In vitro evaluation of promiscuity and toxicity of a small molecule targeting wild and T164I β2-adrenergic receptors

Inhaled β2 agonists form the first-line treatment for bronchial asthma due to their superior bronchodilator effects. In our previous studies, we have identified a significant association of Thr164Ile (T164I) polymorphism (C491T, rs1800088) in the β2 Adrenergic Receptor (β2AR) with refractoriness to β2 agonists like salbutamol. Utilizing molecular modeling approaches, we have demonstrated the suboptimal binding of salbutamol as a plausible rationale behind the refractoriness observed in asthmatics. Furthermore, our high throughput virtual screening studies led to the identification of high-affinity agonist, (5-[[2-[4-(4-hydroxyphenyl)-3,6-dihydro-2H-pyridin-1-yl]acetyl]amino]benzene-1,3-dicarboxamide), referred to as "CACPD2011a-0001278239" in the Phase database. This compound exhibited promiscuous and high-affinity binding to both the wild type and T164I β2AR variants. The present study aims to evaluate the efficacy and toxicity of CACPD2011a-0001278239 in In vitro systems. Promiscuous and high-affinity properties of CACPD2011a-0001278239 were assessed in MRC-5 lung fibroblasts expressing wild β2AR and T164Iβ2AR transfected CHW-1102 cells. In both cells, CACPD2011a-0001278239 exhibited superior potency with appreciable EC₅₀ values compared to salbutamol, indicating its promiscuity and superior binding potential for β2AR isoforms. Further, toxicity studies of CACPD2011a-0001278239 were conducted by assessing the cytotoxicity (MTT assay), mutagenicity (reverse-mutation assay), and genotoxicity (chromosomal aberrations and micronucleus test). In all these investigations, CACPD2011a-0001278239 demonstrated non-cytotoxic, non-mutagenic, and non-genotoxic properties. In conclusion, the present biological investigations confirm that CACPD2011a-0001278239 possesses high affinity and promiscuous binding properties while exhibiting a favorable safety profile. These findings suggest CACPD2011a-0001278239 as a potential promiscuous agonist for addressing refractoriness in asthmatics, regardless of whether they harbor wild type or T164I β2AR variant.

Bronchial asthma; Promiscuity testing; T164I β2AR; Toxicity assessment; β2 agonist.