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

Abstract

The Kir inward rectifying potassium channels have a broad tissue distribution and are implicated in a variety of functional roles. At least seven classes (Kir1 – Kir7) 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 Kir2.1 inhibitors, we performed a high-throughput screen (HTS) of more than 300,000 small molecules within the MLPCN for modulators of Kir2.1 function. Here we report one potent Kir2.1 inhibitor, ML133, which inhibits Kir2.1 with IC50 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 Kir1.1 (IC50 > 300 μM), and displays weak activity for Kir4.1 (76 μM) and Kir7.1 (33 μM), making ML133 the most selective small molecule inhibitor of the Kir family reported to date. Due to the high homology within the Kir 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 Kir inhibitors. Using chimeric channels between Kir2.1 and Kir1.1 and site-directed mutagenesis, we have identified D172 and I176 within M2 segment of Kir2.1 as molecular determinants critical for the potency of ML133 mediated inhibition. Double mutation of the corresponding residues of Kir1.1 to those of Kir2.1 (N171D and C175I) transplants ML133 inhibition to Kir1.1. Together, the combination of a potent, Kir2 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 Kir2 inward rectifier potassium channels.

The K + channel K IR 2.1 functions in tandem with proton influx to mediate sour taste transduction

“…Moreover, we observed an increase in the rate of inhibition by ML133 when the pH of the extracellular solution was raised to 8.5, as previously described for K IR 2.1 ( Fig. S2) (34). Thus, based on sensitivity to Ba 2+ , Cs + , and ML133, we conclude that the resting K + current in PKD2L1 cells is mediated by K IR 2.1.…”

“…3 C and D), but considerably different from the IC 50 of either K IR 2.2 or K IR 4.2 (0.23 ± 0.06 and 5.8 ± 0.8 μM, respectively; P < 0.0001 and P < 0.01 by one-way ANOVA followed by Tukey's multiple-comparison test). Finally, we tested the K IR 2-specific blocker ML133, which has a reported IC 50 of 1.9 μM for K IR 2.1 (34). ML133 (50 μM) blocked the resting K + current in PKD2L1 cells by ∼90%, similar to its effect on K IR 2.1 and K IR 2.2, whereas K IR 4.2 was virtually insensitive to ML133 ( Fig.…”

The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver

“…ML133 binds to residues on the inner face of the pore-lining M2 helix and presumably blocks the K ϩ permeation pathway at that level (54). In the same conditions we employed ([K ϩ ] o ϭ 140 mM, V m ϭ Ϫ100 mV, and pH 7.4), ML133 inhibits Kir2 channels with K 1/2 values in the range of ϳ2-4 M, while other Kir subfamilies (Kir1, Kir4, Kir6, and Kir7) exhibit K 1/2 values of ϳ8 M or higher (54).…”

Cardiac and renal inward rectifier potassium channel pharmacology: emerging tools for integrative physiology and therapeutics

“…Min Li and colleagues took a modern drug discovery approach to develop a potent and selective small-molecule inhibitor of Kir2.1 [28]. Using a fluorescence-based thallium (Tl + ) flux assay [29,30], the investigators screened approximately 300,000 structurally diverse small molecules from the NIH Molecular Libraries Small-Molecule Repository for chemical modulators of Kir2.1.…”