Selective inhibition of the K _ir2 family of inward rectifier potassium channels by a small molecule probe: The discovery, SAR, and pharmacological characterization of ML133

The K _ir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (K _ir1-K _ir7) of structurally related inward rectifier potassium channels are known, and there are no selective small molecule tools to study their function. In an effort to develop selective K _ir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of K _ir2.1 function. Here we report one potent K _ir2.1 inhibitor, ML133, which inhibits K _ir2.1 with an IC ₅₀ of 1.8 μM at pH 7.4 and 290 nM at pH 8.5 but exhibits little selectivity against other members of Kir2.x family channels. However, ML133 has no effect on K _ir1.1 (IC ₅₀ > 300 μM) and displays weak activity for K _ir4.1 (76 μM) and K _ir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the K _ir family reported to date. Because of the high homology within the K _ir2 family-the channels share a common design of a pore region flanked by two transmembrane domains-identification of site(s) critical for isoform specificity would be an important basis for future development of more specific and potent K _ir inhibitors. Using chimeric channels between K _ir2.1 and K _ir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of K _ir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of K _ir1.1 to those of K _ir2.1 (N171D and C175I) transplants ML133 inhibition to K _ir1.1. Together, the combination of a potent, K _ir2 family selective inhibitor and identification of molecular determinants for the specificity provides both a tool and a model system to enable further mechanistic studies of modulation of K _ir2 inward rectifier potassium channels.