Within the framework of work package 1 of the project, we developed new synthetic pathways for the parallel synthesis of modifies analogues of our initial leads. We conducted in vitro and ex vivo tests to assess the effectiveness of optimized derivatives on activated fibroblasts as well as macrophages, leading to the identification of two predominant advanced lead compounds. These compounds significantly reduced the levels of inflammatory chemokines at both a preventive and therapeutic level, greatly enhancing the action of the initial compound without inducing cytotoxicity. We also established predictive models of quantitative structure-activity relationships (SAR), which allowed us to predict the binding mode of the most active derivatives to target proteins. Finally, we examined the ADMET properties (Absorption, Distribution, Metabolism, Excretion, Toxicity) of representative compounds to study the mechanism of action of this series and for in vivo studies.

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In the context of work package 2, our focus was on studying the mode of action (MoA) of the new compounds using chemoproteomics and gene expression analysis (RNA sequencing) to identify the their molecular targets of these compounds in arthritic fibroblasts and investigate the involved molecular pathways. From the chemoproteomic analysis of the initial compound GKL09, five potential targets emerged, with two of them being confirmed as the predominant targets through validation experiments. Similarly, for compounds 18 and 19, we identified two possible protein targets.

 

In work package 3, we performed the in vivo evaluation of advanced leads prioritised from in vitro/cellular assays of  WP1 and 2, to assess their pharmacological properties and efficacy as effective inhibitors of pathological mechanisms and/or the action of the target proteins. The limited quantities of experimental drugs and the requirement for long-term experiments led to the use of short-duration protocols employing induced animal models such as the DSS colitis model, CAIA arthritis model, and DTH cellular immunity mechanism control model. In these models, a restricted number of compounds were tested, and therapeutic effects were observed in the case of DSS colitis.

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Based on these very encouraging results, we proceeded to secure the intellectual property rights related to the selection, synthesis, and use of the new molecules as active substances that suppress the pathogenic activation of fibroblasts and as potential drugs for the treatment of chronic inflammatory and fibrotic diseases. The novel family of small molecules developed in Drug.Art has significant commercial interest for the pharmaceutical industry as it introduces a new pharmacological approach to chronic inflammatory and fibrotic diseases by specifically targeting activated, pathogenic fibroblasts. We are currently promoting the commercial exploitation of this patent with the aim to ultimately introduce a new drug against inflammation and fibrosis.