechanism for cisplatin

The HSQC NMR spectrum of 15N-cisplatin in cell growth media shows resonances corresponding to the monocarbonato complex, cis-[Pt(NH3)2(CO3)Cl](-), 4, and the dicarbonato complex, cis-[Pt(NH3)2(CO3)2](-2), 5, in addition to cisplatin itself, cis-[Pt(NH3)2Cl2], 1. The presence of Jurkat cells reduces the amount of detectable carbonato species by (2.8+/-0.7) fmol per cell and has little effect on species 1. Jurkat cells made resistant to cisplatin reduce the amount of detectable carbonato species by (7.9+/-5.6) fmol per cell and also reduce the amount of 1 by (3.4+/-0.9) fmol per cell. The amount of detectable carbonato species is also reduced by addition of the drug to medium that has previously been in contact with normal Jurkat cells (cells removed); the reduction is greater when drug is added to medium previously in contact with resistant Jurkat cells (cells removed). This shows that the platinum species are modified by a cell-produced substance that is released to the medium. Since the modified species have been shown not to enter or bind to cells, and since resistant cells modify more than non-resistant cells, the modification constitutes a new extracellular mechanism for cisplatin resistance which merits further attention.

Structure Refinement for a 24-Nucleotide RNA Hairpin

Refinement of the NMR‐based structure of an RNA hairpin that binds the coat protein of bacteriophage R17 is presented. Properties of the upper stem and loop suggest rapid conformational exchange: (i) the G10,1 and G16,1 imino signals are broad, (ii) residues G7–G16 have a substantial fraction of S‐puckered ribose rings and (iii) a primary tetraloop conformation satisfies the distance restraints, but other forms occurred in our molecular dynamics simulations that might be in fast exchange with the primary conformation. Unusual methods were used to assign all of the non‐exchangeable protons in the molecule. The results show that structure determinations of 20–30‐mer RNAs are practical without isotope labeling. The analysis depended on time‐domain deconvolution of 2D NOE spectra, the resolving power of 3D 2‐quantum correlation/NOE spectra and dispersively phased 1H–31P‐COSY spectra. This work corrects some assignment errors in G1–A4, and reports a full‐relaxation matrix refinement (averaging >20 distance restraints per residue). The resolution of the new structure confirms that the unpaired A8 residue is stacked in the stem. It also shows the presence of a two‐residue pyrimidine turn and a possible A11·A14 non‐canonical pair in the tetra‐loop. Finally, it determines an unusual structure for the molecules strand ends. The latter involves stacking of G3/G2/A23/U24, a probable G3·U22 pair and G1 lies in the major groove. The strand‐end structure raises interesting possibilities for unique structures at the junctions of double helical RNA with unpaired regions.

Solution Structure and Constrained Molecular Dynamics Study of Vitamin B12 Conjugates of the Anorectic Peptide PYY(3-36).

Vitamin B12–peptide conjugates have considerable therapeutic potential through improved pharmacokinetic and/or pharmacodynamic properties imparted on the peptide upon covalent attachment to vitamin B12 (B12). There remains a lack of structural studies investigating the effects of B12 conjugation on peptide secondary structure. Determining the solution structure of a B12–peptide conjugate or conjugates and measuring functions of the conjugate(s) at the target peptide receptor may offer considerable insight concerning the future design of fully optimized conjugates. This methodology is especially useful in tandem with constrained molecular dynamics (MD) studies, such that predictions may be made about conjugates not yet synthesized. Focusing on two B12 conjugates of the anorectic peptide PYY(3–36), one of which was previously demonstrated to have improved food intake reduction compared with PYY(3–36), we performed NMR structural analyses and used the information to conduct MD simulations. The study provides rare structural insight into vitamin B12 conjugates and validates the fact that B12 can be conjugated to a peptide without markedly affecting peptide secondary structure.