Se rendre au contenu

Identfication and Characterization of Synthetic Small Molecule Macrocycle Antagonists of Human IL-17A David Livingston, Sethu Alexander, Julian Bond, Timothy Briggs, Andrew Fraley, Stephen Hale, Tanya Landsman, Richard Mar2nelli, Kelley Shortsleeves, Nick TerreQ and Nathan Walsh

Background/Purpose: IL-17A has been demonstrated to be a key pro-inflammatory cytokine in human rheumatoid arthriFs and in several rodent models of arthriFs. SyntheFc macrocycles are more amenable to opFmizaFon for metabolic stability and oral absorpFon than biotherapeuFcs. The aim of this invesFgaFon was to idenFfy high-affinity macrocycle binders of human IL-17A, to quanFfy their inhibitory potency against cytokine producFon in human cells, and to determine if acFve compounds could inhibit a delayed-type hypersensiFvity response in mice. Methods: DNA programmed chemistry (DPC) libraries were generated to synthesize in vitro libraries of non-pepFdic syntheFc macrocycles of molecular weight 600– 1000 kDa. Compounds binding to immobilized IL-17A were idenFfied by PCR and DNA sequencing. Two compounds were resynthesized and characterized by 1) compeFFve ELISA to determine affinity for human IL-17A, 2) inhibiFon of IL-17A-driven IL-6 producFon in human rheumatoid arthriFs synovial fibroblasts (RASF) and human HT-29 adenocarcinoma cells, 3) inhibiFon of other proinflammatory human cytokine acFviFes, such as IL-1β, IL-6, IL-22, and TNFα, and 4) efficacy in a delayed-type hypersensiFvity (DTH) mouse model. The DTH model used a 1-fluoro-2,4- dinitrobenzene (DNFB) sensiFzer, which was applied to the animals at day 0. On day 7, compounds dissolved in DMSO were dosed by intraperitoneal (i.p.) injecFon at a dose of 10 mg/ kg. A second applicaFon of DNFB was performed on the lei ear 30 min aier compound dosing. Aier 24 hours, lei ear edema was measured by change in ear weight compared to the right ear, and levels of INF-γ in ear Fssue homogenates were quanFfied by ELISA. Results: Two syntheFc macrocycles idenFfied in this invesFgaFon, E-34935 and E-35018, were characterized by a compeFFon ELISA with human IL-17A, and determined to have a dissociaFon constant (Kd) = 2 nM. E-34935 and E-35018 were found to inhibit IL-17A with EC50 of 2.0 and 2.1 μM in RASF, and 45 and 20 nM in HT29 cells, respecFvely. Both compounds were inacFve (EC50 > 25 μM) in a banery of cellular assays for the human cytokines IL-1β, IL-6, IL22, and TNFα. A single i.p. dose of 10 mg/kg of E-34935 or E-35018 in the murine DTH model suppressed edema vs. vehicle control by 50 or 54% respecFvely (p < 0.05 vs. vehicle control). In comparison, a rat anF-mouse IL-17A IgG1 (5 mg/kg, i.p.) resulted in 76% inhibiFon of edema. INF-γ levels in Fssue homogenates were also suppressed by E-34935, E-35018, or anF-IL-17A Ab vs. vehicle control by 72%, 62% or 75%, respecFvely (p < 0.05 for all groups vs. vehicle control group). Conclusion: Our data provide evidence that syntheFc macrocycles can be idenFfied that bind potently and specifically to human IL-17A, and act as inhibitors of IL-17A-sFmulated IL-6 producFon in RASF and HT29 cells. These compounds are also anF-inflammatory in an IL-17- directed murine DTH model. Prior to this invesFgaFon, such specific inhibitors of the IL-17AIL17receptor interacFon were limited to polypepFdes.