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Deregulation of the Tumor Necrosis Factor (TNF) pathway is responsible for the pathological onset of various autoimmune disorders and the perpetuation of chronic inflammation that adversely affects more than 50 million people worldwide. The current standard of care for a broad spectrum of chronic inflammatory conditions consists of using TNF blockers that directly prevent TNFα binding with both TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). However, total TNF signaling inhibition often leads to severe side effects, mainly caused by the impairment of TNFR2-mediated homeostatic signaling. Indeed, achieving the selective inhibition of TNFR1 over TNFR2 would address a challenging, unmet medical need in the field of autoimmunity by mitigating the risk associated with non-selective anti-TNF therapies. My Ph.D project aims at exploring new pharmacological approaches that would exploit the intracellular structural difference between TNFR isoforms, namely the presence of a Death Domain (DD) in TNFR1, to mediate its selective inactivation and/or degradation. This new mechanism of action would prevent the pathological overstimulation of the NF-κB-mediated pathway leading to chronic inflammatory conditions while sparing TNFR2-mediated protective response to avoid undesired off-target effects. No therapeutic agent is currently reported as a selective antagonist of the TNFR1-DD and, considering the lack of information on the presence of potential binding hot spots within this particular region, TNFR1-DD targeting could not be addressed via rational inhibitor drug design. Indeed, we chose to employ electrophilic fragments as covalent probes for the biochemical investigation of TNFR1-DD activity, in the perspective of discovering new druggable sites within unexplored cryptic or shallow pockets. By combining multiple binding affinity techniques for the screening of an in-house fragment library, with organic chemistry synthesis of structural analogs and their in-cell functional characterization (including proximity-based assays, protein NMR and ad-hoc investigational tools of TNFR1-pathway specific components), we discovered low molecular weight molecules (MW