In silico and In vitro Discovery of Plant-Based Pro-Inflammatory Cytokine Inhibitors

Abstract

Purpose: Dry eye disease (DED) is a prevalent condition range between 44.2% to 62.9% of the population, with female sex and aging being the greatest risk factors. The pathogenesis of DED is based on the dysregulation of tear film homeostasis, resulting in a vicious circle of desiccating stress, inflammation, and tissue damage. Mild DED is managed with artificial tears and warm compresses, whereas moderate-to-severe DED require additional anti-inflammatory therapies to break the vicious circle. Anti-inflammatory drugs such as corticosteroids are effective treatments but are associated with adverse side effects including increased risk of cataracts and glaucoma. Discovering novel anti-inflammatory therapeutics with minimal adverse side effects would greatly benefit DED patients who depend on anti-inflammatory treatment to mitigate symptoms. Flavonoids are a class of chemicals found in all vegetation; some are known for their anti-inflammatory effects. The numerous members of the flavonoid class could be leveraged to discover novel inhibitors against inflammatory DED targets. The purpose of this thesis was to discover a novel flavonoid that could act as an anti-inflammatory agent to treat DED.

Methods: This thesis employed a hybrid approach using network analysis, computational modelling, and in vitro validation. Network analysis was employed to select DED-associated protein targets and candidate flavonoids. “Dry Eye Syndrome” proteins were extracted from the STRING-DISEASE database and were cross-referenced with drug databanks using the functional enrichment analysis tool, ToppFun, for flavonoids. A network analysis tool, Cytoscape (v 3.9.1.), was utilized to visualize and rank DED-associated proteins and flavonoids by number of edge interactions. Two proteins were selected for their distinct roles in the immune response, and two flavonoids were selected for their association with multiple pro-inflammatory DED-associated proteins. Molecular docking and molecular dynamic (MD) simulations modelled and simulated the protein-ligand complex of both protein targets with each flavonoid molecule. Four protein-ligand complexes were assessed for stability and specific residue-ligand interactions. The inhibitory effects of both flavonoids were assessed in vitro with ELISAs against both protein targets. The biocompatibility of the most effective flavonoid inhibitor was assessed with human corneal epithelial cells (HCEC) using an Alamar Blue metabolic activity assay; viability was assessed by evaluating the proportion of live, apoptotic, and dead cells.

Results: The network analysis generated a protein-protein interaction network of 64 DED-associated proteins and a drug-protein network of 108 flavonoids. Tumour necrosis factor (TNF) 𝛂 interactions and interleukin (IL) 17A (14 interactions) were amongst the 5 highly ranked proteins and selected for their roles in the innate and adaptive immune response, respectively. Luteolin (19 interactions) and rutin (17 interactions) were the highest ranked flavonoids associated with DED-associated proteins. Molecular docking and MD simulations predicted that both flavonoids could bind directly to the receptor binding sites of both cytokines. However, ELISAs showed that neither luteolin nor rutin could inhibit TNF-𝛂 and TNF receptor interaction (all p≥0.07). On the other hand, both luteolin (IC50 = 16.54 mM) and rutin (IC50 = 8.73 mM) had inhibitory effects against IL-17A and IL-17A receptor interaction (all p<0.01). Metabolic activity and viability of HCEC remained high (all ≥ 85.95%) below 3.0 mM rutin; no significant difference in metabolic activity and viability were detected in all concentrations of rutin compared to the control (all p>0.06).

Conclusion: This thesis identified rutin as a novel flavonoid inhibitor of IL-17A. These findings suggest that rutin may play a role in anti-inflammatory therapeutics for DED.