ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels

Abstract

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based highthroughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of porelining amino acid residues identified Glutamate 149 and Alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC 50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/ SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ionchannels and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CL HEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CL HEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (C max = 0.20 µM and T max = 3 hours) and favorable CNS distribution (mouse brain:plasma K p of 10.9 to support in vivo studies for in vivo studies.

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992

“…Stably transfected monoclonal T-Rex-human embryonic kidney 293 (HEK-293) cell lines expressing Kir1.1, Kir2.2, Kir2.3, Kir4.1, Kir4.2, Kir6.2/sulfonylurea receptor 1 (SUR1), or Kir7.1-M125R from a tetracycline-inducible promoter were generated as described previously (Lewis et al, 2009;Raphemot et al, 2011Raphemot et al, , 2013Swale et al, 2016). To promote cell surface expression and the function of Kir4.2, lysine 110 was mutated to asparagine and the most distal 22 amino acids were deleted from the C terminus (Kir4.2-K110N-D22), as described by Pearson et al (2006).…”

“…Tl 1 flux assays were performed as described previously (Lewis et al, 2009;Raphemot et al, 2011Raphemot et al, , 2013Swale et al, 2016) with modifications to reagents optimized per cell line as noted below. Briefly, cells cultured overnight 37°C and 5% CO 2 in 384-well plates were loaded with the thallium-sensitive dye Thallos-AM (TEFLabs, Austin, TX) in ambient conditions, washing before and after dye using Hanks' balanced salt solution/20 mM HEPES (assay buffer).…”

“…VU0134992 Selectivity. The selectivity of VU0134992 for Kir4.1 versus nine other members of the Kir channel family was evaluated at concentrations ranging from 0.3 nM to 30 mM in 11-point CRC experiments, using established Tl 1 flux assays (Swale et al, 2016). The results are summarized in Table 1.…”

Plight of the pore polar bar(rier)

“…1). This is not unique to VU590 since the T153C mutation also increases Kir7.1 sensitivity to VU714, a 1.5 mM IC 50 Kir7.1 inhibitor described previously by our group that also interacts with E149 and S150 7 . Interestingly, however, the potency of ML418, a higher affinity (IC 50 D 310 nM) analog of VU714, is unaffected by the T153C mutation.…”

“…In a recent HTS of several thousand compounds for small-molecule modulators of Kir7.1, we identified surprisingly few tractable hits (0.2% hit rate) that can be moved forward to lead optimization. 7 Based on the findings described above, we speculate that the pore polar barrier might be at least in part to blame. If this is correct, performing a HTS against the Kir7.1-T153C mutant should yield more chemically tractable hits for optimization.…”