Final Report Summary - TARKINAID (Targeting Src-family tyrosine kinases in chronic autoimmune and inflammatory diseases)
Executive Summary:
Chronic autoimmune and inflammatory diseases such as rheumatoid arthritis or chronic inflammatory lung diseases are severe and devastating conditions that strongly impair the quality of life of the affected individuals and are a major burden to health care budgets and the entire society. Despite significant efforts aimed at the understanding and treating those diseases, current therapeutic options are still very limited due to major side effects and very high costs. Therefore, development of novel therapeutic strategies for the treatment of chronic autoimmune and inflammatory diseases is a highly important research priority.
Src-family kinases are nonreceptor tyrosine kinases which are involved in tumor development and progression and therefore are major targets of existing anti-leukemic therapies in the clinic. However, they are also present in immune cells and preliminary findings of the consortium had indicated that they are also involved in the development of inflammatory diseases in experimental animals. The consortium members had also previously developed Src-family kinases inhibitors which inhibited various inflammation-related functions of human leukocytes. Based on those prior studies, the TARKINAID consortium aimed to develop novel anti-inflammatory drug candidates and to improve our understanding of the role of Src-family kinases in the inflammation process. The most significant achievements within the three major aims of the project were as follows:
1) Development of novel Src-family inhibitors with anti-inflammatory properties. We have developed a large number of novel and patentable small molecule inhibitors of Src-family kinases and tested their effect in various inflammation-related in vitro and in vivo assays. After several rounds of structural optimization, we have developed a set of structurally related drug candidates which inhibited Src-family kinases in cell-free assays at low nanomolar concentrations, completely blocked in vitro neutrophil functions, were orally bioavailable, inhibited in vivo signs of neutrophil, B-cell and T-cell activation, and strongly reduced inflammatory responses in the reverse passive Arthus reaction. Importantly, their effects exceeded that of the clinically used tyrosine kinase inhibitor dasatinib in several in vivo and in vitro assay systems. Further optimization of these molecules are expected to lead to preclinical drug candidates suitable for clinical drug development.
2) Identification of novel anti-inflammatory effects of clinically used Src-family inhibitors. We have revealed unexpectedly diverse novel effects of the anti-leukemic agent dasatinib in various inflammatory disease models. The drug had inhibitory effects on neutrophils, macrophages and osteoclasts in vitro. Its in vivo effects included attenuation of immune complex-induced arthritis and dermatitis, protection from lung damage in LPS-induced acute lung injury and allergic asthma, protection from lethal sepsis and malaria infection, and attenuation of experimental silicosis. Dasatinib also reduced the dose of TNF antagonists required to treat human TNF transgene-induced arthritis in mice. Those results point to interesting potential novel therapeutic indications of dasatinib-related molecules in the future.
3) Identification of novel roles of Src-family kinases in inflammatory diseases. Using gene-targeted mice, we identified a number of novel functions of the myeloid Src-family kinases Hck, Fgr and Lyn, including critical roles in immune complex-induced arthritis and dermatitis and LPS-induced acute lung injury. We have revealed important mechanistic details of their role in inflammation and identified several other components in the downstream signaling pathway.
Taken together, the TARKINAID consortium has developed several novel anti-inflammatory drug candidates and generated major scientific discoveries related to the role of Src-family kinases in inflammatory diseases.
Project Context and Objectives:
Chronic autoimmune and inflammatory diseases such as rheumatoid arthritis or chronic inflammatory lung diseases are severe and devastating conditions that strongly impair the quality of life of the affected individuals and are a major burden to health care budgets and the entire society. Despite significant efforts aimed at the understanding and treating those diseases, current therapeutic options are still very limited due to major side effects and very high costs. Though anti-inflammatory agents are available since the development of aspirin more than 100 years ago, the research aimed at identifying new anti-inflammatory drugs is still ongoing and very active. Although the classical non-steroidal anti-inflammatory drugs (NSAIDs) as well as steroids are very potent in inhibiting acute inflammatory responses, they all have significant side effects which emerge during the long term treatment of chronic inflammatory and autoimmune diseases. The progress in the understanding of the cellular and molecular basis of the inflammatory process is highlighted by a few new strategies aimed to control inflammation. Among these strategies, the most promising approaches include antibodies that bind and inactivate cytokines (TNF, IL-6) or their receptors. However, all those approaches require a parenteral route of drug delivery and have many other limitations including a very high cost, causing a major burden for the national health systems in the developed world. Therefore, development of novel therapeutic strategies for the treatment of chronic autoimmune and inflammatory diseases is a highly important research priority. In particular, the search for small, orally bioavailable drugs is highly needed and deserves major efforts from both the scientific community and the pharmaceutical industry.
Src-family kinases are nonreceptor tyrosine kinases involved in diverse biological functions. Their role in cellular proliferation and invasion, the high frequency of activating mutations in Src-family kinase genes in tumors and the presence of their oncogenic variants in oncogenic viruses indicated a role for those kinases in malignancies. Those findings also triggered significant efforts aimed at therapeutic targeting of Src-family kinases in human cancer and resulted in the development of several small-molecule Src-family kinase inhibitors that showed very positive effect in human oncology patients during the last several years.
Src-family kinases are also involved in various functions of immune cells but their role in inflammatory diseases and the possibility of their therapeutic targeting in those diseases have not yet been widely appreciated. Using genetically modified mice, members of the consortium had previously found that Src-family kinases in leukocytes play an indispensable role in animal models of various chronic autoimmune inflammatory diseases. Members of the consortium had also developed several small molecule inhibitors that strongly inhibited Src-family kinases and also blocked various inflammation-related in vitro functional responses of human leukocytes. Building on those then mainly unpublished studies, the aim of the TARKINAID project was to develop novel small-molecule inhibitors of Src-family kinases for the therapy of chronic autoimmune and inflammatory diseases.
It should be mentioned that targeting components of intracellular signal transduction pathways is not an entirely novel pharmacological approach to treat inflammation. For example, drugs inhibiting the mitogen-activated protein kinase p38 or phosphoinositide 3-kinases (PI3Ks) have been generated and tested both in animals and humans. Tofacitinib, an inhibitor of Jak-family kinases has been approved for use in rheumatoid arthritis patients in the USA (though not yet in Europe). An orally available Syk inhibitor reached late Phase 3 clinical trials, although further progress to marketing development has been halted. On the other hand, targeting Src-family kinases in inflammatory diseases has not yet received wider appreciation.
During the proposed project, small molecule inhibitors of Src-family kinases with in vitro inhibitory effect on leukocytes were further tested on diverse in vitro assay systems, in vivo safety and on a number of in vivo models of inflammation. After several rounds of structural optimization, the most promising lead molecules were subjected to further analysis in animal models of autoimmune and inflammatory diseases. These experiments were expected to result in novel anti-inflammatory molecules with a novel mechanism of action.
Besides the above preclinical drug-development studies, the consortium also planned to perform experiments to test the hypothesis (again supported by our unpublished results) that Src-family inhibitor anti-tumor agents (dasatinib and bosutinib) also exert anti-inflammatory effects. Finally, the consortium aimed to perform additional experiments on existing gene knockout mice to obtain more detailed insight into the role of Src-family kinases in in vivo autoimmune inflammatory diseases.
The consortium members had extensive experience with the development of small molecule kinase inhibitors, inflammation-related in vitro functional studies on leukocytes and the analysis of in vivo inflammatory disease models in experimental animals. The above mentioned preliminary findings provided the “proof of principle” for the consortium’s activities during the preclinical drug development process.
The consortium consisted of eleven members (including four SMEs) from six different countries. To develop a network between European and Brazilian immunologists, two group leaders were from Brazil. To promote research in Eastern-European countries, the coordinator and one SME were from Hungary.
To our knowledge this project has been the first comprehensive approach to validate the use of drugs inhibiting members of the Src-family, a group of intracellular tyrosine kinases acting as signal transducers for different surface receptors in myeloid leukocytes. Using a whole variety of specific chronic and autoimmune disease models including rheumatoid arthritis, autoimmune skin disease, allergic and acute lung inflammation and fibrosis, the project was set out to identify Src- family kinase inhibitors as anti-inflammatory drugs including inhibitors already in use for the treatment of cancer or inhibitors emerging from the planned activities. We also hoped to be able to provide a formal proof that inhibition of Src-family kinases represents a new strategy to control the inflammation process.
The project has been expected to provide potential benefit to biomedical science and society through various routes:
1) Development of entirely novel small-molecule inhibitors as oral anti-inflammatory agents
2) Identification of novel anti-inflammatory indications for the use of Src-family kinase inhibitors already in the clinic for treating malignant diseases
3) Novel understanding of the disease pathogenesis of autoimmune and inflammatory diseases, leading to better diagnosis and therapeutic approaches in the future
4) Capacity-building aspects leading to better trained scientists, novel research infrastructure, novel collaborations and interactions with emerging regions such as Eastern Europe and Brazil.
Project Results:
Within the overall subject of the role of Src-family kinases in inflammation, the three main objectives of the project were:
1) Development of novel Src-family inhibitors with anti-inflammatory properties.
2) Identification of novel anti-inflammatory effects of clinically used Src-family inhibitors.
3) Identification of novel roles of Src-family kinases in inflammatory diseases.
The major achievements towards those three objectives are provided below.
1) DEVELOPMENT OF NOVEL SRC-FAMILY INHIBITORS WITH ANTI-INFLAMMATORY PROPERTIES
The aim of this part of the project was to develop several novel small-molecule Src-family kinase inhibitors that have substantial in vivo anti-inflammatory effect. This part of the project was based on prior studies from two consortium partners (Semmelweis and Vichem) who had previously developed tyrosine kinase inhibitors that inhibited various in vitro functions of human neutrophils. During the TARKINAID project, we have further developed those initial set of molecules and performed substantial structural optimization. We have developed a sublibrary of more than 1000 structural analogs of the initial compounds and selected the best secondary hits based on their in vitro efficacy and patentability features, followed by a further lead optimization strategy. Chemical synthesis, structural manipulation and cell-free kinase assays were performed by Vichem. The biological effect of the initial molecules was screened by several members of the consortium whereas the latest drug candidates (of which only very small quantities were available) have been tested primarily by Semmelweis. The latest drug candidate molecules were structurally novel and patentable, i. e. their structure has not been reported before in the scientific or patent literature.
In general the most effective drug candidates strongly inhibited the Src-family kinases Hck, Fgr, Lyn and Src in cell-free assay systems. They also blocked integrin- and Fc-receptor-mediated functional responses of human neutrophils at submicromolar concentrations but did not have any consistent effect on the migration of neutrophils in in vitro chemotaxis assays. Most tested compounds also reduced osteoclast development and various macrophage functions but did not have consistent effect on macrophage migration and related responses. During the course of several rounds of structural optimization, we have been able to develop small molecule inhibitors that were able to inhibit Src-family kinases in cell-free assays at low nanomolar concentrations. They also inhibited in vitro neutrophil functions at mid-nanomolar concentrations and blocked osteoclast development in a low nanomolar range.
We have also tested the in vivo effect of various novel Src-family kinase inhibitors. We have developed a novel assay to test the in vivo effect of the inhibitors on neutrophils, B-cells and T-cells. Many tested inhibitors had clear inhibitory effects on neutrophils and T-cells and, to a lesser extent, on B-cells. We have also performed additional experiments on inflammatory responses of the novel inhibitors. We have shown that one of the inhibitors attenuated the contact hypersensitivity response in experimental mice. In addition, the most advanced two inhibitors blocked inflammation and edema formation in the reverse passive Arthus reaction. Importantly, most tested inhibitors proved to be orally bioavailable. The effect of several novel compounds surpassed that of the clinically used tyrosine kinase inhibitor dasatinib. It should be mentioned that the most efficacious novel compounds also showed signs of toxicity upon long-term administration at higher doses, indicating that their therapeutic window may be limited and further structural optimization may be needed to reduce potential side effects.
Taken together, the above studies allowed us to develop structurally novel, patentable, orally bioavailable tyrosine kinase inhibitors that showed strong inhibitory effect in cell-free and in vitro cell-based assay systems, as well as in biochemical and functional assays of in vivo inflammation upon oral administration in experimental mice.
2) IDENTIFICATION OF NOVEL ANTI-INFLAMMATORY EFFECTS OF CLINICALLY USED SRC-FAMILY INHIBITORS
The aim of this part of the project was to test whether Src-family kinase inhibitors already used in the clinic for the therapy of imatinib-resistant chronic myeloid leukemia and other applications also have effects on cells of the immune system and the in vivo inflammatory reaction. While most of our efforts were focused on the analysis of dasatinib, a dual inhibitor of Src-family and Abl-family tyrosine kinases, we also performed limited studies on bosutinib, another clinically used Src-family tyrosine kinase inhibitor.
Our first studies on in vitro functional responses of immune cells and related lineages have revealed dramatic effects of dasatinib on various functional responses of human neutrophils including several cellular and biochemical responses triggered by integrin or Fc-receptor ligation. Most of those functions were inhibited by mid-nanomolar concentrations of dasatinib. On the other hand, dasatinib had little effect on the chemotactic activity of human neutrophils towards fMLP or IL-8 (except for studies in Zigmond-chamber assays) and had limited effect on functional responses triggered by other agonists such as various G-protein-coupled receptor agonists or innate immune ligands. Importantly, dasatinib exerted its effect on neutrophils at concentrations, and under conditions, expected to occur during human anti-lekemia therapy and it also inhibited neutrophil functions upon oral administration to experimental mice.
Additional in vitro studies on dasatinib have revealed that it inhibited activation and certain migratory functions of macrophages, and it dramatically reduced osteoclast development at very low nanomolar concentrations.
We have also performed several additional in vivo studies with dasatinib. The drug strongly reduced inflammatory responses in two different readouts of the reverse passive Arthus reaction and reduced arthritis development in the autoantibody-induced K/B×N serum-transfer model and, to a lesser extent, in the autoantibody-induced dermatitis process. Dasatinib showed synergistic effect with TNF antagonists in a human TNF transgene-induced arthritis model, dramatically reducing the effective therapeutic dose of TNF antagonists. This effect was likely due to the fact that dasatinib strongly reduced circulating TNF levels in this model. Dasatinib also protected mice from lung damage in LPS-induced acute lung injury, allergic asthma and experimental silicosis models. In addition, dasatinib strongly improved the survival of septic animals and mice infected with malaria parasites. Though some of those effects were only seen in a narrow concentration range or at higher dasatinib doses, they nevertheless point to exciting potential novel directions of the therapeutic exploitation of Src-family kinase inhibitors in various inflammation-related disease processes.
We have also tested the effect of bosutinib in some of the same assay systems, however that compound had much fewer and weaker effects than dasatinib on the inflammatory processes tested.
3) IDENTIFICATION OF NOVEL ROLES OF SRC-FAMILY KINASES IN INFLAMMATORY DISEASES
The aim of these experiments was to reveal novel functions of Src-family kinases in various in vivo and in vitro models of inflammation and to identify the molecular and cellular mechanisms involved. Most of those experiments were performed using mice genetically deficient for one or more of the Src-family kinases expressed in myeloid-lineage cells, Hck, Fgr and Lyn.
Using a number of in vitro studies, we have found that triple-knockout neutrophils lacking Hck, Fgr and Lyn were strongly defective in their responses to stimulation by immobilized immune complexes or β2 integrin-mediated activation. Defective functional responses included respiratory burst, cell spreading, degranulation, release of chemokines/cytokines and lipid (LTB4) mediators, as well as several intracellular biochemical events. Analysis of single and double knockout neutrophils revealed substantial overlap between Hck, Fgr and Lyn since all three kinases had to be deleted to achieve complete abrogation of the functional responses tested. Double knockout neutrophils lacking Hck and Fgr also showed significant reduction of certain chemokine and cytokine responses upon activation by innate immune ligands such as LPS or Pam3CSK4.
Further analyses revealed that triple knockout macrophages lacking Hck, Fgr and Lyn were also strongly defective in functional responses such as respiratory burst and chemokine/cytokine release. In addition, myeloid Src-family kinases were also involved in certain aspects of macrophage migration, as well as the organization of the macrophage and osteoclast cytoskeleton.
Additional in vivo studies have revealed that Src-family kinases are critically involved in autoantibody-induced arthritis and dermatitis processes, as well as in LPS-induced acute lung injury. Again, analysis of single and double knockout mice revealed substantial functional overlap between Hck, Fgr and Lyn. Src-family kinases were required for the accumulation of myeloid cells at the site of injury in all cases tested during the project. Interestingly, however, this was not due to a cell-autonomous role of Src-family kinases in leukocyte migration but instead to a role for Hck, Fgr and/or Lyn in the release of proinflammatory mediators from neutrophils and/or macrophages to the inflammatory environment. Those studies have revealed a critical role for Src-family kinase in the development of a chemoattractant extracellular milieu during an in vivo inflammation process.
We have also performed additional studies to characterize the molecular pathways acting downstream of Src-family kinases during in vitro and in vivo inflammatory reactions. We have shown that Src-family kinases are critically involved in the phosphorylation of the ITAM tyrosines of the Fc-receptor common γ-chain, providing a molecular link to signaling by Fcγ-receptors. In addition, we have identified the CARD9 adapter molecule as one of the downstream molecules of myeloid Src-family kinases in autoantibody-induced arthritis and dermatitis models. CARD9 acted downstream of Src-family kinases, Syk and PLCγ2 and triggered NFκB activation through IκBα phosphorylation and degradation. CARD9 was required for gene expression changes and chemokine and cytokine but not lipid mediator (LTB4) release from neutrophils and macrophages, indicating that CARD9 acts as a divergence point downstream of Src-family kinases during autoantibody-induced inflammation. Importantly, neutrophil-specific deletion was sufficient to attenuate autoantibody-induced arthiritis and dermatitis, providing the most direct evidence so far for the important role of neutrophil gene expression changes in an in vivo inflammation process.
Taken together, the TARKINAID project resulted in significant and unexpected scientific discoveries related to the role of Src-family kinases in inflammation, which is expected to lead to improved understanding, diagnosis and therapy of inflammatory diseases in the future. The project also allowed us to establish fruitful collaborations between European and Brazilian scientists.
Potential Impact:
POTENTIAL IMPACT
The TARKINAID project intended to fulfil the challenge of the call, i.e. “new therapeutic approaches in chronic inflammatory and autoimmune diseases” focusing on the use of Src family kinase inhibitors (some of which are already in clinical use for the treatment of cancer) as inhibitors of chronic inflammatory pathologies. The strength of our consortium is reflected by the fact that the involved European laboratories provided a major contribution to the understanding of tyrosine kinase signaling in regulating myeloid and other leukocytic cell functions. Another strength of the consortium was the coverage of research expertise necessary to bring a newly developed drug from its initial generation to in vitro and experimental in vivo preclinical animal studies.
Our project also offered the opportunity to achieve another objective of the call, i.e. “..to lead to more links and to closer cooperation between Member States, Associated countries and Brazil..”. Indeed the project includes two Brazilian laboratories whose expertise (chronic lung inflammation and infection) complemented that of the member state laboratories. An added value was the inclusion of three innovative biotech companies that fulfilled different tasks (generation of new anti-kinase drugs, studies of chronic fibrotic inflammation and rheumatoid arthritis) and supplied the consortium with essential disease models and innovative new drugs.
Taken together, the project has been expected to provide potential benefit to biomedical science and society through various routes:
1) Development of entirely novel small-molecule inhibitors as oral anti-inflammatory agents
To this end, we have generated a large number of novel tyrosine kinase inhibitors and tested their effect in cell-free assays, in vitro cellular functional studies and in vivo animal models. Through a constant interaction between the biological assays and chemical synthesis, we have been able to perform several steps of functional optimization. As a result, the final molecules developed as a result of this project showed strong inhibition of various Src-family kinases (Hck, Fgr, Lyn) at low nanomolar concentrations, inhibited various immune cells in cell-based in vitro assays at mid-nanomolar concentrations, were orally bioavailable, showed clear inhibitory effect on leukocytes in vivo and provided inhibitory effect in in vivo models of inflammation. Most of the effects of the developed inhibitors exceeded those of the clinically used tyrosine kinase inhibitor dasatinib. It is expected that further development of the most promising compounds may lead to anti-inflammatory compounds ready for administration in human clinical trials. The most critical aspects of the further development is the reduction of side effect and therefore the widening of the therapeutic window of the drug candidates. After further development, the drug candidates are planned to be submitted for commercial patent applications.
2) Identification of novel anti-inflammatory indications for the use of Src-family kinase inhibitors already in the clinic for treating malignant diseases
To this end, we have performed a very large number of experiments to test the effect of dasatinib and, to a lesser extent, bosutinib on various inflammatory disease models in experimental mice. We have discovered that dasatinib inhibits human neutrophil function even at therapeutic concentrations, and showed that it attenuates the symptoms of autoimmune arthritis, acute lung injury, sepsis, silicosis, asthma and malaria in experimental mice. Those studies may provide important novel information for extending the use of dasatinib and other related molecules to various aspects of inflammatory and inflammation-related diseases. Since dasatinib and bosutinib are extensively covered by pharmaceutical patents, we were not planning to submit patent applications for the coverage of those findings but instead began to share our findings with the scientific community in form of scientific publications to alert the community to the potential additional use of those existing drugs.
3) Novel understanding of the disease pathogenesis of autoimmune and inflammatory diseases, leading to better diagnosis and therapeutic approaches in the future
We have performed a large number of experiments on genetically modified mice to identify the role of Src-family kinases in animal models of chronic autoimmune and inflammatory diseases. We have revealed that Hck, Fgr and Lyn, three Src-family kinases present in myeloid cells play a critical role in autoantibody-induced arthritis and dermatitis models, as well as in the development of acute lung injury, and they also play a major role in atherosclerotic plaque formation. Mechanistically, Src-family kinases were not involved in myeloid cell migration but they were critical for the release of various functional responses of neutrophils and macrophages, most importantly in those related to the release of proinflammatory mediators such as chemokines, cytokines and lipid mediators. Those studies, reported in major scientific journals, strongly improved our understanding of inflammatory disease processes and may have long-term benefit through the development of novel diagnostic and therapeutic strategies.
4) Capacity-building aspects leading to better trained scientists, novel research infrastructure, novel collaborations and interactions with emerging regions such as Eastern Europe and Brazil.
The consortium was led by a scientist in Hungary and one other critical participant was also from Hungary, ensuring the integration of Central-Eastern European countries to the scientific activity of the EU. We have also strongly collaborated with Brazilian partners who have made essential contributions to the analysis of sophisticated lung disease models and various infectious diseases in experimental animals. The extensive interaction between developed and emerging countries strongly contributed to the improvement of scientific activities both within Europe and worldwide.
DISSEMINATION ACTIVITIES
Since the beginning of the project, the dissemination activities were aimed at enhancing visibility of the consortium and of the project, mainly for informing the scientific community about the project objectives and for communicating the value of the research funded by the European Community through FP7.
The main dissemination approaches from the project start included:
1) Development of a Project website
The first edition of the portal on TARKINAID and TIMER projects has been completed at Month 3 and the portal website (EUmBRella website: http://www.eumbrella.org) officially launched. The first edition of the portal has been revised and improved during the whole course of the project. The website has been set up and developed by beneficiary 11 ALTA, who has been in charge for updating it until the end of the project. The portal website includes also the individual TARKINAID website (http://www.eumbrella.org/tarkinaid.html).
The portal EUmBRella website includes:
• Public content for TARKINAID project
• Public content for TIMER project
• Public content focused on the EU-Brazil Research Partnership in Chronic Inflammatory and Autoimmune Diseases
• Dissemination and training
• Link to TRIAD (independent website)
• Link to PODIO social network (restricted area for the participants of TIMER, TARKINAID and TRIAD)
2) Development of Project information material
A Brochure of the project was prepared by beneficiary 11 ALTA, which was distributed at the 15th International Congress of Immunology (22-27 August 2013, Milan, Italy), at the 11th World Congress on Inflammation (21-25 September 2013, Natal, Brazil), at Semmelweis Symposium (7-9 November 2013, Budapest, Hungary), and in occasion of project meetings.
ALTA has also designed the logo of the EUmBRella website and of TARKINAID website (see below) that visually identifies the project and that will be used in occasion of meetings, posters, and any communication activities
3) Project logos
These have been designed by ALTA.
4) Participation at scientific events
The presentation of the TARKINAID results at several national and international congresses and conferences (as listed in Template A2).
5) Press releases
The activation of TARKINAID project has been communicated to the media via publication on the Semmelweis University website. The starting of TARKINAID project has been communicated also via the Biomedcode Hellas SA and Freiburg University website. In additiona a number of press coverage has occurred during major publications arising from the project.
6) Publications/chapter of books/reviews
The publications citing TARKINAID as a funding source have been published during the whole course of the project (as listed in Template A1). A number of those publications appeared in leading scientific journals such as Journal of Experimental Medicine or Nature Communications. Several other papers and reviews are in various stages of the submission and peer review process.
EXPLOITATION ACTIVITIES
Besides the generation of novel scientific knowledge that can be exploited by the wider scientific community, the project has also led to several discoveries that can be directly exploited commercially. The two most directly exploitable results are the following:
1) Development of novel anti-inflammatory drug candidates.
We have developed a number of novel tyrosine kinase inhibitors that have clear potential for commercial exploitation. Those molecules are patentable novel compounds that have not been described in the scientific or patent literature before. They have very strong inhibitory effects on their target molecules (Src-family kinases including Hck, Fgr and Lyn) at low nanomolar concentrations, inhibit inflammatory cell function in vitro, are orally bioavailable, and inhibit in vivo inflammatory processes. We believe that further structural optimization (in particular to reduce potential side effects) will allow us to submit patent applications for their use as anti-inflammatory therapeutic drug candidates in the future.
2) We have developed a novel assay system for the rapid in vivo analysis of the effect of orally administered tyrosine kinase inhibitors without the need for killing the experimental animal. This assay may prove to be useful for the rapid assessment of the efficacy of tyrosine kinase inhibitors in vivo. We have made initial preparatory steps for the patenting this assay as a novel rapid in vivo screening tool.
List of Websites:
PROJECT WEBSITE
The project website is www.eumbrella.org/tarkinaid.html
The TARKINAID website is included within the EUmBRella portal website (www.eumbrella.org) which covers the 3 EU-Brazil research projects (TIMER, TARKINAID, TRIAD) funded under the 7th European Framework Programme (Health 2012 Programme) for Research in the field of chronic inflammatory/autoimmune diseases, within the bilateral S&T cooperation between Europe and Brazil.
CONTRACTORS INVOLVED
1) Semmelweis Egyetem (HUNGARY): Attila Mocsai and Tibor Vantus
2) Ludwig-Maximilians Universität München (GERMANY): Markus Sperandio and Barbara Walzog
3) Università degli Studi di Verona (ITALY): Giorgio Berton
4) Centre National de la Recherche Scientifique (FRANCE): Isabelle Maridonneau-Parini
5) Vichem Chemie Research Ltd. (HUNGARY): Gyorgy Keri
6) Universitätsklinikum Freiburg (GERMANY): Cassian Sitaru
7) Universidade Federal do Rio de Janeiro (BRAZIL): Patricia Rocco
8) Biomedcode Hellas SA (GREECE): Maria Denis
9) Ambiotis SAS (FRANCE): Marc Dubourdeau
10) Fundacao Oswaldo Cruz (BRAZIL): Hugo Castro Faria Neto
11) ALTA Ricerca e Svluppo in Biotecnologie S.r.l.u. (ITALY): Paola Cesaroni
COORDINATOR CONTACT DETAILS
Dr. Attila Mócsai
Professor of Physiology
Department of Physiology
Semmelweis University School of Medicine
Tűzoltó utca 37-47.
1094 Budapest, Hungary
E-mail: mocsai.attila@med.semmelweis-univ.hu
Tel.: +36-1-459-1500 x60-409
Fax: +36-1-266-7480
Chronic autoimmune and inflammatory diseases such as rheumatoid arthritis or chronic inflammatory lung diseases are severe and devastating conditions that strongly impair the quality of life of the affected individuals and are a major burden to health care budgets and the entire society. Despite significant efforts aimed at the understanding and treating those diseases, current therapeutic options are still very limited due to major side effects and very high costs. Therefore, development of novel therapeutic strategies for the treatment of chronic autoimmune and inflammatory diseases is a highly important research priority.
Src-family kinases are nonreceptor tyrosine kinases which are involved in tumor development and progression and therefore are major targets of existing anti-leukemic therapies in the clinic. However, they are also present in immune cells and preliminary findings of the consortium had indicated that they are also involved in the development of inflammatory diseases in experimental animals. The consortium members had also previously developed Src-family kinases inhibitors which inhibited various inflammation-related functions of human leukocytes. Based on those prior studies, the TARKINAID consortium aimed to develop novel anti-inflammatory drug candidates and to improve our understanding of the role of Src-family kinases in the inflammation process. The most significant achievements within the three major aims of the project were as follows:
1) Development of novel Src-family inhibitors with anti-inflammatory properties. We have developed a large number of novel and patentable small molecule inhibitors of Src-family kinases and tested their effect in various inflammation-related in vitro and in vivo assays. After several rounds of structural optimization, we have developed a set of structurally related drug candidates which inhibited Src-family kinases in cell-free assays at low nanomolar concentrations, completely blocked in vitro neutrophil functions, were orally bioavailable, inhibited in vivo signs of neutrophil, B-cell and T-cell activation, and strongly reduced inflammatory responses in the reverse passive Arthus reaction. Importantly, their effects exceeded that of the clinically used tyrosine kinase inhibitor dasatinib in several in vivo and in vitro assay systems. Further optimization of these molecules are expected to lead to preclinical drug candidates suitable for clinical drug development.
2) Identification of novel anti-inflammatory effects of clinically used Src-family inhibitors. We have revealed unexpectedly diverse novel effects of the anti-leukemic agent dasatinib in various inflammatory disease models. The drug had inhibitory effects on neutrophils, macrophages and osteoclasts in vitro. Its in vivo effects included attenuation of immune complex-induced arthritis and dermatitis, protection from lung damage in LPS-induced acute lung injury and allergic asthma, protection from lethal sepsis and malaria infection, and attenuation of experimental silicosis. Dasatinib also reduced the dose of TNF antagonists required to treat human TNF transgene-induced arthritis in mice. Those results point to interesting potential novel therapeutic indications of dasatinib-related molecules in the future.
3) Identification of novel roles of Src-family kinases in inflammatory diseases. Using gene-targeted mice, we identified a number of novel functions of the myeloid Src-family kinases Hck, Fgr and Lyn, including critical roles in immune complex-induced arthritis and dermatitis and LPS-induced acute lung injury. We have revealed important mechanistic details of their role in inflammation and identified several other components in the downstream signaling pathway.
Taken together, the TARKINAID consortium has developed several novel anti-inflammatory drug candidates and generated major scientific discoveries related to the role of Src-family kinases in inflammatory diseases.
Project Context and Objectives:
Chronic autoimmune and inflammatory diseases such as rheumatoid arthritis or chronic inflammatory lung diseases are severe and devastating conditions that strongly impair the quality of life of the affected individuals and are a major burden to health care budgets and the entire society. Despite significant efforts aimed at the understanding and treating those diseases, current therapeutic options are still very limited due to major side effects and very high costs. Though anti-inflammatory agents are available since the development of aspirin more than 100 years ago, the research aimed at identifying new anti-inflammatory drugs is still ongoing and very active. Although the classical non-steroidal anti-inflammatory drugs (NSAIDs) as well as steroids are very potent in inhibiting acute inflammatory responses, they all have significant side effects which emerge during the long term treatment of chronic inflammatory and autoimmune diseases. The progress in the understanding of the cellular and molecular basis of the inflammatory process is highlighted by a few new strategies aimed to control inflammation. Among these strategies, the most promising approaches include antibodies that bind and inactivate cytokines (TNF, IL-6) or their receptors. However, all those approaches require a parenteral route of drug delivery and have many other limitations including a very high cost, causing a major burden for the national health systems in the developed world. Therefore, development of novel therapeutic strategies for the treatment of chronic autoimmune and inflammatory diseases is a highly important research priority. In particular, the search for small, orally bioavailable drugs is highly needed and deserves major efforts from both the scientific community and the pharmaceutical industry.
Src-family kinases are nonreceptor tyrosine kinases involved in diverse biological functions. Their role in cellular proliferation and invasion, the high frequency of activating mutations in Src-family kinase genes in tumors and the presence of their oncogenic variants in oncogenic viruses indicated a role for those kinases in malignancies. Those findings also triggered significant efforts aimed at therapeutic targeting of Src-family kinases in human cancer and resulted in the development of several small-molecule Src-family kinase inhibitors that showed very positive effect in human oncology patients during the last several years.
Src-family kinases are also involved in various functions of immune cells but their role in inflammatory diseases and the possibility of their therapeutic targeting in those diseases have not yet been widely appreciated. Using genetically modified mice, members of the consortium had previously found that Src-family kinases in leukocytes play an indispensable role in animal models of various chronic autoimmune inflammatory diseases. Members of the consortium had also developed several small molecule inhibitors that strongly inhibited Src-family kinases and also blocked various inflammation-related in vitro functional responses of human leukocytes. Building on those then mainly unpublished studies, the aim of the TARKINAID project was to develop novel small-molecule inhibitors of Src-family kinases for the therapy of chronic autoimmune and inflammatory diseases.
It should be mentioned that targeting components of intracellular signal transduction pathways is not an entirely novel pharmacological approach to treat inflammation. For example, drugs inhibiting the mitogen-activated protein kinase p38 or phosphoinositide 3-kinases (PI3Ks) have been generated and tested both in animals and humans. Tofacitinib, an inhibitor of Jak-family kinases has been approved for use in rheumatoid arthritis patients in the USA (though not yet in Europe). An orally available Syk inhibitor reached late Phase 3 clinical trials, although further progress to marketing development has been halted. On the other hand, targeting Src-family kinases in inflammatory diseases has not yet received wider appreciation.
During the proposed project, small molecule inhibitors of Src-family kinases with in vitro inhibitory effect on leukocytes were further tested on diverse in vitro assay systems, in vivo safety and on a number of in vivo models of inflammation. After several rounds of structural optimization, the most promising lead molecules were subjected to further analysis in animal models of autoimmune and inflammatory diseases. These experiments were expected to result in novel anti-inflammatory molecules with a novel mechanism of action.
Besides the above preclinical drug-development studies, the consortium also planned to perform experiments to test the hypothesis (again supported by our unpublished results) that Src-family inhibitor anti-tumor agents (dasatinib and bosutinib) also exert anti-inflammatory effects. Finally, the consortium aimed to perform additional experiments on existing gene knockout mice to obtain more detailed insight into the role of Src-family kinases in in vivo autoimmune inflammatory diseases.
The consortium members had extensive experience with the development of small molecule kinase inhibitors, inflammation-related in vitro functional studies on leukocytes and the analysis of in vivo inflammatory disease models in experimental animals. The above mentioned preliminary findings provided the “proof of principle” for the consortium’s activities during the preclinical drug development process.
The consortium consisted of eleven members (including four SMEs) from six different countries. To develop a network between European and Brazilian immunologists, two group leaders were from Brazil. To promote research in Eastern-European countries, the coordinator and one SME were from Hungary.
To our knowledge this project has been the first comprehensive approach to validate the use of drugs inhibiting members of the Src-family, a group of intracellular tyrosine kinases acting as signal transducers for different surface receptors in myeloid leukocytes. Using a whole variety of specific chronic and autoimmune disease models including rheumatoid arthritis, autoimmune skin disease, allergic and acute lung inflammation and fibrosis, the project was set out to identify Src- family kinase inhibitors as anti-inflammatory drugs including inhibitors already in use for the treatment of cancer or inhibitors emerging from the planned activities. We also hoped to be able to provide a formal proof that inhibition of Src-family kinases represents a new strategy to control the inflammation process.
The project has been expected to provide potential benefit to biomedical science and society through various routes:
1) Development of entirely novel small-molecule inhibitors as oral anti-inflammatory agents
2) Identification of novel anti-inflammatory indications for the use of Src-family kinase inhibitors already in the clinic for treating malignant diseases
3) Novel understanding of the disease pathogenesis of autoimmune and inflammatory diseases, leading to better diagnosis and therapeutic approaches in the future
4) Capacity-building aspects leading to better trained scientists, novel research infrastructure, novel collaborations and interactions with emerging regions such as Eastern Europe and Brazil.
Project Results:
Within the overall subject of the role of Src-family kinases in inflammation, the three main objectives of the project were:
1) Development of novel Src-family inhibitors with anti-inflammatory properties.
2) Identification of novel anti-inflammatory effects of clinically used Src-family inhibitors.
3) Identification of novel roles of Src-family kinases in inflammatory diseases.
The major achievements towards those three objectives are provided below.
1) DEVELOPMENT OF NOVEL SRC-FAMILY INHIBITORS WITH ANTI-INFLAMMATORY PROPERTIES
The aim of this part of the project was to develop several novel small-molecule Src-family kinase inhibitors that have substantial in vivo anti-inflammatory effect. This part of the project was based on prior studies from two consortium partners (Semmelweis and Vichem) who had previously developed tyrosine kinase inhibitors that inhibited various in vitro functions of human neutrophils. During the TARKINAID project, we have further developed those initial set of molecules and performed substantial structural optimization. We have developed a sublibrary of more than 1000 structural analogs of the initial compounds and selected the best secondary hits based on their in vitro efficacy and patentability features, followed by a further lead optimization strategy. Chemical synthesis, structural manipulation and cell-free kinase assays were performed by Vichem. The biological effect of the initial molecules was screened by several members of the consortium whereas the latest drug candidates (of which only very small quantities were available) have been tested primarily by Semmelweis. The latest drug candidate molecules were structurally novel and patentable, i. e. their structure has not been reported before in the scientific or patent literature.
In general the most effective drug candidates strongly inhibited the Src-family kinases Hck, Fgr, Lyn and Src in cell-free assay systems. They also blocked integrin- and Fc-receptor-mediated functional responses of human neutrophils at submicromolar concentrations but did not have any consistent effect on the migration of neutrophils in in vitro chemotaxis assays. Most tested compounds also reduced osteoclast development and various macrophage functions but did not have consistent effect on macrophage migration and related responses. During the course of several rounds of structural optimization, we have been able to develop small molecule inhibitors that were able to inhibit Src-family kinases in cell-free assays at low nanomolar concentrations. They also inhibited in vitro neutrophil functions at mid-nanomolar concentrations and blocked osteoclast development in a low nanomolar range.
We have also tested the in vivo effect of various novel Src-family kinase inhibitors. We have developed a novel assay to test the in vivo effect of the inhibitors on neutrophils, B-cells and T-cells. Many tested inhibitors had clear inhibitory effects on neutrophils and T-cells and, to a lesser extent, on B-cells. We have also performed additional experiments on inflammatory responses of the novel inhibitors. We have shown that one of the inhibitors attenuated the contact hypersensitivity response in experimental mice. In addition, the most advanced two inhibitors blocked inflammation and edema formation in the reverse passive Arthus reaction. Importantly, most tested inhibitors proved to be orally bioavailable. The effect of several novel compounds surpassed that of the clinically used tyrosine kinase inhibitor dasatinib. It should be mentioned that the most efficacious novel compounds also showed signs of toxicity upon long-term administration at higher doses, indicating that their therapeutic window may be limited and further structural optimization may be needed to reduce potential side effects.
Taken together, the above studies allowed us to develop structurally novel, patentable, orally bioavailable tyrosine kinase inhibitors that showed strong inhibitory effect in cell-free and in vitro cell-based assay systems, as well as in biochemical and functional assays of in vivo inflammation upon oral administration in experimental mice.
2) IDENTIFICATION OF NOVEL ANTI-INFLAMMATORY EFFECTS OF CLINICALLY USED SRC-FAMILY INHIBITORS
The aim of this part of the project was to test whether Src-family kinase inhibitors already used in the clinic for the therapy of imatinib-resistant chronic myeloid leukemia and other applications also have effects on cells of the immune system and the in vivo inflammatory reaction. While most of our efforts were focused on the analysis of dasatinib, a dual inhibitor of Src-family and Abl-family tyrosine kinases, we also performed limited studies on bosutinib, another clinically used Src-family tyrosine kinase inhibitor.
Our first studies on in vitro functional responses of immune cells and related lineages have revealed dramatic effects of dasatinib on various functional responses of human neutrophils including several cellular and biochemical responses triggered by integrin or Fc-receptor ligation. Most of those functions were inhibited by mid-nanomolar concentrations of dasatinib. On the other hand, dasatinib had little effect on the chemotactic activity of human neutrophils towards fMLP or IL-8 (except for studies in Zigmond-chamber assays) and had limited effect on functional responses triggered by other agonists such as various G-protein-coupled receptor agonists or innate immune ligands. Importantly, dasatinib exerted its effect on neutrophils at concentrations, and under conditions, expected to occur during human anti-lekemia therapy and it also inhibited neutrophil functions upon oral administration to experimental mice.
Additional in vitro studies on dasatinib have revealed that it inhibited activation and certain migratory functions of macrophages, and it dramatically reduced osteoclast development at very low nanomolar concentrations.
We have also performed several additional in vivo studies with dasatinib. The drug strongly reduced inflammatory responses in two different readouts of the reverse passive Arthus reaction and reduced arthritis development in the autoantibody-induced K/B×N serum-transfer model and, to a lesser extent, in the autoantibody-induced dermatitis process. Dasatinib showed synergistic effect with TNF antagonists in a human TNF transgene-induced arthritis model, dramatically reducing the effective therapeutic dose of TNF antagonists. This effect was likely due to the fact that dasatinib strongly reduced circulating TNF levels in this model. Dasatinib also protected mice from lung damage in LPS-induced acute lung injury, allergic asthma and experimental silicosis models. In addition, dasatinib strongly improved the survival of septic animals and mice infected with malaria parasites. Though some of those effects were only seen in a narrow concentration range or at higher dasatinib doses, they nevertheless point to exciting potential novel directions of the therapeutic exploitation of Src-family kinase inhibitors in various inflammation-related disease processes.
We have also tested the effect of bosutinib in some of the same assay systems, however that compound had much fewer and weaker effects than dasatinib on the inflammatory processes tested.
3) IDENTIFICATION OF NOVEL ROLES OF SRC-FAMILY KINASES IN INFLAMMATORY DISEASES
The aim of these experiments was to reveal novel functions of Src-family kinases in various in vivo and in vitro models of inflammation and to identify the molecular and cellular mechanisms involved. Most of those experiments were performed using mice genetically deficient for one or more of the Src-family kinases expressed in myeloid-lineage cells, Hck, Fgr and Lyn.
Using a number of in vitro studies, we have found that triple-knockout neutrophils lacking Hck, Fgr and Lyn were strongly defective in their responses to stimulation by immobilized immune complexes or β2 integrin-mediated activation. Defective functional responses included respiratory burst, cell spreading, degranulation, release of chemokines/cytokines and lipid (LTB4) mediators, as well as several intracellular biochemical events. Analysis of single and double knockout neutrophils revealed substantial overlap between Hck, Fgr and Lyn since all three kinases had to be deleted to achieve complete abrogation of the functional responses tested. Double knockout neutrophils lacking Hck and Fgr also showed significant reduction of certain chemokine and cytokine responses upon activation by innate immune ligands such as LPS or Pam3CSK4.
Further analyses revealed that triple knockout macrophages lacking Hck, Fgr and Lyn were also strongly defective in functional responses such as respiratory burst and chemokine/cytokine release. In addition, myeloid Src-family kinases were also involved in certain aspects of macrophage migration, as well as the organization of the macrophage and osteoclast cytoskeleton.
Additional in vivo studies have revealed that Src-family kinases are critically involved in autoantibody-induced arthritis and dermatitis processes, as well as in LPS-induced acute lung injury. Again, analysis of single and double knockout mice revealed substantial functional overlap between Hck, Fgr and Lyn. Src-family kinases were required for the accumulation of myeloid cells at the site of injury in all cases tested during the project. Interestingly, however, this was not due to a cell-autonomous role of Src-family kinases in leukocyte migration but instead to a role for Hck, Fgr and/or Lyn in the release of proinflammatory mediators from neutrophils and/or macrophages to the inflammatory environment. Those studies have revealed a critical role for Src-family kinase in the development of a chemoattractant extracellular milieu during an in vivo inflammation process.
We have also performed additional studies to characterize the molecular pathways acting downstream of Src-family kinases during in vitro and in vivo inflammatory reactions. We have shown that Src-family kinases are critically involved in the phosphorylation of the ITAM tyrosines of the Fc-receptor common γ-chain, providing a molecular link to signaling by Fcγ-receptors. In addition, we have identified the CARD9 adapter molecule as one of the downstream molecules of myeloid Src-family kinases in autoantibody-induced arthritis and dermatitis models. CARD9 acted downstream of Src-family kinases, Syk and PLCγ2 and triggered NFκB activation through IκBα phosphorylation and degradation. CARD9 was required for gene expression changes and chemokine and cytokine but not lipid mediator (LTB4) release from neutrophils and macrophages, indicating that CARD9 acts as a divergence point downstream of Src-family kinases during autoantibody-induced inflammation. Importantly, neutrophil-specific deletion was sufficient to attenuate autoantibody-induced arthiritis and dermatitis, providing the most direct evidence so far for the important role of neutrophil gene expression changes in an in vivo inflammation process.
Taken together, the TARKINAID project resulted in significant and unexpected scientific discoveries related to the role of Src-family kinases in inflammation, which is expected to lead to improved understanding, diagnosis and therapy of inflammatory diseases in the future. The project also allowed us to establish fruitful collaborations between European and Brazilian scientists.
Potential Impact:
POTENTIAL IMPACT
The TARKINAID project intended to fulfil the challenge of the call, i.e. “new therapeutic approaches in chronic inflammatory and autoimmune diseases” focusing on the use of Src family kinase inhibitors (some of which are already in clinical use for the treatment of cancer) as inhibitors of chronic inflammatory pathologies. The strength of our consortium is reflected by the fact that the involved European laboratories provided a major contribution to the understanding of tyrosine kinase signaling in regulating myeloid and other leukocytic cell functions. Another strength of the consortium was the coverage of research expertise necessary to bring a newly developed drug from its initial generation to in vitro and experimental in vivo preclinical animal studies.
Our project also offered the opportunity to achieve another objective of the call, i.e. “..to lead to more links and to closer cooperation between Member States, Associated countries and Brazil..”. Indeed the project includes two Brazilian laboratories whose expertise (chronic lung inflammation and infection) complemented that of the member state laboratories. An added value was the inclusion of three innovative biotech companies that fulfilled different tasks (generation of new anti-kinase drugs, studies of chronic fibrotic inflammation and rheumatoid arthritis) and supplied the consortium with essential disease models and innovative new drugs.
Taken together, the project has been expected to provide potential benefit to biomedical science and society through various routes:
1) Development of entirely novel small-molecule inhibitors as oral anti-inflammatory agents
To this end, we have generated a large number of novel tyrosine kinase inhibitors and tested their effect in cell-free assays, in vitro cellular functional studies and in vivo animal models. Through a constant interaction between the biological assays and chemical synthesis, we have been able to perform several steps of functional optimization. As a result, the final molecules developed as a result of this project showed strong inhibition of various Src-family kinases (Hck, Fgr, Lyn) at low nanomolar concentrations, inhibited various immune cells in cell-based in vitro assays at mid-nanomolar concentrations, were orally bioavailable, showed clear inhibitory effect on leukocytes in vivo and provided inhibitory effect in in vivo models of inflammation. Most of the effects of the developed inhibitors exceeded those of the clinically used tyrosine kinase inhibitor dasatinib. It is expected that further development of the most promising compounds may lead to anti-inflammatory compounds ready for administration in human clinical trials. The most critical aspects of the further development is the reduction of side effect and therefore the widening of the therapeutic window of the drug candidates. After further development, the drug candidates are planned to be submitted for commercial patent applications.
2) Identification of novel anti-inflammatory indications for the use of Src-family kinase inhibitors already in the clinic for treating malignant diseases
To this end, we have performed a very large number of experiments to test the effect of dasatinib and, to a lesser extent, bosutinib on various inflammatory disease models in experimental mice. We have discovered that dasatinib inhibits human neutrophil function even at therapeutic concentrations, and showed that it attenuates the symptoms of autoimmune arthritis, acute lung injury, sepsis, silicosis, asthma and malaria in experimental mice. Those studies may provide important novel information for extending the use of dasatinib and other related molecules to various aspects of inflammatory and inflammation-related diseases. Since dasatinib and bosutinib are extensively covered by pharmaceutical patents, we were not planning to submit patent applications for the coverage of those findings but instead began to share our findings with the scientific community in form of scientific publications to alert the community to the potential additional use of those existing drugs.
3) Novel understanding of the disease pathogenesis of autoimmune and inflammatory diseases, leading to better diagnosis and therapeutic approaches in the future
We have performed a large number of experiments on genetically modified mice to identify the role of Src-family kinases in animal models of chronic autoimmune and inflammatory diseases. We have revealed that Hck, Fgr and Lyn, three Src-family kinases present in myeloid cells play a critical role in autoantibody-induced arthritis and dermatitis models, as well as in the development of acute lung injury, and they also play a major role in atherosclerotic plaque formation. Mechanistically, Src-family kinases were not involved in myeloid cell migration but they were critical for the release of various functional responses of neutrophils and macrophages, most importantly in those related to the release of proinflammatory mediators such as chemokines, cytokines and lipid mediators. Those studies, reported in major scientific journals, strongly improved our understanding of inflammatory disease processes and may have long-term benefit through the development of novel diagnostic and therapeutic strategies.
4) Capacity-building aspects leading to better trained scientists, novel research infrastructure, novel collaborations and interactions with emerging regions such as Eastern Europe and Brazil.
The consortium was led by a scientist in Hungary and one other critical participant was also from Hungary, ensuring the integration of Central-Eastern European countries to the scientific activity of the EU. We have also strongly collaborated with Brazilian partners who have made essential contributions to the analysis of sophisticated lung disease models and various infectious diseases in experimental animals. The extensive interaction between developed and emerging countries strongly contributed to the improvement of scientific activities both within Europe and worldwide.
DISSEMINATION ACTIVITIES
Since the beginning of the project, the dissemination activities were aimed at enhancing visibility of the consortium and of the project, mainly for informing the scientific community about the project objectives and for communicating the value of the research funded by the European Community through FP7.
The main dissemination approaches from the project start included:
1) Development of a Project website
The first edition of the portal on TARKINAID and TIMER projects has been completed at Month 3 and the portal website (EUmBRella website: http://www.eumbrella.org) officially launched. The first edition of the portal has been revised and improved during the whole course of the project. The website has been set up and developed by beneficiary 11 ALTA, who has been in charge for updating it until the end of the project. The portal website includes also the individual TARKINAID website (http://www.eumbrella.org/tarkinaid.html).
The portal EUmBRella website includes:
• Public content for TARKINAID project
• Public content for TIMER project
• Public content focused on the EU-Brazil Research Partnership in Chronic Inflammatory and Autoimmune Diseases
• Dissemination and training
• Link to TRIAD (independent website)
• Link to PODIO social network (restricted area for the participants of TIMER, TARKINAID and TRIAD)
2) Development of Project information material
A Brochure of the project was prepared by beneficiary 11 ALTA, which was distributed at the 15th International Congress of Immunology (22-27 August 2013, Milan, Italy), at the 11th World Congress on Inflammation (21-25 September 2013, Natal, Brazil), at Semmelweis Symposium (7-9 November 2013, Budapest, Hungary), and in occasion of project meetings.
ALTA has also designed the logo of the EUmBRella website and of TARKINAID website (see below) that visually identifies the project and that will be used in occasion of meetings, posters, and any communication activities
3) Project logos
These have been designed by ALTA.
4) Participation at scientific events
The presentation of the TARKINAID results at several national and international congresses and conferences (as listed in Template A2).
5) Press releases
The activation of TARKINAID project has been communicated to the media via publication on the Semmelweis University website. The starting of TARKINAID project has been communicated also via the Biomedcode Hellas SA and Freiburg University website. In additiona a number of press coverage has occurred during major publications arising from the project.
6) Publications/chapter of books/reviews
The publications citing TARKINAID as a funding source have been published during the whole course of the project (as listed in Template A1). A number of those publications appeared in leading scientific journals such as Journal of Experimental Medicine or Nature Communications. Several other papers and reviews are in various stages of the submission and peer review process.
EXPLOITATION ACTIVITIES
Besides the generation of novel scientific knowledge that can be exploited by the wider scientific community, the project has also led to several discoveries that can be directly exploited commercially. The two most directly exploitable results are the following:
1) Development of novel anti-inflammatory drug candidates.
We have developed a number of novel tyrosine kinase inhibitors that have clear potential for commercial exploitation. Those molecules are patentable novel compounds that have not been described in the scientific or patent literature before. They have very strong inhibitory effects on their target molecules (Src-family kinases including Hck, Fgr and Lyn) at low nanomolar concentrations, inhibit inflammatory cell function in vitro, are orally bioavailable, and inhibit in vivo inflammatory processes. We believe that further structural optimization (in particular to reduce potential side effects) will allow us to submit patent applications for their use as anti-inflammatory therapeutic drug candidates in the future.
2) We have developed a novel assay system for the rapid in vivo analysis of the effect of orally administered tyrosine kinase inhibitors without the need for killing the experimental animal. This assay may prove to be useful for the rapid assessment of the efficacy of tyrosine kinase inhibitors in vivo. We have made initial preparatory steps for the patenting this assay as a novel rapid in vivo screening tool.
List of Websites:
PROJECT WEBSITE
The project website is www.eumbrella.org/tarkinaid.html
The TARKINAID website is included within the EUmBRella portal website (www.eumbrella.org) which covers the 3 EU-Brazil research projects (TIMER, TARKINAID, TRIAD) funded under the 7th European Framework Programme (Health 2012 Programme) for Research in the field of chronic inflammatory/autoimmune diseases, within the bilateral S&T cooperation between Europe and Brazil.
CONTRACTORS INVOLVED
1) Semmelweis Egyetem (HUNGARY): Attila Mocsai and Tibor Vantus
2) Ludwig-Maximilians Universität München (GERMANY): Markus Sperandio and Barbara Walzog
3) Università degli Studi di Verona (ITALY): Giorgio Berton
4) Centre National de la Recherche Scientifique (FRANCE): Isabelle Maridonneau-Parini
5) Vichem Chemie Research Ltd. (HUNGARY): Gyorgy Keri
6) Universitätsklinikum Freiburg (GERMANY): Cassian Sitaru
7) Universidade Federal do Rio de Janeiro (BRAZIL): Patricia Rocco
8) Biomedcode Hellas SA (GREECE): Maria Denis
9) Ambiotis SAS (FRANCE): Marc Dubourdeau
10) Fundacao Oswaldo Cruz (BRAZIL): Hugo Castro Faria Neto
11) ALTA Ricerca e Svluppo in Biotecnologie S.r.l.u. (ITALY): Paola Cesaroni
COORDINATOR CONTACT DETAILS
Dr. Attila Mócsai
Professor of Physiology
Department of Physiology
Semmelweis University School of Medicine
Tűzoltó utca 37-47.
1094 Budapest, Hungary
E-mail: mocsai.attila@med.semmelweis-univ.hu
Tel.: +36-1-459-1500 x60-409
Fax: +36-1-266-7480