Abstract

TMEM16A/Anoctamin-1 (ANO1) is a protein widely expressed in mammalian tissues and has the properties of the classical calcium-activated chloride channel (CaCC). This protein has been implicated in numerous major physiological functions. However, the lack of effective and selective blockers has hindered a detailed study of the physiological functions of this channel. In this study, we have developed a potent and selective blocker for endogenous ANO1 in Xenopus laevis oocytes (xANO1) using a drug screening method we had previously established (Oh et al., 2008). We have synthesized a number of anthranilic acid derivatives and have determined the correlation between biological activity and the nature and position of substituents in these derived compounds. A structure-activity relationship (SAR) revealed novel chemical classes of xANO1 blockers. The derivatives contain an -NO₂ group on position 5 of a naphthyl group substituted anthranilic acid, and they fully blocked xANO1 chloride currents with an IC₅₀ < 10 μM. The most potent blocker, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA) had an IC₅₀ of 0.08 μM for xANO1. Selectivity tests revealed that other chloride channels such as bestrophin-1, CLC2 and cystic fibrosis transmembrane conductance regulator (CFTR) were not appreciably blocked by 10 - 30 μM MONNA. The potent and selective blockers for ANO1 identified here should permit pharmacological dissection of ANO1/CaCC function and serve as potential candidates for drug therapy of related diseases such as hypertension, cystic fibrosis, bronchitis, asthma and hyperalgesia.