Discovery, Characterization, and Structure–Activity Relationships of an Inhibitor of Inward Rectifier Potassium (Kir) Channels with Preference for Kir2.3, Kir3.X, and Kir7.1

The inward rectifier family of potassium (Kir) channels is comprised of at least 16 family members exhibiting broad and often overlapping cellular, tissue, or organ distributions. The discovery of disease-causing mutations in humans and experiments on knockout mice has underscored the importance of Kir channels in physiology and in some cases raised questions about their potential as drug targets. However, the paucity of potent and selective small-molecule modulators targeting specific family members has with few exceptions mired efforts to understand their physiology and assess their therapeutic potential. A growing body of evidence suggests that G protein-coupled inward rectifier K (GIRK) channels of the Kir3.X subfamily may represent novel targets for the treatment of atrial fibrillation. In an effort to expand the molecular pharmacology of GIRK, we performed a thallium (Tl+) flux-based high-throughput screen of a Kir1.1 inhibitor library for modulators of GIRK. One compound, termed VU573, exhibited 10-fold selectivity for GIRK over Kir1.1 (IC50 = 1.9 and 19 μM, respectively) and was therefore selected for further study. In electrophysiological experiments performed on Xenopus laevis oocytes and mammalian cells, VU573 inhibited Kir3.1/3.2 (neuronal GIRK) and Kir3.1/3.4 (cardiac GIRK) channels with equal potency and preferentially inhibited GIRK, Kir2.3, and Kir7.1 over Kir1.1 and Kir2.1.Tl+ flux assays were established for Kir2.3 and the M125R pore mutant of Kir7.1 to support medicinal chemistry efforts to develop more potent and selective analogs for these channels. The structure–activity relationships of VU573 revealed few analogs with improved potency, however two compounds retained most of their activity toward GIRK and Kir2.3 and lost activity toward Kir7.1. We anticipate that the VU573 series will be useful for exploring the physiology and structure–function relationships of these Kir channels

Genetic Reduction or Negative Modulation of mGlu₇ Does Not Impact Anxiety and Fear Learning Phenotypes in a Mouse Model of <i>MECP2</i> Duplication Syndrome

Rett syndrome and <i>MECP2</i> Duplication syndrome are neurodevelopmental disorders attributed to loss-of-function mutations in, or duplication of, the gene encoding methyl-CpG-binding protein 2 (MeCP2), respectively. We recently reported decreased expression and function of the metabotropic glutamate receptor 7 (mGlu₇) in a mouse model of Rett syndrome. Positive allosteric modulation of mGlu₇ activity was sufficient to improve several disease phenotypes including cognition. Here, we tested the hypothesis that mGlu₇ expression would be reciprocally regulated in a mouse model of <i>MECP2</i> Duplication syndrome, such that negative modulation of mGlu₇ activity would exert therapeutic benefit. To the contrary, we report that mGlu₇ is not functionally increased in mice overexpressing MeCP2 and that neither genetic nor pharmacological reduction of mGlu₇ activity impacts phenotypes that are antiparallel to those observed in Rett syndrome model mice. These data expand our understanding of how mGlu₇ expression and function is affected by changes in MeCP2 dosage and have important implications for the therapeutic development of mGlu₇ modulators

A GRM7 mutation associated with developmental delay reduces mGlu7 expression and produces neurological phenotypes

The metabotropic glutamate receptor 7 (mGlu7) is a G protein–coupled receptor that has been recently linked to neurodevelopmental disorders. This association is supported by the identification of GRM7 variants in patients with autism spectrum disorder, attention deficit hyperactivity disorder, and severe developmental delay. One GRM7 mutation previously reported in 2 patients results in a single amino acid change, I154T, within the mGlu7 ligand-binding domain. Here, we report 2 new patients with this mutation who present with severe developmental delay and epilepsy. Functional studies of the mGlu7-I154T mutant reveal that this substitution resulted in significant loss of mGlu7 protein expression in HEK293A cells and in mice. We show that this occurred posttranscriptionally at the level of protein expression and trafficking. Similar to mGlu7–global KO mice, mGlu7-I154T animals exhibited reduced motor coordination, deficits in contextual fear learning, and seizures. This provides functional evidence that a disease-associated mutation affecting the mGlu7 receptor was sufficient to cause neurological dysfunction in mice and further validates GRM7 as a disease-causing gene in the human population

Eliciting Renal Failure in Mosquitoes with a Small-Molecule Inhibitor of Inward-Rectifying Potassium Channels

<div><p>Mosquito-borne diseases such as malaria and dengue fever take a large toll on global health. The primary chemical agents used for controlling mosquitoes are insecticides that target the nervous system. However, the emergence of resistance in mosquito populations is reducing the efficacy of available insecticides. The development of new insecticides is therefore urgent. Here we show that VU573, a small-molecule inhibitor of mammalian inward-rectifying potassium (Kir) channels, inhibits a Kir channel cloned from the renal (Malpighian) tubules of <i>Aedes aegypti</i> (<i>Ae</i>Kir1). Injection of VU573 into the hemolymph of adult female mosquitoes (<i>Ae. aegypti</i>) disrupts the production and excretion of urine in a manner consistent with channel block of <i>Ae</i>Kir1 and renders the mosquitoes incapacitated (flightless or dead) within 24 hours. Moreover, the toxicity of VU573 in mosquitoes (<i>Ae. aegypti</i>) is exacerbated when hemolymph potassium levels are elevated, suggesting that Kir channels are essential for maintenance of whole-animal potassium homeostasis. Our study demonstrates that renal failure is a promising mechanism of action for killing mosquitoes, and motivates the discovery of selective small-molecule inhibitors of mosquito Kir channels for use as insecticides.</p></div

High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein–Protein Interaction

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in <b>MIV</b>-<b>6<i>R</i></b>, which inhibits the menin–MLL interaction with IC₅₀ = 56 nM. Treatment with <b>MIV</b>-<b>6</b> demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules