Raavi, ; Quinnell, S. P.; Casiraghi, A.; Leifer, B.; Leu, B.; Stilgenbauer, M.; Wang, F.; Hou, H.; Koehler, A. N.; Vegas, A. J.

Cytokines are key mediators of inflammation and are prominently involved in immune-mediated disorders, playing key roles in the pathogenesis of diseases such as rheumatoid arthritis, asthma, cancer, and systemic lupus erythematosus. Currently, cytokines are a challenging class of protein targets for traditional small-molecule drug discovery efforts. Biologic-based inhibitors have achieved clinical success, but the current suite of biologics therapies is limited, lack oral bioavailability, and have numerous side effects and compliance challenges. The development of small-molecule therapeutics is an attractive alternative that could further expand our therapeutic modulation of these targets. Here, we profiled a panel of 32 disease-relevant human cytokines to identify small-molecule ligands and inhibitors to survey their tractability for small-molecule modulation. Using a binding-first, small-molecule microarray-based approach we probed the binding preferences of each cytokine against a collection of 65,000 drug and lead-like compounds. We have identified 864 key chemical chemotypes that define structural motifs that bias for binding to specific cytokines. We further validated these chemotypes in a thermal denaturation sensitivity assay, resulting in 296 validated cytokine binders. We then prioritized three cytokines and established that novel, first-in-class inhibitors can be identified from these binders with potency ranging from single-digit to double-digit micromolar in reporter cellular assays. Boltz-2 predictions further delineated the binding landscape, underscoring how these inhibitors engage cytokine surfaces with defined structural complementarity. For the first time, our studies show that cytokines are indeed broadly amenable to small-molecule binding and inhibition with key insights into the chemical structures that can enable the inhibition of specific cytokines.