Background/Purpose: :  IL17A has been demonstrated to be a key pro-inflammatory cytokine in human rheumatoid arthritis and in several rodent models of arthritis.  Synthetic macrocycles are more amenable to optimization for metabolic stability and oral absorption than biotherapeutics.  The aim of this investigation was to identify high-affinity macromolecule binders of human IL17A, to quantify their inhibitory potency against IL17A-dependent cytokine production in human cells, and to determine if active compounds could inhibit a delayed-type hypersensitivity response in mice. 

Methods: DNA programmed chemistry (DPC) libraries were generated to synthesize in vitro libraries of non-peptidic synthetic macrocycles of molecular weight 600– 1000 kDa.  Compounds binding to immobilized IL17A were identified by PCR and DNA sequencing.  Two compounds were resynthesized and characterized by 1) competitive ELISA to determine affinity for human IL17A, 2) inhibition of IL17A-driven IL-6 production in human rheumatoid arthritis synovial fibroblasts (RASF) and human HT-29 adenocarcinoma cells, 3) inhibition of other pro-inflammatory human cytokine activities, such as IL-1β, IL-6, IL-22, and TNFα, and 4) efficacy in a delayed-type hypersensitivity (DTH) mouse model.  The DTH model used a 1-fluoro-2,4-dinitrobenzene (DNFB) sensitizer, which was applied to the animals at day 0.  On day 7, compounds dissolved in DMSO were dosed by intraperitoneal (i.p.) injection at a dose of 10 mg/kg.  A second application of DNFB was performed on the left ear 30 min after compound dosing.  After 24 hours, left ear edema was measured by change in ear weight compared to the right ear, and levels of INF-γ in ear tissue homogenates were quantified by ELISA. 

Results: Two synthetic macromolecules identified in this investigation, E-34935 and E-35018, were characterized by a competition ELISA with human IL17A, and determined to have a dissociation constant (Kd) = 2 nM.   E-34935 and E-35018 were found to inhibit IL17A with EC50 of 2.0 and 2.1 μM in RASF, and 45 and 20 nM in HT29 cells, respectively.  Both compounds were inactive  (EC50 > 25 μM) in a battery 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% respectively (p < 0.05 vs. vehicle control).   In comparison, a rat anti-mouse IL17A IgG1 (5 mg/kg, i.p.) resulted in 76% inhibition of edema.   INF-γ levels in tissue homogenates were also suppressed by E-34935, E-35018, or anti-IL17A Ab vs. vehicle control by 72%, 62% or 75%, respectively (p < 0.05 for all groups vs. vehicle control group). 

Conclusion: Our data provide evidence that synthetic macrocycles can be identified that bind potently and specifically to human IL17A, and act as inhibitors of IL17A-stimulated IL-6 production in RASF and HT29 cells.  These compounds are also anti-inflammatory in an IL17-directed murine DTH model.   Prior to this investigation, such specific inhibitors of the IL17A-IL17receptor interaction were limited to polypeptides.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-and-characterization-of-synthetic-small-molecule-macrocycle-antagonists-of-human-il17a/