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Effects of noncovalent platinum drug-protein interactions on drug efficacy: use of fluorescent conjugates as probes for drug metabolism.

ABSTRACT: The overall efficacy of platinum based drugs is limited by metabolic deactivation through covalent drug-protein binding. In this study the factors affecting cytotoxicity in the presence of glutathione, human serum albumin (HSA) and whole serum binding with cisplatin, BBR3464, and TriplatinNC, a "noncovalent" derivative of BBR3464, were investigated. Upon treatment with buthionine sulfoximine (BSO), to reduce cellular glutathione levels, cisplatin and BBR3464-induced apoptosis was augmented whereas TriplatinNC-induced cytotoxicity was unaltered. Treatment of A2780 ovarian carcinoma cells with HSA-bound cisplatin (cisplatin/HSA) and cisplatin preincubated with whole serum showed dramatic decreases in cytotoxicity, cellular accumulation, and DNA adduct formation compared to treatment with cisplatin alone. Similar effects are seen with BBR3464. In contrast, TriplatinNC, the HSA-bound derivative (TriplatinNC/HSA), and TriplatinNC pretreated with whole serum retained identical cytotoxic profiles and equal levels of cellular accumulation at all time points. Confocal microscopy of both TriplatinNC-NBD, a fluorescent derivative of TriplatinNC, and TriplatinNC-NBD/HSA showed nuclear/nucleolar localization patterns, distinctly different from the lysosomal localization pattern seen with HSA. Cisplatin-NBD, a fluorescent derivative of cisplatin, was shown to accumulate in the nucleus and throughout the cytoplasm while the localization of cisplatin-NBD/HSA was limited to lysosomal regions of the cytoplasm. The results suggest that TriplatinNC can avoid high levels of metabolic deactivation currently seen with clinical platinum chemotherapeutics, and therefore retain a unique cytotoxic profile after cellular administration.

SUBMITTER: Benedetti BT

PROVIDER: S-EPMC3341405 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Effects of noncovalent platinum drug-protein interactions on drug efficacy: use of fluorescent conjugates as probes for drug metabolism.

Benedetti Brad T BT Peterson Erica J EJ Kabolizadeh Peyman P Martínez Alberto A Kipping Ralph R Farrell Nicholas P NP

Molecular pharmaceutics 20110516 3

The overall efficacy of platinum based drugs is limited by metabolic deactivation through covalent drug-protein binding. In this study the factors affecting cytotoxicity in the presence of glutathione, human serum albumin (HSA) and whole serum binding with cisplatin, BBR3464, and TriplatinNC, a "noncovalent" derivative of BBR3464, were investigated. Upon treatment with buthionine sulfoximine (BSO), to reduce cellular glutathione levels, cisplatin and BBR3464-induced apoptosis was augmented where ...[more]

PMID: 21548575

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Project description:Fluorescent proteins contain an internal chromophore constituted of amino acids or an external chromophore covalently bonded to the protein. To increase their fluorescence intensities, many research groups have attempted to mutate amino acids within or near the chromophore. Recently, a new type of fluorescent protein, called UnaG, in which the ligand binds to the protein through many noncovalent interactions was discovered. Later, a series of mutants of the UnaG protein were introduced, which include eUnaG with valine 2 mutated to leucine emitting significantly stronger fluorescence than the wild type and V2T mutant, in which valine 2 is mutated to threonine, emitting weaker fluorescence than the wild type. Interestingly, the single mutation sites of both eUnaG and V2T mutants are distant from the fluorophore, bilirubin, which renders the mechanism of such fluorescence enhancement or reduction unclear. To elucidate the origin of fluorescence intensity changes induced by the single mutations, we carried out extensive analyses on MD simulations for the original UnaG, eUnaG and V2T, and found that the bilirubin ligand bound to eUnaG is conformationally more rigid than the wild-type, particularly in the skeletal dihedral angles, possibly resulting in the increase of quantum yield through a reduction of non-radiative decay. On the other hand, the bilirubin bound to the V2T appears to be flexible than that in the UnaG. Furthermore, examining the structural correlations between the ligand and proteins, we found evidence that the bilirubin ligand is encapsulated in different environments composed of protein residues and water molecules that increase or decrease the stability of the ligand. The changed protein stability affects the mobility and confinement of water molecules captured between bilirubin and the protein. Since the flexible ligand contains multiple hydrogen bond (H-bond) donors and acceptors, the H-bonding structure and dynamics of bound water molecules are highly correlated with the rigidity of the bound ligand. Our results suggest that, to understand the fluorescence properties of protein mutants, especially the ones with noncovalently bound fluorophores with internal rotations, the interaction network among protein residues, ligand, and water molecules within the binding cavity should be investigated rather than focusing on the local structure near the fluorescing moiety. Our in-depth simulation study may offer a foundation for the design principles for engineering this new class of fluorescent proteins.

Project description:BackgroundBoth ganglioside GD2-targeted immunotherapy and antibody-drug conjugates (ADCs) have demonstrated clinical success as solid tumor therapies in recent years, yet no research has been carried out to develop anti-GD2 ADCs against solid tumors. This is the first study to analyze cytotoxic activity of clinically relevant anti-GD2 ADCs in a wide panel of cell lines with varying GD2 expression and their effects in mouse models of GD2-positive solid cancer.MethodsAnti-GD2 ADCs were generated based on the GD2-specific antibody ch14.18 approved for the treatment of neuroblastoma and commonly used drugs monomethyl auristatin E (MMAE) or F (MMAF), conjugated via a cleavable linker by thiol-maleimide chemistry. The antibody was produced in a mammalian expression system, and its specific binding to GD2 was analyzed. Antigen-binding properties and biodistribution of the ADCs in mice were studied in comparison with the parent antibody. Cytotoxic effects of the ADCs were evaluated in a wide panel of GD2-positive and GD2-negative tumor cell lines of neuroblastoma, glioma, sarcoma, melanoma, and breast cancer. Their antitumor effects were studied in the B78-D14 melanoma and EL-4 lymphoma syngeneic mouse models.ResultsThe ch14.18-MMAE and ch14.18-MMAF ADCs retained antigen-binding properties of the parent antibody. Direct dependence of the cytotoxic effect on the level of GD2 expression was observed in cell lines of different origin for both ADCs, with IC50 below 1 nM for the cells with high GD2 expression and no cytotoxic effect for GD2-negative cells. Within the analyzed cell lines, ch14.18-MMAF was more effective in the cells overexpressing GD2, while ch14.18-MMAE had more prominent activity in the cells expressing low GD2 levels. The ADCs had a similar biodistribution profile in the B78-D14 melanoma model compared with the parent antibody, reaching 7.7% ID/g in the tumor at 48 hours postinjection. The average tumor size in groups treated with ch14.18-MMAE or ch14.18-MMAF was 2.6 times and 3.8 times smaller, respectively, compared with the control group. Antitumor effects of the anti-GD2 ADCs were also confirmed in the EL-4 lymphoma model.ConclusionThese findings validate the potential of ADCs targeting ganglioside GD2 in treating multiple GD2-expressing solid tumors.

Dataset Information

Effects of noncovalent platinum drug-protein interactions on drug efficacy: use of fluorescent conjugates as probes for drug metabolism.

Publications

Effects of noncovalent platinum drug-protein interactions on drug efficacy: use of fluorescent conjugates as probes for drug metabolism.

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