Sulfonylureas uncouple glucose-dependence for GPR40-mediated enhancement of insulin secretion from INS-1E cells.

Activation of GPR40 is reported to enhance insulin secretion in the presence of glucose. We determined whether sulfonylureas could replace glucose for GPR40-mediated enhancement of insulin secretion and investigated underlying mechanisms using INS-1E cells. GW9508, a specific agonist of GPR40, significantly enhanced insulin secretion in the presence of high concentrations of glucose. In contrast, sulfonylureas increased insulin secretion in the absence of glucose. In the presence of sulfonylureas, activation of GPR40 significantly enhanced insulin secretion. The L-type calcium channel (LTCC) activator S-(-)-Bay K8644 also concentration-dependently increased insulin secretion in the absence of glucose. In the presence of 10 micromol/L S-(-)-Bay K8644, GW9508 significantly increased insulin secretion. On the other hand, the LTCC blocker nifedipine significantly inhibited insulin secretion mediated by either glucose, glipizide or glucose plus GW9508. Thus, sulfonylureas could replace glucose to support GPR40-mediated enhancement of insulin secretion, whereas blockage of LTCC reduced both glucose and sulfonylurea-mediated insulin secretion.

Characterization of the potency, selectivity, and pharmacokinetic profile for six adenosine A2A receptor antagonists.

Antagonists of adenosine A2A receptors (A2A -antagonists) with different chemical structures have been developed by several pharmaceutical companies for the potential treatment of Parkinson's disease. Pharmacological characterization of these antagonists was incomplete, and different assay conditions were used in different labs. Therefore, we characterized the potencies, selectivities, and pharmacokinetic profiles of six prototypical A2A -antagonists. Displacements of [3H]MSX-2 and of [3H]CGS21680 binding to the human cloned and rat A2A receptors were performed. The rank order of potency of antagonists to displace [(3)H]MSX-2 binding to the human A2A was SCH58261 > or = Biogen-34 > or = Ver-6623 > or = MSX-2 > KW-6002 > > DMPX. For the rat striatal A2A, the order of potency was Biogen-34 > or = SCH58261 > or = Ver-6623 > or = MSX-2 > or = KW-6002 > > DMPX. SCH58261 was the most potent antagonist of the human A2A with a K(i) value of 4 nM, whereas Biogen-34 was the most potent antagonist of the rat A2A with a K(i) value of 1.2 nM. Similar results were obtained from cAMP assays. Selectivities of A2A-antagonists were determined using radioligands [3H]DPCPX, [3H]ZM241385, and [125I]-AB-MECA for A1, A2B, and A3 receptors, respectively. KW-6002 and Biogen-34 exhibited the highest selectivity for A2A vs A1 (human and rat), respectively. The pharmacokinetic profiles of antagonists were evaluated in vivo in rats. DMPX and KW-6002 had the greatest oral bioavailability. In contrast, SCH58261, MSX-2, and Ver-6623 had low or poor oral bioavailability. In summary, SCH58261, Biogen-34, MSX-2, and Ver-6623 had high affinities for both human and rat A2A receptors, with reasonable selectivity for A2A over A1 and A2B receptors. They are suitable as A2A -antagonists for in vitro pharmacological studies. Among the six A2A-antagonists, KW-6002 is the best for use in in vivo animal studies, particularly for a CNS target, based on its bioavailability, half life, and brain penetration.

Selective inhibition of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus infection.

Edema factor (EF), a key virulence factor in anthrax pathogenesis, has calmodulin (CaM)-activated adenylyl cyclase activity. We have found that adefovir dipivoxil, a drug approved to treat chronic infection of hepatitis B virus, effectively inhibits EF-induced cAMP accumulation and changes in cytokine production in mouse primary macrophages. Adefovir diphosphate (PMEApp), the active cellular metabolite of adefovir dipivoxil, inhibits the adenylyl cyclase activity of EF in vitro with high affinity (K(i) = 27 nM). A crystal structure of EF-CaM-PMEApp reveals that the catalytic site of EF forms better van der Waals contacts and more hydrogen bonds with PMEApp than with its endogenous substrate, ATP, providing an explanation for the approximately 10,000-fold higher affinity EF-CaM has for PMEApp versus ATP. Adefovir dipivoxil is a clinically approved drug that can block the action of an anthrax toxin. It can be used to address the role of EF in anthrax pathogenesis.

Calcium dependence of the interaction between calmodulin and anthrax edema factor.

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.

Structure-based inhibitor discovery against adenylyl cyclase toxins from pathogenic bacteria that cause anthrax and whooping cough.

Edema factor (EF) and CyaA are adenylyl cyclase toxins secreted by pathogenic bacteria that cause anthrax and whooping cough, respectively. Using the structure of the catalytic site of EF, we screened a data base of commercially available, small molecular weight chemicals for those that could specifically inhibit adenylyl cyclase activity of EF. From 24 compounds tested, we have identified one quinazoline compound, ethyl 5-aminopyrazolo[1,5-a]quinazoline-3-carboxylate, that specifically inhibits adenylyl cyclase activity of EF and CyaA with approximately 20 microm Ki. This compound neither affects the activity of host resident adenylyl cyclases type I, II, and V nor exhibits promiscuous inhibition. The compound is a competitive inhibitor, consistent with the prediction that it binds to the adenine portion of the ATP binding site on EF. EF is activated by the host calcium sensor, calmodulin. Surface plasmon resonance spectroscopic analysis shows that this compound does not affect the binding of calmodulin to EF. This compound is dissimilar from a previously described, non-nucleoside inhibitor of host adenylyl cyclase. It may serve as a lead to design antitoxins to address the role of adenylyl cyclase toxins in bacterial pathogenesis and to fight against anthrax and whooping cough.

Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins.

Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase exotoxins involved in the pathogenesis of anthrax and whooping cough, respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance spectroscopy analyses, we show that low Ca(2+) concentrations increase the affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+) concentrations directly inhibit catalysis. Both events occur in a physiologically relevant range of Ca(2+) concentrations. Despite the similarity in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N- and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by either terminal domain of CaM while EF cannot. EF consists of a catalytic core and a helical domain, and both are required for CaM activation of EF. Mutations that decrease the interaction of the helical domain with the catalytic core create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA is fully activated by CaM without the domain corresponding to the helical domain of EF.

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin.

Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.