The Novel Diazoxide Analog 3-Isopropylamino-7-Methoxy-<b>
                     <i>4H</i>
                  </b>-1,2,4-Benzothiadiazine 1,1-Dioxide Is a Selective Kir6.2/SUR1 Channel Opener

Dabrowski, Michael; Ashcroft, Frances M.; Ashfield, Rebecca; Lebrun, Philippe; Pirotte, Bernard; Egebjerg, Jan; Hansen, John Bondo; Wahl, Philip

doi:10.2337/diabetes.51.6.1896

Cited by 34 publications

(38 citation statements)

References 65 publications

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“…Nonetheless, direct competitive inhibition is unlikely because of the inability of increasing concentrations of NNC55-0462 to overcome the reduced efficacy caused by syntaxin-1A. The SUR1 domains critical to the actions of a closely related compound, NNC55-9216, were mapped out to transmembrane domains 8 -11 and both nucleotide binding folds (24). Because syntaxin-1A can bind both nucleotide binding folds of SUR1 (33,34), such interactions may induce negative allosteric modulation onto the K ATP channel opener binding sites of SUR1, thereby decreasing the K ATP channel opener binding affinity.…”

Section: Discussionmentioning

confidence: 99%

“…HEK-293 cells stably expressing human Kir6.2/SUR1 (BA8 cells) have been previously characterized (24,27), and cell culture and transfection were performed as previously described (33)(34)(35). For a detailed description, refer to the supplementary appendix, available online at http://dx.doi.org/10.2337/db07-0030.…”

Section: Methodsmentioning

confidence: 99%

“…K ATP channel openers have negative allosteric effects on glibenclamide binding to SUR (36,37). In fact, such SUR1-specific K ATP channel openers could displace 3 H-glibenclamide binding (24), which, taken along with the high affinity of NNC55-0462 for SUR1, led us to use a higher concentration of tolbutamide to inhibit the channels.…”

Section: Syntaxin-1a Action On Nnc55-0462 On Sur1mentioning

confidence: 99%

“…Recently, a class of islet ␤-cell-specific K ATP channel openers (thiadiazine dioxides) being developed (24 -27) have been shown to be capable of inducing ␤-cell rest in rodent (10,28,29) and human islets (30,31), improving insulin stores and secretory responses, and protecting against autoimmune injury in an animal model of type 1 diabetes (32). These ␤-cell-specific K ATP channel openers interact with cytoplasmic and transmembrane domains of the SUR1 subunit and displace 3 H-glibenclamide binding to HEK cells stably expressing human Kir6.2/SUR1 (BA8 cells) (24,27). Their precise molecular mechanism of action, however, remains unknown (13).…”

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confidence: 99%

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The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Kang

Elias

et al. 2007

Diabetes

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Islet ␤-cell-specific ATP-sensitive K ؉ (K ATP ) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in ␤-cells to inhibit K ATP channels. As a strategy to elucidate the molecular mechanism of action of these K ATP channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet ␤-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K ATP channels previously activated by NNC55-0462. This action on K ATP channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K ATP channel openers NNC55-0462 and diazoxide on K ATP channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K ATP channel openers. Diabetes

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Syntaxin-1a Action On Nnc55-0462 On Sur1mentioning

confidence: 99%

mentioning

confidence: 99%

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The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Kang

Elias

et al. 2007

Diabetes

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“…21 DMEM with supplements as above plus 44 µg/mL hygromycin and 0.6 mg/mL G418 as selection medium was used for the HEK 293 cells with recombinant expression of human Kir6.2/SUR1. 22 All cell lines were cultured at 37°C, 5% CO 2 in humidified air using standard culture ware from Nunc A/S, Denmark.…”

Section: Cell Culturementioning

confidence: 99%

Establishment and Application of in Vitro Membrane Potential Assays in Cell Lines with Endogenous or Recombinant Expression of ATP-Sensitive Potassium Channels (Kir6.2/SUR1) Using a Fluorescent Probe Kit

Arkhammar¹,

Wahl²,

Gerlach³

et al. 2004

SLAS Discovery

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The flow of current through the adenosine triphosphate (ATP)-sensitive potassium channel (K ATP ) of the isoform Kir6.2/ SUR1 regulates the resting membrane potential in the pancreatic β-cell. In combination with the cellular glucose metabolism, it is an important minute-to-minute regulator of insulin secretion and whole-body glucose homeostasis. The same K ATP isoform is further reported to be present in glucagon-secreting α-cells, intestinal L-cells, and glucose-responsive neurons in the hypothalamus. All in all, this makes Kir6.2/SUR1 an interesting drug target. Using a commercially available fluorescent membrane potential probe kit and a conventional 96-well fluorescence plate reader, the authors have developed and established qualitative membrane potential assays used to screen for potassium channel closers (KCCs) and openers (KCOs) in insulin-and glucagon-secreting cell lines as well as in cells with recombinant expression of the human Kir6.2/ SUR1 channel complex. Both glucose-and KCC-induced depolarization could be demonstrated. The magnitudes of these responses and KCO-induced repolarization at high glucose displayed some variation between the different cell lines but a similar rank order of test compounds. Some cell types required the presence of a KCC, such as tolbutamide, to display significant effects of KCOs. The authors find that robust and reliable functional in vitro assays compatible with medium-throughput screening and high-throughput screening can be developed as a base for finding new, more potent, and isoform-selective

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Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels

et al. 2009

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4H-1,2,4-Benzothiadiazine-1,1-dioxides with various substituents in positions 3, 5, and 7 were synthesized and tested as K(ATP) channel agonists in artificial cell systems (CHO cells transfected with SUR1/Kir6.2, and HEK 293 transfected with SUR2B/Kir6.1) as model systems for insulin-secreting pancreatic beta-cells and for smooth muscle cells, respectively. The effects of agonists were tested in intact cells using DiBAC4(3) [bis-(1,3-dibarbituric acid)trimethine oxanol] as a membrane potential dye, and the results compared with their binding affinity for the SUR2B-type K(ATP) channels using the radioligand [(3)H]P1075. Compounds with cycloalkyl and (cycloalkyl)methyl side chains in position 3 had higher affinities towards the SUR2B/Kir6.1 receptor compared with the parent compound diazoxide (1 a). Compounds with bulky, nonpolar residues in position 3 exhibited remarkable selectivity for SUR2B-type K(ATP) channels. The compound substituted with a bulky (1-adamantyl)methyl residue exhibited micromolar affinity and activity on SUR2B-type K(ATP) channels without being able to activate the SUR1-type K(ATP) channels.

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The Novel Diazoxide Analog 3-Isopropylamino-7-Methoxy- 4H -1,2,4-Benzothiadiazine 1,1-Dioxide Is a Selective Kir6.2/SUR1 Channel Opener

Cited by 34 publications

References 65 publications

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Establishment and Application of in Vitro Membrane Potential Assays in Cell Lines with Endogenous or Recombinant Expression of ATP-Sensitive Potassium Channels (Kir6.2/SUR1) Using a Fluorescent Probe Kit

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels

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The Novel Diazoxide Analog 3-Isopropylamino-7-Methoxy- 4H -1,2,4-Benzothiadiazine 1,1-Dioxide Is a Selective Kir6.2/SUR1 Channel Opener

Cited by 34 publications

References 65 publications

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Establishment and Application of in Vitro Membrane Potential Assays in Cell Lines with Endogenous or Recombinant Expression of ATP-Sensitive Potassium Channels (Kir6.2/SUR1) Using a Fluorescent Probe Kit

Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of KATP Channels

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Product

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Synthesis of Modified 4H‐1,2,4‐Benzothiadiazine‐1,1‐dioxides and Determination of their Affinity and Selectivity for Different Types of K_ATP Channels