Design, Synthesis, Molecular Modeling and Anti-Hyperglycemic Evaluation of Quinazoline-Sulfonylurea Hybrids as Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Sulfonylurea Receptor (SUR) Agonists.

New quinazoline-sulfonylurea hybrids were prepared and examined for their in vivo anti-hyperglycemic activities in STZ-induced hyperglycemic rats using glibenclamide as a reference drug. Compounds VI-6-a, V, IV-4, VI-4-c, IV-6, VI-2-a, IV-1, and IV-2 were more potent than the reference glibenclamide. They induced significant reduction in the blood glucose levels of diabetic rats: 78.2, 73.9, 71.4, 67.3, 62, 60.7, 58.4, and 55.9%, respectively, while the reference glibenclamide had 55.4%. Compounds IV-1, VI-2-a, IV-2, V, and IV-6 showed more prolonged antidiabetic activity than glibenclamide. Moreover, molecular docking and pharmacokinetic studies were performed to examine binding modes of the prepared compounds against peroxisome proliferator-activated receptor gamma (PPARγ). The highest active compounds exhibited good binding affinity with high free energy of binding against PPARγ. In silico absorption, distribution, metabolism, elimination and toxicity (ADMET) studies were performed to investigate pharmacokinetics and safety of the synthesized compounds. They showed considerable human intestinal absorption with low toxicity profile.

Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of phthalimide-sulfonylurea hybrids as PPARγ and SUR agonists.

New series of phthalimide-sulfonylurea hybrids were prepared and examined for their in vivo anti-hyperglycemic activities in STZ-induced hyperglycemic rats using glibenclamide as a reference drug. Compounds 6c, 6d, 6g, 6h, 6j and 6k induced significant reduction in the blood glucose levels of diabetic rats ranging from 24.43 to 21.43%. Moreover, molecular docking and pharmacophore approaches were carried out to examine binding modes and fit values of the prepared compounds against PPARγ and SUR, respectively. Compounds 6c, 6d, 6j and 6m exhibited the highest binding free energies against PPARγ. Compounds 6c, 6j, 6k, 6l, and 6n showed the highest fit values against the generated pharmacophore model. Also, QSAR technique was carried out to estimate the proposed PPARγ binding affinities and insulin-secreting abilities. The synthesized compounds showed promising estimated activities. In-silico ADMET studies were performed to investigate pharmacokinetics of the synthesized compounds. They showed considerable human intestinal absorption with low BBB penetration.

Modulation of SUR1 KATP Channel Subunit Activity in the Peripheral Nervous System Reduces Mechanical Hyperalgesia after Nerve Injury in Mice.

The ATP-sensitive K+ channel (KATP) is involved in hypersensitivity during chronic pain and is presumed to be a downstream target of mu opioid receptors. Multiple subtypes of KATP channels exist in the peripheral and central nervous system and their activity may be inversely correlated to chronic pain phenotypes in rodents. In this study, we investigated the different KATP channel subunits that could be involved in neuropathic pain in mice. In chronic pain models utilizing spinal nerve ligation, SUR1 and Kir6.2 subunits were found to be significantly downregulated in dorsal root ganglia and the spinal cord. Local or intrathecal administration of SUR1-KATP channel subtype agonists resulted in analgesia after spinal nerve ligation but not SUR2 agonists. In ex-vivo nerve recordings, administration of the SUR1 agonist diazoxide to peripheral nerve terminals decreased mechanically evoked potentials. Genetic knockdown of SUR1 through an associated adenoviral strategy resulted in mechanical hyperalgesia but not thermal hyperalgesia compared to control mice. Behavioral data from neuropathic mice indicate that local reductions in SUR1-subtype KATP channel activity can exacerbate neuropathic pain symptoms. Since neuropathic pain is of major clinical relevance, potassium channels present a target for analgesic therapies, especially since they are expressed in nociceptors and could play an essential role in regulating the excitability of neurons involved in pain-transmission.

SUR2B/Kir6.1 channel openers correct endothelial dysfunction in chronic heart failure via the miR-1-3p/ET-1 pathway.

The SUR2B/Kir6.1 channel openers iptakalim and natakalim reverse cardiac remodeling and ameliorate endothelial dysfunction by re-establishing the balance between the nitric oxide and endothelin systems. In this study, we investigated the microRNAs (miRs) involved in the molecular mechanisms of SUR2B/Kir6.1 channel opening in chronic heart failure. Both iptakalim and natakalim significantly upregulated the expression of miR-1-3p, suggesting that this miR is closely associated with the therapeutic effects against chronic heart failure. Bioinformatic analysis showed that many of the 183 target genes of miR-1-3p are involved in cardiovascular diseases, suggesting that miR-1-3p plays a vital role in such diseases and vascular remodeling. Target gene prediction showed that miR-1-3p combines with the 3' untranslated region (UTR) of endothelin-1 (ET-1) mRNA. Iptakalim and natakalim upregulated miR-1-3p expression and downregulated ET-1 mRNA expression in vitro. The dual luciferase assay confirmed that there is a complementary binding sequence between miR-1-3p and the 3' UTR 158-165 sequence of ET-1 mRNA. To verify the effect of miR-1-3p on ET-1, lentiviral vectors overexpressing or inhibiting miR-1-3p were constructed for the transduction of rat primary cardiac microvascular endothelial cells. The results showed that natakalim enhanced the miR-1-3p level. miR-1-3p overexpression downregulated the expression of ET-1, whereas miR-1-3p inhibition had the opposite effect. Therefore, we verified that SUR2B/Kir6.1 channel openers could correct endothelial imbalance and ameliorate chronic heart failure through the miR-1-3p/ET-1 pathway in endothelial cells. Our study provides comprehensive insights into the molecular mechanisms behind the SUR2B/Kir6.1 channel's activity against chronic heart failure.

Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels.

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic ß cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of quinazolin-4(3H)-one derivatives as potential PPARγ and SUR agonists.

Peroxisome proliferator-activated receptor gamma (PPARγ) and sulfonylurea receptor (SUR) play crucial roles in management of type-2 diabetes mellitus. In this study, a series of novel quinazoline-4(3H)-one-sulfonylurea hybrids were designed and synthesized as dual PPARγ and SUR agonists. The synthesized compounds were evaluated for their in vivo anti-hyperglycemic activities against STZ-induced hyperglycemic rats. Four compounds (19a, 19d, 19f and 25g) demonstrated potent activities with reduction in blood glucose levels of 40.43, 46.42, 41.23 and 42.50 %, respectively. The most active ten compounds were further evaluated in vitro for their PPARγ binding affinities and insulin-secreting abilities. Compounds 19b, 19d, 19f, 25f and 25g exhibited the highest affinities against PPARγ with IC50 values of 0.371, 0.350, 0.369, 0.408 and 0.353µM, respectively. In addition, compounds 19d, 19f, and 25d showed the highest insulin-secreting activities with EC50 values of 0.97, 1.01 and 1.15µM, respectively. Furthermore, molecular docking and pharmacophore generation techniques were carried out to investigate binding patterns and fit values of the designed compounds with PPARγ and SUR, respectively. Also, two QSAR models were generated to explore the structural requirements controlling the different biological activities of the synthesized compounds against PPARγ and SUR.

Design, synthesis, molecular modeling and anti-hyperglycemic evaluation of novel quinoxaline derivatives as potential PPARγ and SUR agonists.

In our effort to develop potent anti-hyperglycemic agents with potential agonistic activities toward PPARγ and SUR, three novel series of quinoxaline derivatives bearing sulfonylurea or sulfonylthiourea moieties with different linkers were designed and synthesized. Some of the newly synthesized compounds were evaluated in vivo for their anti-hyperglycemic activities in STZ-induced hyperglycemic rats. Compounds 15a, 15e, 19b and 24a exhibited the highest anti-hyperglycemic activities with % reduction in blood glucose level of (50.58, 43.84, 45.10 and 49.62, respectively). Additionally, eight compounds revealed potent anti-hyperglycemic activities were further evaluated in vitro for their PPARγ binding affinity and insulin-secreting ability as potential mechanisms for anti-hyperglycemic activity. Four compounds (15a, 15b, 15d and 15e) significantly bound to PPARγ with IC50 values of 0.482, 0.491, 0.350 and 0.369µM, respectively. Moreover, Compounds 15a and 15b have demonstrated induction of insulin-secretion with EC50 values of 0.92 and 0.98µM, respectively. Furthermore, molecular docking and pharmacophore generation techniques were carried out to investigate binding patterns and fit values of the designed compounds with PPARγ and SUR, respectively.

Role of Glibenclamide in Brain Injury After Intracerebral Hemorrhage.

Brain edema following intracerebral hemorrhage (ICH) causes severe secondary brain injury, and no efficient pharmacological preventions are available. The present study was designed to demonstrate the neuroprotective effects of glibenclamide on brain edema and key factors of the blood-brain barrier (BBB). The study was divided into two parts. First, we utilized an autoblood-induced rat model to investigate the expression of sulfonylurea receptor 1 (Sur1). Second, rats were randomized into sham, vehicle, and glibenclamide groups. Neurological scores, brain water content, Evans blue extravasation, Morris water maze test, western blots, and immunofluorescence were used to study the effects of glibenclamide. The expression of the Sur1-Trpm4 channel but not the Sur1-KATP channel was increased in the perihematomal tissue following ICH. Glibenclamide administration significantly decreased the brain water content, restored the BBB, and reduced the expression of MMPs. In addition, glibenclamide improved long-term cognitive deficits following ICH. Glibenclamide protected BBB integrity and improved neurological outcomes after ICH by inhibiting the Sur1-Trpm4 channel, which reduces the expression of MMPs and thereby increases BBB tight-junction protein levels. Glibenclamide may have potential to protect the BBB after ICH.

Pharmacological evidence: a new therapeutic approach to the treatment of chronic heart failure through SUR2B/Kir6.1 channel in endothelial cells.

Both iptakalim (Ipt) and natakalim (Nat) activate the SUR2B/Kir6.1 channel, an ATP-sensitive potassium channel (KATP) subtype, with high selectivity. In this study we investigated the therapeutic effects of Ipt and Nat against isoproterenol-induced chronic heart failure (ISO-CHF) in rats, and demonstrated a new therapeutic approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells. In ISO-CHF rats, oral administration of Nat (1, 3, 9 mg·kg-1·d-1) or Ipt (3 mg·kg-1·d-1) for 60 days significantly improved cardiac dysfunction, reversed cardiac remodeling, significantly attenuated the pathological increases in BNP levels, and improved endothelial dysfunction by adjusting the balance between endothelin and NO systems. The therapeutic effects of Nat were prevented by the selective KATP blocker glibenclamine (Gli, 50 mg·kg-1·d-1), confirming that these effects were mediated through activation of the SUR2B/Kir6.1 channel in endothelial cells. The molecular mechanisms underlying the therapeutic effects of Nat were further addressed using proteomic methods. We identified 724 proteins in the plasma of ISO-CHF rats; 55 proteins were related to Nat. These differentially expressed proteins were mainly involved in single-organism processes and the regulation of biological quality relative to CHF, including proteasome (Psm) and ATP protein clusters. We screened out PRKAR2ß, GAS6/eNOS/NO and NO/PKG/VASP pathways involved in the amelioration of CHF among the 24 enriched pathways. We further confirmed 6 protein candidates, including PRKAR2ß, GAS6 and VASP, which were involved in the endothelial mechanisms, and ATP, TIMP3 and AGT, which contributed to its cardiovascular actions. This study demonstrates a new pharmacological approach to the treatment of CHF through activation of the SUR2B/Kir6.1 channel in endothelial cells, and that the eNOS/VASP pathways are involved in its signaling mechanisms.

Myorelaxant action of fluorine-containing pinacidil analog, flocalin, in bladder smooth muscle is mediated by inhibition of L-type calcium channels rather than activation of KATP channels.

Flocalin (FLO) is a new ATP-sensitive K(+) (KATP) channel opener (KCO) derived from pinacidil (PIN) by adding fluorine group to the drug's structure. FLO acts as a potent cardioprotector against ischemia-reperfusion damage in isolated heart and whole animal models primarily via activating cardiac-specific Kir6.2/SUR2A KATP channels. Given that FLO also confers relaxation on several types of smooth muscles and can partially inhibit L-type Ca(2+) channels, in this study, we asked what is the mechanism of FLO action in bladder detrusor smooth muscle (DSM). The actions of FLO and PIN on contractility of rat and guinea pig DSM strips and membrane currents of isolated DSM cells were compared by tensiometry and patch clamp. Kir6 and SUR subunit expression in rat DSM was assayed by reverse transcription PCR (RT-PCR). In contrast to PIN (10 µM), FLO (10 µM) did not produce glibenclamide-sensitive DSM strips' relaxation and inhibition of spontaneous and electrically evoked contractions. However, FLO, but not PIN, inhibited contractions evoked by high K(+) depolarization. FLO (40 µM) did not change the level of isolated DSM cell's background K(+) current, but suppressed by 20 % L-type Ca(2+) current. Determining various Kir6 and SUR messenger RNA (mRNA) expressions in rat DSM by RT-PCR indicated that dominant KATP channel in rat DSM is of vascular type involving association of Kir6.1 and SUR2B subunits. Myorelaxant effects of FLO in bladder DSM are explained by partial blockade of L-type Ca(2+) channel-mediated Ca(2+) influx rather than by hyperpolarization associated with increased K(+) permeability. Thus, insertion of fluorine group in PIN's structure made the drug more discriminative between Kir6.2/SUR2A cardiac- and Kir6.1/SUR2B vascular-type KATP channels and rendered it partial L-type Ca(2+) channel-blocking potency.

ABCC9/SUR2 in the brain: Implications for hippocampal sclerosis of aging and a potential therapeutic target.

The ABCC9 gene and its polypeptide product, SUR2, are increasingly implicated in human neurologic disease, including prevalent diseases of the aged brain. SUR2 proteins are a component of the ATP-sensitive potassium ("KATP") channel, a metabolic sensor for stress and/or hypoxia that has been shown to change in aging. The KATP channel also helps regulate the neurovascular unit. Most brain cell types express SUR2, including neurons, astrocytes, oligodendrocytes, microglia, vascular smooth muscle, pericytes, and endothelial cells. Thus it is not surprising that ABCC9 gene variants are associated with risk for human brain diseases. For example, Cantu syndrome is a result of ABCC9 mutations; we discuss neurologic manifestations of this genetic syndrome. More common brain disorders linked to ABCC9 gene variants include hippocampal sclerosis of aging (HS-Aging), sleep disorders, and depression. HS-Aging is a prevalent neurological disease with pathologic features of both neurodegenerative (aberrant TDP-43) and cerebrovascular (arteriolosclerosis) disease. As to potential therapeutic intervention, the human pharmacopeia features both SUR2 agonists and antagonists, so ABCC9/SUR2 may provide a "druggable target", relevant perhaps to both HS-Aging and Alzheimer's disease. We conclude that more work is required to better understand the roles of ABCC9/SUR2 in the human brain during health and disease conditions.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Sulfonylureas, which stimulate insulin secretion from pancreatic ß-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on ß-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on ß-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

Direct activation of ß-cell KATP channels with a novel xanthine derivative.

ATP-regulated potassium (KATP) channel complexes of inward rectifier potassium channel (Kir) 6.2 and sulfonylurea receptor (SUR) 1 critically regulate pancreatic islet ß-cell membrane potential, calcium influx, and insulin secretion, and consequently, represent important drug targets for metabolic disorders of glucose homeostasis. The KATP channel opener diazoxide is used clinically to treat intractable hypoglycemia caused by excessive insulin secretion, but its use is limited by off-target effects due to lack of potency and selectivity. Some progress has been made in developing improved Kir6.2/SUR1 agonists from existing chemical scaffolds and compound screening, but there are surprisingly few distinct chemotypes that are specific for SUR1-containing KATP channels. Here we report the serendipitous discovery in a high-throughput screen of a novel activator of Kir6.2/SUR1: VU0071063 [7-(4-(tert-butyl)benzyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione]. The xanthine derivative rapidly and dose-dependently activates Kir6.2/SUR1 with a half-effective concentration (EC50) of approximately 7 µM, is more efficacious than diazoxide at low micromolar concentrations, directly activates the channel in excised membrane patches, and is selective for SUR1- over SUR2A-containing Kir6.1 or Kir6.2 channels, as well as Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and voltage-gated potassium channel 2.1. Finally, we show that VU0071063 activates native Kir6.2/SUR1 channels, thereby inhibiting glucose-stimulated calcium entry in isolated mouse pancreatic ß cells. VU0071063 represents a novel tool/compound for investigating ß-cell physiology, KATP channel gating, and a new chemical scaffold for developing improved activators with medicinal chemistry.

D-chiro-inositol glycan stimulates insulin secretion in pancreatic ß cells.

Insulin has been shown to act on pancreatic ß cells to regulate its own secretion. Currently the mechanism underlying this effect is unclear. INS-2, a novel inositol glycan pseudo-disaccharide containing D-chiro-inositol and galactosamine, has been shown to function as an insulin mimetic and a putative insulin mediator. In the present study we found that INS-2 stimulates insulin secretion in MIN6 ß cells and potentiates glucose stimulated insulin secretion in isolated mouse islets. Importantly, INS-2 failed to potentiate insulin secretion induced by tolbutamide, which stimulates insulin release by closing ATP sensitive potassium channels (KATP). Electrophysiological studies showed that INS-2 inhibited sulfonylurea-sensitive KATP conductance. The effect of INS-2 on inhibiting KATP channel is mediated by protein phosphatase 2C (PP2C), as knocking down PP2C expression in MIN6 cells by PP2C small hairpin RNA completely abolished the effect of INS-2 on KATP and consequently attenuated INS-2 induced insulin secretion. In conclusion, the present study identifies a novel mechanism involving PP2C in regulating KATP channel activity and consequently insulin secretion.

ATP-sensitive K(+) channels in rat colonic epithelium.

ATP-sensitive K(+) (KATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that KATP channels contribute to epithelial K(+) currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I sc) induced by the KATP channel opener pinacidil (5 10(-4) mol l(-1)) were measured in Ussing chambers under control conditions and in the presence of different blockers of KATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K(+) channel opener, when administered at the serosal side, induced an increase in I sc consistent with the induction of transepithelial Cl(-) secretion after activation of basolateral K(+) channels, whereas mucosal administration of pinacidil resulted in a negative I sc. The increase in I sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10(-4) mol l(-1)) and gliclazide (10(-6) mol l(-1)), but was resistant even against a high concentration (10(-2) mol l(-1)) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl(-) currents across apical Cl(-) channels in basolaterally depolarized epithelia indicating that the negative I sc induced by mucosal pinacidil is due to a transient inhibition of Cl(-) secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1 and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, KATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.