6, 725C734 [PMC free article] [PubMed] [Google Scholar] 16. to design a Mevastatin targeted library of small molecules predicted to disrupt the TWEAK-Fn14 conversation. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven study revealing key structural elements of the TWEAK-Fn14 conversation, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor conversation. Our results validate the TWEAK-Fn14 conversation as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers. and invading cells (17). Fn14 expression increases with increasing tumor grade with the highest expression observed in glioblastoma multiforme (grade IV). In contrast, the expression of Fn14 is usually minimal to absent in normal brain tissue. Moreover, TWEAK binding to Fn14 triggers glioma cell invasion and survival (17). TWEAK-Fn14 signaling plays a key role in various disease states and therefore holds significant therapeutic potential as a novel molecular target for developing anti-cancer and anti-autoimmune therapeutic agents in humans. It has been shown that this conversation plays a pivotal role in various immunological conditions like rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, renal injury, ischemic stroke, as well as cardiac dysfunction and failure (18C20). Several studies have confirmed the therapeutic potential Mevastatin of this pathway in human esophageal and pancreatic cancers (21), autoimmune disorders (22), muscle atrophy and injury (23), and chemokine-dependent inflammatory kidney disease (24). The ever increasing knowledge and data on various downstream reactions stimulated by TWEAK-Fn14 conversation has recently been compiled into a complete repository (25). This paves the way for identification of yet unknown components of the signaling Mevastatin pathways. To date, there are five anti-TNF antibody-based drugs already on the market, and 16 out Mevastatin of 22 ligand/receptor pairs under clinical development, constituting one of the most successful classes of biological therapeutics (26). These protein-based therapeutics have some notable disadvantages, including problems associated with drug delivery, stability, and cost. However, very few small molecule inhibitors targeting TNFR family members have been identified. Known small molecule inhibitors for the TNFR family act by disrupting trimerization of their respective ligands, as is the case for TNF (27) and CD40 (28). Benicchi (29) SOCS2 have also focused on the development of a homogeneous time-resolved fluorescence assay for identification of small molecule inhibitors for the TWEAK-Fn14 conversation and reported the identification of hits at a rate of 0.007%. Currently, the potential therapeutic benefit of inhibiting key nodes of the TWEAK-Fn14 signaling pathway remains unclear and untapped due to the absence of small molecule tools to interrogate this pathway. In this study, we initiated the discovery of small molecules targeting the TWEAK-Fn14 pathway by determining the molecular basis of the conversation between TWEAK and Fn14 and elucidating key structural elements of this conversation. The ultimate goal of this work is to employ the structural information on TWEAK-Fn14 binding to identify potential inhibitors of this conversation. The importance of the Fn14 CRD has been established utilizing an NMR answer structure of this domain and functional mutation studies (10). To further characterize the TWEAK-Fn14 conversation, six structural models of TWEAK were built based on experimental structures of low homology templates from the TNF superfamily. Protein-protein docking, followed by data-driven prioritization, yielded two promising TWEAK-Fn14 binding hypotheses. Site-directed mutagenesis confirmed one hypothesis providing a structural basis for target-based identification of small molecule inhibitors of the TWEAK-FN14 Mevastatin conversation. Validated models served as a.
6, 725C734 [PMC free article] [PubMed] [Google Scholar] 16