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TOLLerant Report Summary

Project ID: 642157
Funded under: H2020-EU.1.3.1.

Periodic Reporting for period 1 - TOLLerant (Toll-Like Receptor 4 activation and function in diseases: an integrated chemical-biology approach.)

Reporting period: 2015-01-01 to 2016-12-31

Summary of the context and overall objectives of the project

Diseases of the immune and repair systems of the body comprise a vast array of maladies that impose high social and economic burden in developed and developing countries. Besides infectious diseases chronic inflammatory diseases, such as the autoimmune syndromes, contribute an increasing share to this problem.
Toll-like Receptors (TLRs), expressed on immunity cells in humans and mammalians, are the “sensors” of invading pathogens. TLRs detect minute amounts of molecules deriving from pathogens thus starting innate immunity response and inflammation. The abnormal activation of TLRs, in particular of TLR4, by microbial factors and endogenous molecules with excessive production of inflammatory mediators, mainly cytokines, has emerged as a common molecular mechanism associated to a wide array of inflammatory and autoimmune diseases. Small molecule TLR4 blockers (antagonists) can stop the inflammatory and autoimmune cascade at the very beginning and are potentially more specific and potent anti-inflammatory agents than currently used biological drugs (antibodies) that neutralize cytokines. In addition to inhibition of TLR4, due to its important role in innate immune system, stimulation of activity of TLR4 is also of considerable therapeutic interest, particularly for vaccine adjuvants and for cancer immunotherapy. The project TOLLerant long-term scientific goal is to develop specific drug candidates based on small molecules targeting TLRs (in particular TLR4) and to exploit different types of their delivery using advanced nanoparticle delivery platforms. The academic sector has a fundamental role in the discovery of new drug especially in the field of inflammation and autoimmunity. However, the collaboration between academic and industrial groups is still the driving force for the discovery of new drugs. In this perspective, the second general aim of project TOLLerant is to facilitate the collaboration among industrial and academic groups with the main objective to train 13 young scientists to work in a multidisciplinary environment and to develop specific scientific, industrial and soft skills as the future leaders of the field.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first two years of the project new small molecules were projected with the potential to bind to TLR4 coreceptors MD-2 and CD14, and therefore to interfere in the TLR4 activation process. Once synthesized, some of these molecules showed to inhibit the TLR4-dependent production of inflammatory mediators (cytokines) in mammalian or mouse cells. Nuclear magnetic Resonance (NMR) experiments were performed to determine the aggregation state of the molecules in solution that seems to influence their biological activity. The interaction of synthetic TLR4 antagonists with cationic peptides that potentiate their anti-TLR4 activity was studied by NMR methods as well.
The consortium is also interested in TLR4 modulators derived from natural sources. The chemical structures of natural lipopolysaccharides (LPS) from different pathogenic and non-pathogenic bacteria were characterized by NMR, and the activity of these natural LPS variants on TLR4 (agonism or antagonism) was evaluated in cultured cells .
A second important milestone of the project is the presentation and delivery of TLR4 antagonists and agonists by nano-carriers. Biocompatible nanoparticles that can be imaged by optical methods and radiochemistry were functionalized with TLR4 modulators and the biological activity on cells has been assessed. In a parallel and synergic research, designed tetrahedric protein nanostructures were investigated as new nano-scaffolds for the multivalent presentation of TLR4 modulators. New drug delivery formulations of TLR4 ligands by liposome incorporation were also developed.
A third milestone and deliverable of the project is the production of the MD-2 protein, that directly interacts with the natural and synthetic TLR4 modulators, for in vitro binding studies. MD-2 was expressed in three different hosts: mammalian cells (human HEK293 cells), yeast cells (P. pastoris) and bacteria (E. coli). The expressed MD-2 was shown to fold correctly and to be functional, proteins from mammalian and yeast cells being more active than those from bacteria. A mutant MD-2 has been expressed and its folding and LPS binding properties have been studied by computational methods. The action of these novel small molecules or nanoparticles able to bind to MD-2 and block or activate TLR4 signaling in cells was then studied. Microscopy studies aimed at understanding the cellular localization and compartmentalization of both receptor and ligands during the TLR4 activation process. Cells transfected with TLR4 conjugated to yellow fluorescent protein (YFP) were used for microscopy co-localization studies together with fluorescently labeled LPS (the natural TLR4 ligand).
The activity of synthetic molecules as well as natural LPS variants and nanoparticles as TLR4 agonists or antagonists was studied in different cells expressing TLR4, and the effect of TLR4 modulation on the downstream intracellular signaling is currently under investigation. The capacity of extracellular vesicles containing oxidized phospholipids produced in patients with inflammatory diseases to activate TLR4 as endogenous activators relevant to autoinflammation in cells is also under investigation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Some scientific results obtained in the first two years of the project represent an important progress beyond the state of the art and are also exploitable for the industrial development.
New exploitable results are: 1) Computational methods; 2) New synthetic TLR4 modulators delivered in the project will be patented and published by the consortium; 3) New nanoparticles also incorporating TLR4 ligands, 4) HEK cells expressing TLR4-YFP for microscopy studies; 5) New nanoparticles (also based on designed tetrahedral proteins) for TLR4 ligands presentation and delivery in cells and in vivo; 6) microscopy techniques used to visualize TLR4 and track LPS molecules in cells are beyond the state of the art, and represent an important tool for studying the TLR4 activation/signaling in a dynamic way; 7) new structures of bacterial LPS have been elucidated and the TLR4 activity of LPS variants studied. 8) Nanoparticles and protein nanostructures 9) The role of natural oxidized lipids and their aggregates in TLR4 stimulation and inflammation induction has been outlined 10) NMR studies provided information on the interaction in solution between glycolipid-based TLR4 modulators and cationic peptides: a potential role of co-administration of these molecules to increase the efficacy of TLR4 modulation is under study.
Dissemination and socio-economic impact
TOLLerant website ( is the main public communication channel of the project. It is constantly being updated with the newest information of the projects: event realization, recent publications, and research advancements. Significant dissemination activities are: 1) the publication of research papers and reviews in peer reviewed international journals, 2) the presentation of posters and oral communication to international conferences, 3) the promotion of successful initiatives to increase visibility and awareness of scientific research (European Researchers Nights initiatives, National or local Science Weeks, hands-on workshop for elementary and high school students, “Marie-Curie Ambassadors” initiatives, public open days).
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