Development of mass spectrometric methods to understand the mechanism of action of transition metal-based anticancer compounds

Fook Seng Ronald Lee
EPFL, 2016
EPFL thesis

Abstract

Metal based anti-cancer drugs (metallodrugs) such as cisplatin are a cornerstone of cancer chemotherapy. However, development of this class of compound has been hindered by our lack of understanding of how they function. This is mainly because existing method to study the mechanism of action of metallodrugs are lacking as they were either directly adapted from methods to study organic drugs which have very different chemical properties or current limits in technology and knowledge were a bottleneck to the development of suitable methods. Mass spectrometry (MS) has emerged as a powerful tool which can be applied to study the mechanism of action of metallodrugs. Many different MS methods exist which can be adapted to study different aspects of drug action while accounting for the unique chemical nature of metallodrugs. In this dissertation, we develop MS strategies to study how platinum and ruthenium metallodrugs work and study a few important aspects of their function. Firstly, the in vitro distribution of and ligand state of metallodrugs were studied by imaging mass spectrometry. Next, we attempted to find novel protein targets of metallodrugs using a novel protein expression profiling approach. Promising metallodrug protein targets obtained from this study were then validated via biochemical methods. To facilitate the application of MS based protein-metallodrug fragmentation experiments for finding binding sites of metallodrugs on proteins, we developed and optimized web-based tools for automatic processing of complex spectra from these experiment. We then applied these tools to study the interaction of metallodrugs on relevant proteins, which provided insight into the specific binding properties of metallodrugs on proteins.

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Fook Seng Ronald Lee
EPFL, 2016
EPFL thesis

Abstract

Official source

https://infoscience.epfl.ch/record/222790?ln=en

About this result

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Related publications

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

Analysis of expression of PDCP and MAL13P1.308 of Plasmodium falciparum employing a quantitative proteomics approach based on SILAC

Céline Freymond

Plasmodium falciparum is a deadly parasite that causes malaria in humans. This disease causes the death of one million people every year. To find new means to fight this parasite, it is important to learn more about its biology. As the pathways of protein expression are better understood, it becomes easier to find out how to block these mechanisms. The completion of the genome sequencing has opened new perspective of genome wide analysis. One of these studies was done by LaCount et al. who used the yeast1two1hybrid system to map the complete interaction network between proteins of P. falciparum. ¨ From this network of interaction an interesting protein was studied further by Daubenberger et al. which is PDCP. This protein is closely related to MSP11 in this protein network, a protein involved in erythrocyte invasion. PDCP is a CCCH1type zinc finger protein, a family of proteins that are involved in protein1protein interaction, nucleic acid binding and binding in small ligands. It was shown that its expression was dependant on the density of the parasites. In this study we used the SILAC technology to confirm these previous results. A culture is grown with isotopically heavy isoleucine in the medium as a control sample. After a few cell cycles, almost all the natural isoleucines are replaced with the labeled ones. Three cultures with parasitemia of 2, 5 and 10% are grown and mixed with an equal amount of the control culture. When the proteins are analyzed by mass spectrometry, the peptides that contained isoleucine will be detected as two separate peaks. After normalization of each peptide area with this internal control, the quantity of PDCP can be compared between cultures of different parasitemias. 11 peptides of PDCP have been detected by mass spectrometry, proving its existence for the first time. These peptides were labeled to more than 95%, allowing the comparison of PDCP expression between the cultures. But because of difficulty of reproducibility in in vitro cultures, the regulation of PDCP by the parasitemia has not been observed by SILAC. We wanted to study further the protein interaction network in which PDCP is involved. MAL13P1.308, a protein directly interacting with PDCP and MSP11 in the protein network map, was analyzed by IFA. It was found to be expressed during all stages of the asexual blood stage cycle and it was not imported in the host cell. The next step will be to see if it interacts with PDCP by co1localization studies

2008

Current proteomic strategies depend strongly on the development of analytical methodologies and instrumentation. In parallel to the development of mass spectrometry (MS) - based proteomic workflows, microfluidic devices emerged in this field as a flexible tool for rapid and sensitive protein studies. In this context, the present work focuses on the development of miniaturized analytical systems for protein studies, especially by electrospray ionization mass spectrometric detection. Several approaches have been proposed to complement the mass spectrometric analysis of tryptic peptides using chemical tagging to isolate specific subclasses of proteins by affinity baits or for quantitation purposes. Optimization of a chemical tagging reaction in a sandwich mixer-reactor that consists of mixing two solutions providing the reactants in a channel, one central laminar flow being sandwiched between two outer flow solutions, was first investigated by finite-element method. The numerical simulations have highlighted the importance of positioning the reactant presenting the lowest diffusion coefficient close to the sidewall, as well as decreasing the outer flow velocities, to enhance a chemical reaction because of the parabolic flow profile across the microchannel. This optimization is even more relevant for consecutive reactions. As infusion-based electrospray microchips are known to present great advantages in terms of sensitivity compared to standard ionization source, an ESI emitter microchip based on polymer photo-ablation was designed. First, the hyphenation of the chip in a LC-MS workflow was successfully achieved and evaluated by comparison with a standard pneumatically-assisted ESI source. To perform enhanced on-chip post-column derivatization of peptides, a substitute design to the sandwich mixer-reactor was explored to improve reaction in a microchip before ESI – MS analysis. The electrospray micromixer chip includes a passive mixing unit to perturbate the flow along the microchannel. The mixing efficiency was demonstrated by fluorescence imaging, and on-chip chemical derivatization of peptides and kinetic studies before mass spectrometry were achieved. The on-line LC-MS derivatization of cysteinyl peptides was shown to provide a more confident and accurate protein identification by adding information on the peptidic sequence. Within the development of electrospray microchips for mass spectrometry, many efforts have been put forward in the hyphenation of functional units. Immobilization of functionalized stationary phase in microfluidic systems is widely used to achieve protein or peptide isolation, sample cleaning, separation or reaction. In this context, magnetic beads have proven to be quite useful compared to other methods, such as immobilized packed beads or monoliths, because of their high and reversible magnetization, and various surface functionalization. A polymer microchip including a magnetic track array was designed to focus the magnetic field generated by permanent magnets towards precise locations along the microchannel. A multi-plug magnetic bead capture was obtained and a significant increase of bead capture efficiency was demonstrated both numerically and experimentally, which is beneficial for affinity separation applications, as example. The research in microfluidic front-end devices for mass spectrometry is actually oriented to the development of multi-spray interfaces for high-throughput analysis. An electrospray microchip with a multi-track array was developed. The capability of the device to screen alternatively up to six samples and to perform relative quantification was assessed. Soft ionization techniques have revolutionized the analysis of proteins by mass spectrometry. In contrast with the major content of the thesis dealing with microfluidic systems for protein analysis, the last chapter presents a novel ambient ionization method, so-called membrane-desorption electrospray ionization (M-DESI). The principle is similar to the desorption electrospray ionization (DESI) method. But, in the M-DESI approach the sample is desorbed directly from a mesh membrane positioned vertically and in-axis between the ESI emitter and the mass spectrometer inlet. Analysis of peptides and proteins was demonstrated as a proof-of-concept, offering great perspectives for detection after separation on membrane.

EPFL2009