Evaluation of dual potentiality of 2,4,5-trisubstituted oxazole derivatives as aquaporin-4 inhibitors and anti-inflammatory agents in lung cells

Abstract

Inflammation is a multifaceted "second-line" adaptive defense mechanism triggered by exo/endogenous threating stimuli and inter-communicated by various inflammatory key players. Unresolved or dysregulated inflammation in lungs results in manifestation of diseases and leads to irreparable damage. Aquaporins (AQPs) are a ubiquitously expressed superfamily of intrinsic transmembrane water channel proteins that modulate the fluid homeostasis. In addition to their conventional functions, AQPs have clinical relevance to inflammation prevailing under the infectious conditions of various lung diseases and this proclaims them as appropriate biomarkers to be targeted. Hence an endeavor was undertaken to identify potential ligands to target AQP4 for the treatment of lung diseases. Oxazole being a versatile bio-potent core, a series of 2,4,5-trisubstituted oxazoles 3a-j were synthesized by a Lewis acid mediated reaction of aroylmethylidene malonates with nitriles. In silico studies conducted using the protein data bank (PDB) structure 3gd8 for AQP4 revealed that compound 3a would serve as a suitable candidate to inhibit AQP4 in human lung cells (NCI-H460). Further, in vitro studies demonstrated that compound 3a could effectively inhibit AQP4 and inflammatory cytokines in lung cells and hence it may be considered as a viable drug candidate for the treatment of various lung diseases.

Fig. 1. 2D image of interactions between AQP4 and oxazole 3a.

Fig. 2. (A) NCI-H460 cells were treated with different concentrations of LPS for 48 h and cell viability was determined by MTT assay; (B) NCI-H460 cells were treated with 3a at different concentrations for 24 h and IC50 value was found to be 60 μM; (C) effect of 3a at different concentrations (60 μM, 300 μM and 600 μM) on RBC hemolysis. (D) and (E) Effect of LPS on AQP4 gene and protein expression on NCI-H460 cells treated with 30 μg of LPS for 48 h were studied using real time PCR (qRT-PCR) and western blotting; (F) differential expression of cytokines in NCI-H460 cells control (untreated), LPS treated (30 μg, 48 h), 3a treated (60 μM, 24 h) and LPS (30 μg, 48 h) pre-treated with 3a (60 μM, 24 h) groups using real time PCR (q-RTPCR); (G) effect of LPS and 3a in AQP4 gene expression in NCI-H460 cells control (untreated), LPS treated (30 μg, 48 h), 3a treated (60 μM, 24 h) and LPS (30 μg, 48 h) pre-treated with 3a (60 μM, 24 h) groups using real time PCR (q-RTPCR); (H) differential expression of AQP4 and COX-2 protein in NCI-H460 cells treated with LPS (30 μg, 48 h), 3a (60 μM, 24 h), and LPS (30 μg, 48 h) pre-treated with 3a (60 μM, 24 h) using western blotting. All the results were normalized with β-actin as an internal control. Values represent mean ± S.D. for three different experiments. Values are statistically significant at *P < 0.05, **P < 0.01, ***P < 0.001.