As the chip of synthetic biology, enzymes play a vital role in the bio-manufacturing industry. The development of diverse functional enzymes can provide a rich toolbox for the development of synthetic biology. This article reports the construction of an artificial enzyme with the introduction of a non-natural cofactor. By introducing the 4-dimethylaminopyridine (DMAP) cofactor into the optimal protein skeleton via covalent bonds based on a click-chemistry strategy, we successfully constructed a novel artificial enzyme with the DMAP cofactor as the catalytic center. The artificial enzyme successfully catalyzed an unnatural asymmetric Morita-Baylis- Hillman (MBH) reaction between cycloketenone and p-nitrobenzaldehyde, with a conversion rate of 90% and enantioselectivity (e.e.) of 38%. This study not only provides an effective strategy for the design of new artificial enzymes but also establishes a theoretical basis for the development of unnatural biocatalytic MBH reactions.
Biocatalysis ; 4-Aminopyridine/chemistry* ; Enzymes/metabolism* ; Coenzymes/chemistry* ; Benzaldehydes/chemistry* ; Protein Engineering/methods* ; Click Chemistry

To explore new agents of gamma-aminobutyric acid (GABA) derivatives with more potent antiepileptic activity, a series of 4-(2-acetoxybenzoylamino) butyramide derivatives were designed and synthesized. All of the novel compounds (5a-51) were synthesized from GABA as starting material, and their structures were confirmed with IR, 1H NMR, EI-MS and elemental analysis. Preliminary pharmacological test in vitro showed that all target compounds displayed strong antiepileptic activities and were worth for further study. The structure-activity relationship of 4-(2-acetoxybenzoylamino) butyramide derivatives was also discussed preliminarily.
4-Aminopyridine ; Amides ; chemical synthesis ; chemistry ; therapeutic use ; Animals ; Anticonvulsants ; chemical synthesis ; chemistry ; therapeutic use ; Epilepsy ; chemically induced ; drug therapy ; Female ; Male ; Mice ; Molecular Structure ; Random Allocation ; Structure-Activity Relationship ; gamma-Aminobutyric Acid ; chemistry

It has been demonstrated by our previous research that 4-(2-acetoxybenzoylamino) butyramide derivatives exhibited good antiepileptic activities. In this paper, to explore the SAR and improve the antiepileptic activities of these derivatives, a series of novel 4-(2-acetoxybenzoylamino) butyramide heterocyclic compounds (5a-5n) were synthesized and biologically evaluated. Their structures were confirmed by 1H MNR, ESI-MS and elemental analysis. Pharmacological test in vivo showed that target compounds (5f, 5i-5n) displayed strong antiepileptic activities on 4-AP induced epilepsy in mice with ED50 values ranging from 0.3137 to 0.3604 mmol x kg(-1).
4-Aminopyridine ; Amides ; administration & dosage ; chemical synthesis ; chemistry ; therapeutic use ; Animals ; Anticonvulsants ; administration & dosage ; chemical synthesis ; chemistry ; therapeutic use ; Dose-Response Relationship, Drug ; Epilepsy ; chemically induced ; drug therapy ; Female ; Lethal Dose 50 ; Male ; Mice ; Molecular Structure ; Random Allocation ; Receptors, GABA ; metabolism ; Structure-Activity Relationship

A series of 4-(2-acetoxybenzoylamino) butyrate derivatives were designed and synthesized. All of the novel 12 compounds (7a-7k) were synthesized from gamma-aminobutyric acid (1) as starting material, and their structures were confirmed with IR, 1H NMR, EI-MS and elemental analysis. Preliminary pharmacological test in vitro showed that most of these title compounds possessed antiepileptic activity. Compounds 7i-7k displayed strong antiepileptic activity and are worth for further development. Compounds 4, 7d-7h showed moderate antiepileptic activity. The structure-activity relationship of 4-(2-acetoxybenzoylamino) butyrate derivatives is also discussed preliminarily.
4-Aminopyridine ; Animals ; Anticonvulsants ; chemical synthesis ; chemistry ; therapeutic use ; Butyrates ; chemical synthesis ; chemistry ; therapeutic use ; Drug Design ; Epilepsy ; chemically induced ; drug therapy ; Female ; Lethal Dose 50 ; Male ; Mice ; Molecular Structure ; Random Allocation ; Structure-Activity Relationship ; gamma-Aminobutyric Acid ; chemistry

OBJECTIVETo investigate the vasorelaxant effect of puerarin in rat aortic rings and the mechanism.

METHODThe isolated thoracic aortic rings of male Sprague-Dawley rats were mounted on the organ bath and the contractile responses of the vessel were recorded.

RESULTPuerarin completely relaxed the contractions induced by phenylephrine in a concentration-dependent manner in endothelium-intact and endothelium-denuded rat aorta, but it had no effect on those preconstricted by a high concentration of potassium chloride (KCl, 60 mmol x L(-1)). The relaxant effect of puerarin was significantly inhibited by pretreatment of endothelium-denuded aorta with potassium channel antagonists tetraethylammonium, 4-aminopyridine but not glibenclamide.

CONCLUSIONPuerarin induces an endothelium-independent relaxation in rat aortic rings. The mechanisms may involve the reduction in Ca2+ influx through the calcium channels operated by alpha-adrenergic receptor and the activation of the potassium channels (Kv and BKca, but not KATP).

4-Aminopyridine ; pharmacology ; Animals ; Aorta, Thoracic ; drug effects ; physiology ; Endothelium, Vascular ; physiology ; In Vitro Techniques ; Isoflavones ; isolation & purification ; pharmacology ; Male ; Phenylephrine ; antagonists & inhibitors ; Plants, Medicinal ; chemistry ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; drug effects ; Pueraria ; chemistry ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; pharmacology ; Vasoconstriction ; drug effects ; Vasodilation ; drug effects ; Vasodilator Agents ; pharmacology

AIMTo discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.

METHODSA cell-based 96-well format fluorescence assay using DiBAC4 (3) in cultured PC12 cells was described. Cells were loaded with 5 mumol.L-1 DiBAC4 (3) and incubated at 37 degrees C for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC4 (3) fluorescence responce was then detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels.

RESULTSExtracellular high K(+)-induced depolarization and several potassium channel blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC4 (3) fluorescence response. The potassium channel opener, cromakalim, evoked decrease in DiBAC4 (3) fluorescence response. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC4 (3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents.

CONCLUSIONThe DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.

4-Aminopyridine ; pharmacology ; Animals ; Barbiturates ; chemistry ; Calcium Channel Blockers ; pharmacology ; Cromakalim ; pharmacology ; Isoxazoles ; chemistry ; Membrane Potentials ; drug effects ; Nifedipine ; pharmacology ; PC12 Cells ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Potassium Channels ; drug effects ; Pyridines ; pharmacology ; Quinidine ; pharmacology ; Rats

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (<2 mM) of TEA and 4-AP inhibited phasic and tonic contractions induced by 5 micrometer carbachol, whilst the relatively higher concentrations of TEA and 4-AP (>5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.
4-Aminopyridine/pharmacology ; Animals ; Bladder/metabolism ; Calcium/chemistry/metabolism ; Calcium Channel Blockers/pharmacology ; Carbachol/*pharmacology ; Dose-Response Relationship, Drug ; Female ; Guinea Pigs ; Inhibitory Concentration 50 ; Male ; Mice ; Muscle Contraction/*drug effects ; Muscles/drug effects/metabolism/*pathology ; Nicardipine/pharmacology ; Potassium Channel Blockers/*pharmacology ; Protein Binding ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't ; Tetraethylammonium/pharmacology ; Vasodilator Agents/pharmacology