Final Report Summary - LIPOPROT (New chemical tools for profiling protein lipidation in cancer)
In the 21st century cancer is the major cause of death in the developed world. However, most cases could perhaps be cured if only an effective medication could be used. The Marie Curie IEF project “Lipoprot” led by Dr Remigiusz A. Serwa (fellow) and Dr Edward W. Tate (scientist in charge) aimed to develop novel technologies that in the near future could provide the basis for the development of a new generation of anti-cancer agents, and the means for matching individual patients with drugs that are well suited for their treatment. The experiments performed deciphered the molecular composition of malignant cells, in particular the levels of the molecular entities that consist of fats and proteins (lipidated proteins) in cancer cells. The formation of lipidated proteins (protein lipidation) is controlled by the action of enzymes, protein-lipid transferases, and the abnormal activity of these enzymes in cancer cells has been previously reported, suggesting a possibility for a pharmacological down-regulation of cancer-promoting transformations in cells by levelling-down the activities of these enzymes with novel medications. As the understanding of these processes was very limited, highly innovative chemical and biological methods have been developed and applied in this project in order to describe for the first time the levels of lipidated proteins in cancer and their responses to protein-lipid transferase inhibitors.
The three main objectives of the project were: to design and synthesize chemical probes for protein lipidation; to validate methodology for high-throughput profiling of protein lipidation in a model cell line; and to combine these tools for comparative analysis of protein lipidation in representative cancer cell lines. The synthesis of chemical probes was optimised to be cost and time efficient, and physicochemical properties of all novel compounds was well characterised. Furthermore, certain probes designed and synthesized as a result of the project possessed superior specificity and sensitivity over previously developed probes when applied to cell in culture, and are currently under detailed investigation prior to intended commercialisation. Novel lipid probes were applied to a model cell line (cervical cancer HeLa) in culture. Cell feeding conditions (e.g. probe concentration, incubation time) were extensively optimised for each probe, and this led to the discovery of dozens of novel lipidated proteins. In the following series of experiments, ‘smart-design’ protein enrichment reagents were developed that contained innovative structural features which allowed for the first time the discovery of both the identity of lipidated proteins and the exact location and the nature of chemical bonding that link fats and proteins. Next, several cancer cell lines (breast cancer MDA-MB231 and MCF-7, colon cancer HCT 116, cervical cancer HeLa) were cultured and relative protein lipidation efficiencies, on a protein to protein basis, and global enzymatic activities of lipid transferases were measured. These cell lines were further utilised in a series of experiments to examine numerous aspects of protein myristoylation (the incorporation of myristic acid, C13H27COOH, into proteins catalysed by the enzyme N-myristoyl transferase) in cancer. The experiments included phenotypic and biomolecular screening of the effects of N-myristoyl transferase inhibition in cancer cells. Metabolic activity measurements, flow cytometric and quantitative proteomic analyses were conducted in cancer cell lines, in order to provide preliminary support for the hypothesis that N-myristoyl transferase is a valid drug target in cancer. Importantly, the molecules and technologies developed within the “Lipoprot” project are universal, and may be applied to quantify the extent of protein lipidation in other human and animal diseases. Indeed, the molecular probes and analytical methodologies developed within the project could also be straightforwardly applied in studies on biological systems other than cancer. In collaboration with scientists from the London School of Hygiene & Tropical Medicine ( London, UK) and the Wellcome Trust Sanger Institute (Hinxton, UK) the fellow has shown that probes he synthesized are well suited for labelling of proteins in live malaria-causing parasite, Plasmodium falciparum, and that the analytical methodology developed can be adopted for fluorescent visualisation and mass spectrometry-based quantification of the parasitic proteome. These preliminary studies will be followed up in order to assess lipid transferases as drug targets in malaria.
Due to its interdisciplinary nature, the project “Lipoprot” has contributed to European excellence and competitiveness in many scientific disciplines, such as synthetic chemistry, analytical chemistry, biochemistry, and cell biology. Furthermore, since the project relates to the application of chemical methods in cancer research, its findings are expected to immediately aid the research of medicinal chemists and cancer biologists, but ultimately society as a whole will benefit as well. As a result of the current global drug development crisis, major drug companies are interested in the integration of drug discovery with systems biology, particularly the development of innovative new methods to profile the effects of drug candidates across global biological networks. Such methods were developed in this project. In conclusion, chemical proteomic tools reveal details of human biology on an unprecedented scale, and further developments in this emerging field should be considered for immediate attention from the EU. Currently the US leads the world in the fields of chemical biology and disease biomarker development, and it is particularly important that the European Community benefits from the outcomes of projects that will deliver an alternative, more comprehensive methodology for screening these biomarkers.
The three main objectives of the project were: to design and synthesize chemical probes for protein lipidation; to validate methodology for high-throughput profiling of protein lipidation in a model cell line; and to combine these tools for comparative analysis of protein lipidation in representative cancer cell lines. The synthesis of chemical probes was optimised to be cost and time efficient, and physicochemical properties of all novel compounds was well characterised. Furthermore, certain probes designed and synthesized as a result of the project possessed superior specificity and sensitivity over previously developed probes when applied to cell in culture, and are currently under detailed investigation prior to intended commercialisation. Novel lipid probes were applied to a model cell line (cervical cancer HeLa) in culture. Cell feeding conditions (e.g. probe concentration, incubation time) were extensively optimised for each probe, and this led to the discovery of dozens of novel lipidated proteins. In the following series of experiments, ‘smart-design’ protein enrichment reagents were developed that contained innovative structural features which allowed for the first time the discovery of both the identity of lipidated proteins and the exact location and the nature of chemical bonding that link fats and proteins. Next, several cancer cell lines (breast cancer MDA-MB231 and MCF-7, colon cancer HCT 116, cervical cancer HeLa) were cultured and relative protein lipidation efficiencies, on a protein to protein basis, and global enzymatic activities of lipid transferases were measured. These cell lines were further utilised in a series of experiments to examine numerous aspects of protein myristoylation (the incorporation of myristic acid, C13H27COOH, into proteins catalysed by the enzyme N-myristoyl transferase) in cancer. The experiments included phenotypic and biomolecular screening of the effects of N-myristoyl transferase inhibition in cancer cells. Metabolic activity measurements, flow cytometric and quantitative proteomic analyses were conducted in cancer cell lines, in order to provide preliminary support for the hypothesis that N-myristoyl transferase is a valid drug target in cancer. Importantly, the molecules and technologies developed within the “Lipoprot” project are universal, and may be applied to quantify the extent of protein lipidation in other human and animal diseases. Indeed, the molecular probes and analytical methodologies developed within the project could also be straightforwardly applied in studies on biological systems other than cancer. In collaboration with scientists from the London School of Hygiene & Tropical Medicine ( London, UK) and the Wellcome Trust Sanger Institute (Hinxton, UK) the fellow has shown that probes he synthesized are well suited for labelling of proteins in live malaria-causing parasite, Plasmodium falciparum, and that the analytical methodology developed can be adopted for fluorescent visualisation and mass spectrometry-based quantification of the parasitic proteome. These preliminary studies will be followed up in order to assess lipid transferases as drug targets in malaria.
Due to its interdisciplinary nature, the project “Lipoprot” has contributed to European excellence and competitiveness in many scientific disciplines, such as synthetic chemistry, analytical chemistry, biochemistry, and cell biology. Furthermore, since the project relates to the application of chemical methods in cancer research, its findings are expected to immediately aid the research of medicinal chemists and cancer biologists, but ultimately society as a whole will benefit as well. As a result of the current global drug development crisis, major drug companies are interested in the integration of drug discovery with systems biology, particularly the development of innovative new methods to profile the effects of drug candidates across global biological networks. Such methods were developed in this project. In conclusion, chemical proteomic tools reveal details of human biology on an unprecedented scale, and further developments in this emerging field should be considered for immediate attention from the EU. Currently the US leads the world in the fields of chemical biology and disease biomarker development, and it is particularly important that the European Community benefits from the outcomes of projects that will deliver an alternative, more comprehensive methodology for screening these biomarkers.