10 research outputs found
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Elucidation of Plasma-induced Chemical Modifications on Glutathione and Glutathione Disulphide
Cold atmospheric pressure plasmas are gaining increased interest in the medical sector and clinical trials to treat skin diseases are underway. Plasmas are capable of producing several reactive oxygen and nitrogen species (RONS). However, there are open questions how plasma-generated RONS interact on a molecular level in a biological environment, e.g. cells or cell components. The redox pair glutathione (GSH) and glutathione disulphide (GSSG) forms the most important redox buffer in organisms responsible for detoxification of intracellular reactive species. We apply Raman spectroscopy, mass spectrometry, and molecular dynamics simulations to identify the time-dependent chemical modifications on GSH and GSSG that are caused by dielectric barrier discharge under ambient conditions. We find GSSG, S-oxidised glutathione species, and S-nitrosoglutathione as oxidation products with the latter two being the final products, while glutathione sulphenic acid, glutathione sulphinic acid, and GSSG are rather reaction intermediates. Experiments using stabilized pH conditions revealed the same main oxidation products as were found in unbuffered solution, indicating that the dominant oxidative or nitrosative reactions are not influenced by acidic pH. For more complex systems these results indicate that too long treatment times can cause difficult-to-handle modifications to the cellular redox buffer which can impair proper cellular function
Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association
Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances. Here, we present the synthesis and characterization of pro-apoptotic BimBH3 peptides of different lengths carrying cysteines for labeling with nitroxide or gadolinium spin probes. We show by DEER that the length of the peptides modulates their homo-interactions in the absence of other Bcl-2 proteins and solve by X-ray crystallography the structure of a BimBH3 tetramer, revealing the molecular details of the inter-peptide interactions. Finally, we prove that using orthogonal labels and three-channel DEER we can disentangle the Bim-Bim, Bcl-xL-Bcl-xL, and Bim-Bcl-xL interactions in a simplified interactome.This work was funded by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC-2033—Projektnummer 390677874, the DFG Priority Program SPP1601 “New Frontiers in Sensitivity in EPR Spectroscopy” (to E.B.), DFG BO 3000/5-1 (to E.B.), SFB958 – Z04 (to E.B.), DFG grant INST 130/972-1 FUGG (to E.B.). P.E.C. is supported by an Australian NHMRC fellowship (1079700
Metal-peptide bioconjugates for targeted anti-cancer therapy
In der medizinischen Chemie spielen Metallkomplexe eine wichtige Rolle als Antikrebsmittel. Leider zeigen sie keine Selektivität gegenüber Krebszellen. Um den Mangel an Selektivität zu überwinden, werden Metallkomplexe an zell-penetrierende und zell-zielende Peptide gekoppelt. Der schwierigste Aspekt ist jedoch die selektive Synthese solcher Metallpeptid-Biokonjugate. Metallkomplexe mit geeigneten funktionellen Gruppen, die für SPPS geeignet sind, müssen entworfen werden. Das Ziel dieser Arbeit ist die Einführung und Optimierung der neuartigen synthetische Protokolle für die Synthese von Metall-Peptid-Biokonjugaten.Platin(IV)- und Kobalt(III)-Komplexe werden über Amidbindungen an zell-penetrierende Peptide gebunden. Wohingegen, Gold(III)-Komplexe an Peptide gekoppelt werden, die spezifisch auf Brustkrebszellen zielen. Diese Synthese erfolgt in Lösung. Des Weiteren, wird die anti-proliferative Aktivität von Metall-Peptid-Biokonjugate an menschlichen Krebszellen im MTT-Test untersucht.In medicinal chemistry metal complexes display important role as anti-cancer agents. Unfortunately, they exhibit no selectivity to cancer cells. To overcome the lack of selectivity, metal complexes are coupled to cell penetrating and cell targeting peptides. However, the most challenging aspect is the selective synthesis of such metal-peptide bioconjugates. Metal complexes with appropriate functional groups suitable for Solid Phase Peptide Synthesis have to be designed. The aim of this thesis is to introduce and optimize novel synthetic protocols for the synthesis of metal-peptide bioconjugates. Platinum(IV) and cobalt(III) complexes will be linked to cell penetrating peptides through amide bonds. On the contrary, gold(III) complexes will be coupled to breast cancer cell targeting peptides in solution. Furthermore, the anti-proliferative activity of metal complexes, peptides and their metal-peptide bioconjugates against human cancer cells will be evaluated by MTT assay
Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents
Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl2(DMSO)2], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and 1H, 13C, 15N and 195Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC50 = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC50 = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3)
Targeted, Molecular Europium (III) Probes Enable Luminescence-Guided Surgery and 1 Photon Post-Surgical Luminescence Microscopy of Solid Tumors
International audienceDiscrete luminescent lanthanide complexes representa potential alternative to organic chromophores due to theirtunability of optical properties, insensitivity to photobleaching, andlarge pseudo-Stokes shifts. Previously, we demonstrated that thelack of depth penetration of UV excitation required to sensitizediscrete terbium and europium complexes can be overcome usingCherenkov radiation emitted by clinically employed radioisotopesin situ. Here, we show that the second-generation europiumcomplexes [Eu(III)(pcta-PEPA2)] and [Eu(III)(tacn-pic-PEPA2)] (Φ= 57% and 76%, respectively) lower the limit of detection (LoD) to1 nmol in the presence of 10 μCi of Cherenkov emitting isotopes,18F and 68Ga. Bifunctionalization provides access to cysteine-linked peptide conjugates with comparable brightness and LoD. Theconjugate, [Eu(tacn-(pic-PSMA)-PEPA2)], displays high binding affinity to prostate-specific membrane antigen (PSMA)-expressingPC-3 prostate cancer cells in vitro and can be visualized in the membrane-bound state using confocal microscopy. Biodistributionstudies with the [86Y][Y(III)(tacn-(pic-PSMA)-PEPA2)] analogue in a mouse xenograft model were employed to studypharmacokinetics. Systemic administration of the targeted Cherenkov emitter, [68Ga][Ga(III)(PSMA-617)], followed by intratumoralinjection or topical application of 20 or 10 nmol [Eu(III)(tacn-(pic-PSMA)-PEPA2)], respectively, in live mice resulted in statisticallysignificant signal enhancement using conventional small animal imaging (620 nm bandpass filter). Optical imaging informedsuccessful tumor resection. Ex vivo imaging of the fixed tumor tissue with 1 and 2 photon excitation further reveals the accumulationof the administered Eu(III) complex in target tissues. This work represents a significant step toward the application of luminescentlanthanide complexes for optical imaging in a clinical setting
Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures
Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions
Study on chemical modifications of glutathione by cold atmospheric pressure plasma (Cap) operated in air in the presence of Fe(II) and Fe(III) complexes
Cold atmospheric pressure plasma is an attractive new research area in clinical trials to treat skin diseases. However, the principles of plasma modification of biomolecules in aqueous solutions remain elusive. It is intriguing how reactive oxygen and nitrogen species (RONS) produced by plasma interact on a molecular level in a biological environment. Previously, we identified the chemical effects of dielectric barrier discharges (DBD) on the glutathione (GSH) and glutathione disulphide (GSSG) molecules as the most important redox pair in organisms responsible for detoxification of intracellular reactive species. However, in the human body there are also present redox-active metals such as iron, which is the most abundant transition metal in healthy humans. In the present study, the time-dependent chemical modifications on GSH and GSSG in the presence of iron(II) and iron(III) complexes caused by a dielectric barrier discharge (DBD) under ambient conditions were investigated by IR spectroscopy, mass spectrometry and High Performance Liquid Chromatography (HPLC). HPLC chromatograms revealed one clean peak after treatment of both GSH and GSSH with the dielectric barrier discharge (DBD) plasma, which corresponded to glutathione sulfonic acid GSOH. The ESI-MS measurements confirmed the presence of glutathione sulfonic acid. In our experiments, involving either iron(II) or iron(III) complexes, glutathione sulfonic acid GSOH appeared as the main oxidation product. This is in sharp contrast to GSH/GSSG treatment with DBD plasma in the absence of metal ions, which gave a wild mixture of products. Also interesting, no nitrosylation of GSH/GSSG was oberved in the presence of iron complexes, which seems to indicate a preferential oxygen activation chemistry by this transition metal ion
Catalytic oxidation of small organic molecules by cold plasma in solution in the presence of molecular iron complexes†
The plasma-mediated decomposition of volatile organic compounds has previously been investigated in the gas phase with metal oxides as heterogeneous catalysts. While the reactive species in plasma itself are well investigated, very little is known about the influence of metal catalysts in solution. Here, we present initial investigations on the time-dependent plasma-supported oxidation of benzyl alcohol, benzaldehyde and phenol in the presence of molecular iron complexes . Products were identified by HPLC, ESI-MS, FT-IR, and
spectroscopy. Compared to metal-free oxidation of the substrates, which is caused by reactive oxygen species and leads to a mixture of products, the metal-mediated reactions lead to one product cleanly, and faster than in the metal-free reactions. Most noteworthy, even catalytic amounts of metal complexes induce these clean transformations. The findings described here bear important implications for plasma-supported industrial waste transformations, as well as for plasma-mediated applications in biomedicine, given the fact that iron is the most abundant redox-active metal in the human body