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Chemical AMPylomics: targeting a novel host-pathogen interaction

Final Report Summary - CHEMAMP (Chemical AMPylomics: targeting a novel host-pathogen interaction)

In the European Union alone there are 25,000 deaths per annum due to antibiotic-resistant bacterial infection, a figure that is set against a complete absence in recent years of new antibiotics exploiting novel modes of action. Understanding the mechanisms by which bacteria achieve increased virulence against their host, often in diverse and surprising ways, provides our greatest weapon in the fight against these diseases by revealing new classes of antibiotic targets. Virulence is intimately linked to host-pathogen interactions and bacterial manipulation of host cell signaling, particularly through enzymatic post-translational modification (PTM) of proteins. AMPylation (adenosine monophosphorylation) is rapidly emerging as a novel PTM and fundamental mechanism to regulate protein-protein interactions and cell signaling in host-pathogen interactions and in normal cells, providing an opportunity for the discovery of new functional biology and targets for new antibiotics with novel mode of action. However, without robust tools to identify and manipulate both AMP transferases and their AMPylated protein substrates our understanding of this complex signaling network will remain superficial. In this Marie Curie IEF project “ChemAMP” led by Dr Malgorzata Broncel (fellow) and Prof Edward W. Tate (scientist in charge) novel chemical tools and methodologies were developed enabling the first comprehensive exploration of AMPylation phenomenon and its role in normal cell function and in infection.

The proposed objectives were: to design and synthesize chemical probes for protein AMPylation and to validate methodology for high-throughput profiling of protein AMPylation in host and bacteria as well as during infection. Initially, the synthesis of novel AMPylation probes that target proteins that undergo AMPylation was optimised to be cost and time efficient, and physicochemical properties of all novel compounds were well characterised. Pilot experiments clearly demonstrated that certain AMPylation probes possessed superior specificity and sensitivity when tested in model systems. These selected AMPylation probes were further applied to profiling AMPylation networks in both bacterial and eukaryotic context. Experimental conditions (e.g. probe concentration, incubation time) were first optimised based on in-gel fluorescence imaging, and later fine-tuned based on small scale mass spectrometry-based proteomic measurements. Sophisticated proteomic experiments conducted at later stage and in larger scale, led to a confident and unprecedented discovery of dozens novel AMPylated proteins and protein interaction networks in human cells. Similar investigation was performed in human cells undergoing bacterial infection and the outcomes are currently in final stages of validation. In the subsequent series of experiments, a number of novel multifunctional protein capture reagents utilized for derivatization of the AMPylation probe-labeled proteins with secondary tags that facilitate visualization and isolation of these targets, were synthesized. The performance of these smart ‘fishing pols’ for AMPylated proteins was investigated and compared with the best reagents routinely applied in the field. Novel reagents performed very well, and innovative structural features incorporated into these reagents have allowed not only the identity of AMPylated proteins but also the exact location of the AMPylation site on these proteins to be discovered for the first time applying chemical proteomic technology. Importantly, the molecules and technologies developed within the “ChemAMP” project are universal and could also be straightforwardly applied in studies on biological systems other than bacterial infection. Indeed, in collaboration with scientists from University College London and Imperial College London the fellow has shown that the AMPylation probes she synthesized are well suited for profiling eukaryotic AMPylation, moreover, the developed multifunctional enrichment reagents are suitable for fluorescent visualisation and mass spectrometry-based quantification of other PTMs, for example protein myristoylation, a specific type of protein lipidation involved in cancer and the life cycle of Plasmodium falciparum, malaria-causing parasite.

Due to its interdisciplinary nature, the project “ChemAMP” 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 studying host pathogen interactions and bacterial infection, its findings are expected to immediately aid the research of medicinal chemists and bacteriologists 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 deliver alternative, more comprehensive methodologies for screening and validation of novel drug targets.