Periodic Reporting for period 1 - SmallDrugRheuma (Discovery of Immune Therapeutic Targets and Immunomodulators for the Development of Novel Therapies in Rheumatoid Arthritis.) Reporting period: 2018-06-29 to 2020-06-28 Summary of the context and overall objectives of the project Rheumatoid Arthritis (RA) is an autoimmune chronic disease, characterized by inflammation in multiple joints, ultimately leading to joint deformity, pain and swelling. A high percentage of patients do not respond to currently available therapies; these mostly constitute of big-sized molecules incapable of crossing cellular membranes, and therefore do not target intracellular components of immune/inflammation-related pathways, which are altered in RA. Therefore, finding new drugs targeting these other RA-related intracellular components would widen the catalogue of therapies available to treat RA, potentially obtaining a higher success rate in patients. The SmallDrugRheuma project aimed to identify novel small drugs for RA, capable of crossing cellular membranes and therefore targeting intracellular components, using a drug-centric, rather than a target-centric, approach; this is, focusing on first identifying a drug causing the desired effect (anti-inflammatory) and only then asking what the intracellular route and target are, to make a more clever use of human, time and monetary resources in drug discovery processes.We developed an in vitro inflammation model in human primary immune cells, and tested on it 3,000 different small molecules (including both approved and novel lead-like drugs), covering the whole chemical space. We identified a portfolio of small molecules capable of reducing the secretion of proinflammatory cytokines while do not altering cell viability (n=25). We validated them in several donors and at different concentrations. The extensive screening effort led to 2 strong candidates (repurposing drugs), which allow for 30-50% reduction of IL-1β and/or TNF-α secretion by inflamed PBMCs. Further, we validated this reduction in cytokine secretion in PBMCs from newly diagnosed RA patients which have not yet received any treatment, and identified the intracellular pathways being targeted. In summary, we identified novel compounds capable of reducing IL-1β and TNF-α secretion in both in vitro inflamed HD PBMCs and PBMCs from naïve RA patients. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far "We first developed an in vitro inflammation model in primary human immune cells. Next, we selected almost 3,000 compounds from our 60,000-compound chemical library as being representatives of the whole chemical space, and then ran a high throughput screen on the inflammation model, identifying 210 compounds capable of interacting with the inflamed cells. After validation and dose-response assays, we analysed potential compound-induced cell death to discard toxic compounds. To address the immunomodulatory potential of the candidates, we evaluated their capabilities of reducing the secretion of proinflammatory cytokines in the model, identifying 25 candidate molecules with previously undescribed immunomodulatory capabilities. As in this project we aimed to identify compounds specifically reducing inflammation, we focused on molecules reducing 3 or 4 proinflammatory cytokines and prepared a ranked list of candidates. We validated ex vivo the top-5 candidates at several concentrations in several donors and repeated the assays, to confirm the results: we observed that compound #6 consistently reduced IL-1β secretion by the in vitro inflammation model, while compound #13 reduced both IL-1β and TNF-α secretion. We validated these findings through an orthogonal assay, including this time also cells from RA patients which have not yet received any treatment (naïve). To interrogate the mechanism of action of these compounds, we treated cells from both HD and naïve RA patients with compound #6 and #13, and analysed them by flow cytometry. Compound #6 impeded the secretion of IL-1β, while compound #13 interfered with its production. Regarding TNF-α, compound #13 interfered with TNF-α production, upstream of secretion. Therefore, we generated a portfolio of novel immunomodulatory compounds, whose characterization and validation for the treatment of human diseases will follow up in further projects, and identified two drugs capable of reducing the secretion of two key components of inflammation processes (IL-1β and TNF-α), which we validated in vitro for the treatment of RA. Regarding the latter, we are preparing two patents that will be submitted soon, together with a publication that will follow the patent applications." 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) Traditionally, therapy development progresses from intensive work in target identification to low-success target-specific drug discovery. This project applies an original and unconventional drug-centric rather than target-centric approach, making use of repurposing and lead-like libraries, through a phenotypic screening designed to specifically identify novel small-molecule immunotherapeutics. The results of the SmallDrugRheuma project represent an advancement beyond the state of the art, by identifying two drugs with novel immunomodulatory properties and, as they are repurposing drugs, they have already proved to be safe for their use in humans and, therefore, their way towards a real-world use in clinic is much shorter, thanks to our unbiased, highly efficient, drug-centric approach. Even more, the portfolio of immunomodulatory compounds constitute a huge resource for further research and innovation for the treatment of inflammation and immune-related pathologies.The work carried out enhance the innovation capacity of the ERA as a whole, as the anti-inflammatory properties of the identified drugs are completely novel and, once protected, will open up a new way for their potential use in RA patients. The results of the project will also step up the development of novel therapies, increasing the potential of citizens to live longer in good health, pushing Europe 2020 objectives. Equally, these results will contribute in the mid-long term towards lessening the impact of RA on EU economy and business, as RA has been recognised by the WHO as a Non-Communicable Disease that can lead to substantial costs for European healthcare.The results also contribute to Innovation Union and ERA priorities, because addresses the challenge “Health, demographic change and wellbeing” by developing novel drugs for a chronic disease. In particular, these results increase European excellence and benefit the scientific community and society at different levels: (i) Contribution towards science, innovation and generation of knowledge, by providing valuable insights into novel drugs will be useful for specialists in Immunology, but also for those working in a broad range of disciplines and diseases (e.g. cancer); (ii) Contribution to public health and welfare, through the identification of novel drugs, opening the doors to preclinical/clinical trials in chronic diseases and by improving bench-to-bed translation, contributing to the Europe 2020 challenge “Health, demographic change and wellbeing”; (iii) Quick transfer of results to the market through the host’s experience in PPP and IPR planning, bringing rapid benefits to citizens and competitiveness gains.