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Toll-Like Receptor 4 activation and function in diseases: an integrated chemical-biology approach.

Periodic Reporting for period 2 - TOLLerant (Toll-Like Receptor 4 activation and function in diseases: an integrated chemical-biology approach.)

Reporting period: 2017-01-01 to 2018-12-31

The TOLLerant project aimed to address the medical and societal need of new anti-inflammatory drugs against infectious and non-infectious inflammatory diseases. New therapeutic approaches have been explored, based on the use of small-molecular TLR4 modulators, whose production could be scaled to an industrial level. This goal was achieved during the four years of project by the cooperative effort of a multidisciplinary consortium composed of 13 early stage researchers recruited by 8 teams from universities and 2 from companies located in different countries (Italy, Spain, Slovenia, Germany and Belgium).
Conclusions of the action:
The short term scientific objective of the project has been the study of molecular aspects of TLR4 activation and inhibition by using selective agonists and antagonists obtained by chemical synthesis or extracted from natural sources. This objective has been realized. Several New Chemical Entities (NCAs) were synthesized and delivered during the project (new synthetic molecules, new LPS variants from bacteria, nanoparticles functionalized with molecules). Some TLR4 antagonists were patented. The activity of these NCAs on TLR4 has been validated in vitro (binding experiments, microscopy, and experiments on human and murine cells) and in vivo in animal models of sepsis and LPS-induced pneumonia.
The NCAs produced in a collaborative way within the TOLLerant consortium are an important hit for drug development in the field of anti-inflammatory drugs, vaccine adjuvants, and substances for tumor immunotherapy. The delivery of NCAs therefore partially meets the long-term scientific objective of TOLLerant (develop specific drug hits based on small molecules and nanoparticles targeting TLR4).
The educational objective of the project has been to train the 13 early stage researchers hired by the consortium, providing them with scientific knowledge and industrial skills. During the 4 years project, the two companies collaborated with the academic groups of the TOLLerant consortium with the main objective to train 13 young scientists as the future leaders of the field to work in a multidisciplinary environment and to develop specific scientific, industrial and soft skills.
The young ESRs have been the real driving force of the basic science developed during the project. Several new projects and new ideas derived from brainstorming among ESRs and PIs during the network meetings. ESRs also greatly contributed to the spreading of results in the scientific community and to the communication and dissemination activities directed to the general public and to the society.
The first milestone of the project has been the production of panels of small molecule TLR4 antagonists with different chemical structures. These molecules were developed by computer-assisted rational drug design, knowing the structures of TLR4 and of the two co-receptors MD-2 and CD14. The chemical structures of new synthetic TLR4 modulators were patented, and their synthesis and biological properties were eventually published.
The production of functional recombinant MD-2 co-receptor has been the second milestone of the project, and allowed direct binding studies with ligands.
The experimental methods to determine the binding between functional MD-2 and its ligands have been delivered and can be exploited as well for high-throughput selection of TLR4 modulators.
Nuclear magnetic Resonance (NMR) experiments were performed to determine the aggregation state of the molecules in solution, which seems to influence their biological activity.
As third milestone, 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. The chemical structures of LPS variants are original and have been published. These natural compounds are now available for industrial exploitation.
A fourth 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 synthetic and natural TLR4 modulators and their biological activity on cells has been assessed.
The nanoparticles carrying TLR4 antagonists and TLR4 agonists have been published and are now available for industrial development as new TLR4-directed nanotherapeutics.
In a parallel and synergistic research approach, designed tetrahedric protein nanostructures were investigated as new nano-scaffolds for the multivalent presentation of TLR4 modulators.
As a fifth milestone, microscopy studies with fluorescently-labelled lipooligosaccharide (LOS) allowed to track LOS in cells after TLR4 stimulation, collecting important information on the molecular mechanism of TLR4 activation and internalization.
As sixth milestone, the in vivo effect of TLR4 modulation was studied in 3 animal models of sepsis by industrial partners.
Several deliverables of the project are now available to network companies and other companies for industrial development.
Some scientific results obtained during the 4 years of the project represent an important progress beyond the state of the art and are also exploitable for industrial development.
Exploitable results and deliverables of the project:
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 ligand presentation and delivery in cells and in vivo; 6) microscopy techniques used to visualize TLR4 and track LPS molecules in cells as an important tool for studying 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:
The TOLLerant website ( was the main public communication channel of the project. It has been constantly updated with the newest information of the projects: event realization, recent publications, and research advancements. Significant dissemination activities have been: 1) the publication of research papers (about 18) and reviews in peer reviewed international journals, 2) the presentation of posters and oral communications to international conferences (about 60), 3) the promotion of successful initiatives to increase visibility and awareness of scientific research
TOLLerant scientific objectives
The dual role of TLR4
TOLLerant multidisciplinary approach
The TOLLerant team (at the meeting in Napoli, 2017)
The industrial training in TOLlerant
TOLLerant project presentation
Increasing the complexity of the project