Abstract
Purpose :
A safe, fast-acting, and effective non-corticosteroid eye drop for long-term treatment of dry eye disease (DED) and other chronic inflammation-associated eye conditions represents an unmet medical need in ophthalmology. Inhibition of the Janus Kinase (JAK) signaling pathway has been demonstrated to attenuate ocular inflammation in multiple non-clinical models. Herein, we describe a small molecule drug discovery effort to identify a novel, potent, selective, and water-soluble series of JAK inhibitors as immunomodulating/anti-inflammatory agents optimized for topical ocular disposition.
Methods :
Test compounds were evaluated for activity against the JAK enzyme family and a panel of kinases identified to predict promiscuity across the human kinome. Evaluation of IL-6-stimulated STAT3 inhibition was conducted using HEK-Blue IL-6 cells (InvivoGen). Generation of micronuclei was examined as a measure of cytotoxicity using TK6 cells via flow cytometry. Compounds were screened for kinome selectivity at 500 nM. Intrinsic solubility and pKa values were measured.
Results :
A focused library comprised of known 3-(4-(2-(arylamino)pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitriles was constructed and evaluated as a molecular starting point. Structure—activity relationships (SARs) revealed a ligand efficient (LE) and potent JAK inhibitor series with structural features amenable to aqueous solubility. However, subsequent in vitro analysis revealed intrinsic series-associated cytotoxicity. The KINOMEscan™ selectivity profile of 4 indicated the likelihood of widespread series affinity throughout kinases in the human genome, a finding highly correlated with toxicological effects. To mitigate off-target promiscuity, an sp2-to-sp3 structure-guided drug design strategy was used to delete aromatic rings, increase fraction sp3 (Fsp3), and bolster molecular complexity. This led to the discovery of 1.
Conclusions :
Compound 1 demonstrates on-target cellular potency (STAT3(IL-6) IC50 = 162 nM), excellent aqueous solubility (Aq. Sol. = 24904 µM, pH = 6), minimal off-target kinase activity (S(35) = 0.055), and no observable cytotoxicity in the micronucleus assay (3xMN ≥ 50 μM). Based on these attributes—in conjunction with favorable “drug-like” properties and metrics (LE = 0.41)—1 was selected as a lead candidate for further evaluation and optimization.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.