Brandon M. Brown

New Positive Ca2+-Activated K+ Channel Gating Modulators with Selectivity for KCa3.1

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both KCa2 and KCa3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate KCa2.2 and KCa2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for KCa3.1 (EC50 111 ± 27 nM) over KCa2.3 (EC50 13.7 ± 6.9 μM), and SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 (EC50 109 nM ± 14 nM) over KCa2.3 (EC50 4.4 ± 1.6 μM). Both compounds are 200- to 400-fold selective over representative KV (KV1.3, KV2.1, KV3.1, and KV11.1), NaV (NaV1.2, NaV1.4, NaV1.5, and NaV1.7), as well as CaV1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of KCa3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in KCa3.1−/− mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a KCa3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of KCa3.1 activation.

Dyclonine rescues frataxin deficiency in animal models and buccal cells of patients with Friedreich's ataxia

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreichs ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron–sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA.

A novel pan-negative-gating modulator of KCa2/3 channels, fluoro-di-benzoate, RA-2, inhibits endothelium-derived hyperpolarization-type relaxation in coronary artery and produces bradycardia in vivo

Small/intermediate conductance KCa channels (KCa2/3) are Ca2+/calmodulin regulated K+ channels that produce membrane hyperpolarization and shape neurologic, epithelial, cardiovascular, and immunologic functions. Moreover, they emerged as therapeutic targets to treat cardiovascular disease, chronic inflammation, and some cancers. Here, we aimed to generate a new pharmacophore for negative-gating modulation of KCa2/3 channels. We synthesized a series of mono- and dibenzoates and identified three dibenzoates [1,3-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate) (RA-2), 1,2-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate), and 1,4-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate)] with inhibitory efficacy as determined by patch clamp. Among them, RA-2 was the most drug-like and inhibited human KCa3.1 with an IC50 of 17 nM and all three human KCa2 subtypes with similar potencies. RA-2 at 100 nM right-shifted the KCa3.1 concentration-response curve for Ca2+ activation. The positive-gating modulator naphtho[1,2-d]thiazol-2-ylamine (SKA-31) reversed channel inhibition at nanomolar RA-2 concentrations. RA-2 had no considerable blocking effects on distantly related large-conductance KCa1.1, Kv1.2/1.3, Kv7.4, hERG, or inwardly rectifying K+ channels. In isometric myography on porcine coronary arteries, RA-2 inhibited bradykinin-induced endothelium-derived hyperpolarization (EDH)–type relaxation in U46619-precontracted rings. Blood pressure telemetry in mice showed that intraperitoneal application of RA-2 (≤100 mg/kg) did not increase blood pressure or cause gross behavioral deficits. However, RA-2 decreased heart rate by ≈145 beats per minute, which was not seen in KCa3.1−/− mice. In conclusion, we identified the KCa2/3–negative-gating modulator, RA-2, as a new pharmacophore with nanomolar potency. RA-2 may be of use to generate structurally new types of negative-gating modulators that could help to define the physiologic and pathomechanistic roles of KCa2/3 in the vasculature, central nervous system, and during inflammation in vivo.

Development of a QPatch Automated Electrophysiology Assay for Identifying KCa3.1 Inhibitors and Activators

The intermediate-conductance Ca(2+)-activated K(+) channel KCa3.1 (also known as KCNN4, IK1, or the Gárdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca(2+) sensitivity of KCa3.1 was studied using varying intracellular Ca(2+) concentrations. A free Ca(2+) concentration of 1 μM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca(2+) concentration (∼200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121)

Intermediate-conductance (KCa3.1) and small-conductance (KCa2) calcium-activated K+ channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing KCa activators, such as naphtho[1,2-d]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity and identified 5-methylnaphtho[2,1-d]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for KCa3.1 over KCa2.3. To understand why introduction of a single CH3 group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for KCa3.1 over KCa2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the KCa3.1 and KCa2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type KCa activators bind relatively “deep” in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In KCa3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more “forward” and directly hydrogen bonds with R362 in KCa3.1. Mutating R362 to serine, the corresponding residue in KCa2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of KCa activators on KCa3.1. The increase in SKA-121’s KCa3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on KCa3.1 and M72 on CaM at the KCa3.1–CaM-BD/CaM interface.

Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimers disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

Kca3.1 channel modulators as potential therapeutic compounds for glioblastoma

Background The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is widely expressed in cells of the immune system such as T- and B-lymphocytes, mast cells, macrophages and microglia, but also found in dedifferentiated vascular smooth muscle cells, fibroblasts and many cancer cells including pancreatic, prostate, leukemia and glioblastoma. In all these cell types KCa3.1 plays an important role in cellular activation, migration and proliferation by regulating membrane potential and Ca2+ signaling. Methods and Results KCa3.1 therefore constitutes an attractive therapeutic target for diseases involving excessive proliferation or activation of one more of these cell types and researchers both in academia and in the pharmaceutical industry have developed several potent and selective small molecule inhibitors of KCa3.1. This article will briefly review the available compounds (TRAM-34, senicapoc, NS6180), their binding sites and mechanisms of action, and then discuss the potential usefulness of these compounds for the treatment of brain tumors based on their brain penetration and their efficacy in reducing microglia activation in animal models of ischemic stroke and Alzheimer’s disease. Conclusion Senicapoc, which has previously been in Phase III clinical trials, would be available for repurposing, and could be used to quickly translate findings made with other KCa3.1 blocking tool compounds into clinical trials.

Are there superagonists for calcium-activated potassium channels?

Similar to GABAA receptor-channels the calciummediated gating of the small-conductance KCa2 and the intermediate-conductance KCa3.1 channels can be positively or negatively modulated by small molecule drugs, which, in analogy to the GABA field, have been termed positive (PAM) or negative allosteric modulators. While positive gating modulators like EBIO, NS309, SKA-31 and SKA-121 shift the calciumresponse curve of these voltage-independent, calmodulin-gated channels to the left and apparently increase their sensitivity to calcium, negative gating modulators decrease calcium sensitivity. However, in contrast to GABAA receptors, where the binding site for the endogenous ligand GABA is located on the extracellular side and where allosteric modulation by benzodiazepines, neurosteroids and barbiturates has been studied in exquisite detail, only a small number of studies have been performed for KCa channels. One reason is of course the lower level of pharmacological interest. While GABAA receptors are firmly established as clinically used drug targets, no KCa2 or KCa3.1 channel modulators have yet reached the clinic despite their undeniable therapeutic potential for neurological, cardiovascular and inflammatory diseases. Another reason is the technical challenge involved in studying KCa channel gating. The gating apparatus is located at the intracellular C-terminus, where calmodulin, which functions as a calcium-sensing b-subunit, is constitutively associated with the calmodulin binding domain of the channels, necessitating the performance of inside-out patch-clamp recordings when aiming to work at defined intracellular calcium concentrations. Nevertheless, a few studies, including some exquisite X-ray crystallography, have been performed and it is currently hypothesized that KCa channel PAMs bind at the interface between the calmodulin N-lobe and the calmodulin-binding domain of the channels and thus “facilitate” mechanical opening ( D increased open channel probability) at a given Ca2C concentration. Both benzimidazole-type activators like EBIO and NS309 and naphthothiazole/oxazole-type activators like SKA-31 and SKA-121 (Fig. 1) have been shown to bind in this interface pocket either through co-crystallization of calmodulin in complex with the calmodulin-binding domain of KCa2.2, 3,4 or, more recently, by our own group using a combination of electrophysiology and site-directed mutagenesis. The latter study was guided by homology modeling of the KCa2.3 and KCa3.1 interface pocket and docking studies using the RosettaLigand computational modeling software. While the crystallography studies afforded the first insight into the atomistic mechanism of action of KCa activators, our molecular modeling study provides a plausible explanation for why KCa channel activators in general are 5–10-fold more potent in activating KCa3.1 than KCa2 channels. 5 The presence of R362 creates an extensive “background” hydrogen-bond network in the KCa3.1 interface pocket that stabilizes the main contacts NH2-substituted KCa activators make with M51 and E54 in calmodulin (Fig. 1). The three KCa2 channels have shorter N or S residues in the corresponding position and therefore cannot form this hydrogen-bond network. The Rosetta models further suggested an explanation for why the 5-position methyl substituted SKA-121 is more potent on KCa3.1