1.Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype.
Hougaard C1, Hammami S, Eriksen BL, Sørensen US, Jensen ML, Strøbæk D, Christophersen P. Mol Pharmacol. 2012 Feb;81(2):210-9. doi: 10.1124/mol.111.074252. Epub 2011 Nov 1.
We have previously identified Ser293 in transmembrane segment 5 as a determinant for selective K(Ca)2.1 channel activation by GW542573X (4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester). Now we show that Ser293 mediates both activation and inhibition of K(Ca)2.1: CM-TPMF (N-{7-[1-(4-chloro-2-methylphenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N'-methoxy-formamidine) and B-TPMF (N-{7-[1-(4-tert-butyl-phenoxy)ethyl]-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-N'-methoxy-formamidine), two newly identified and structurally related [1,2,4]triazolo[1,5-a]pyrimidines, act either as activators or as inhibitors of the human K(Ca)2.1 channel. Whereas (-)-CM-TPMF activates K(Ca)2.1 with an EC(50) value of 24 nM, (-)-B-TPMF inhibits the channel with an IC(50) value of 31 nM. In contrast, their (+)-enantiomers are 40 to 100 times less active. Both (-)-CM-TPMF and (-)-B-TPMF are subtype-selective, with 10- to 20-fold discrimination toward other K(Ca)2 channels and the K(Ca)3 channel.
2.Microtiter plate-format optode.
Kim SB1, Cho HC, Cha GS, Nam H. Anal Chem. 1998 Nov 15;70(22):4860-3.
Microtiter plate-format optodes could be assembled by casting bulk-response membranes into the standard 96-well polypropylene-based plate or by screen printing them on an optically transparent substrate with 96-well pattern. The compositions of thick optode membranes, especially the ratios of poly(vinyl chloride) (PVC) to plasticizer [bis-(2-ethylhexyl) sebacate (DOS)], were carefully optimized to provide reproducible and rapid response. Adjusting the ratio of PVC to DOS by 1:6, bulk-response membranes containing neutral carrier (4-tert-butyl calix[4]arene tetraacetic acid tetraethyl ester for sodium-selective membrane or valinomycin for potassium-selective membrane) and lipophilic pH indicator (ETH 5294) could exhibit equilibrium response in 5 min. The practical utility of microtiter plate-format optodes has been examined by determining clinically relevant electrolytes in serum samples. It was demonstrated that microtiter plate-format optodes can provide high sample throughput (approximately 100 samples in less than 5 min), analytical performance comparable to that of a potentiometric clinical analyzer, and additional information on electrolytes using the same samples prepared for other colorimetric measurements.
3.Chemistry of 4-alkylaryloxenium ion "precursors": sound and fury signifying something?
Novak M1, Brinster AM, Dickhoff JN, Erb JM, Jones MP, Leopold SH, Vollman AT, Wang YT, Glover SA. J Org Chem. 2007 Dec 21;72(26):9954-62. Epub 2007 Nov 21.
Quinol esters 2b, 2c, and 3b and sulfonamide 4c were investigated as possible precursors to 4-alkylaryloxenium ions, reactive intermediates that have not been previously detected. These compounds exhibit a variety of interesting reactions, but with one possible exception, they do not generate oxenium ions. The 4-isopropyl ester 2b predominantly undergoes ordinary acid- and base-catalyzed ester hydrolysis. The 4-tert-butyl ester 2c decomposes under both acidic and neutral conditions to generate tert-butanol and 1-acetyl-1,4-hydroquinone, 8, apparently by an SN1 mechanism. This is also a minor decomposition pathway for 2b, but the mechanism in that case is not likely to be SN1. Decomposition of 2c in the presence of N3- leads to formation of the explosive 2,3,5,6-tetraazido-1,4-benzoquinone, 14, produced by N3--induced hydrolysis of 8, followed by a series of oxidations and nucleophilic additions by N3-. No products suggestive of N3--trapping of an oxenium ion were detected.
4.Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter.
Bouchon B1, Chambon C, Mounetou E, Papon J, Miot-Noirault E, Gaudreault RC, Madelmont JC, Degoul F. Mol Pharmacol. 2005 Nov;68(5):1415-22. Epub 2005 Aug 12.
We have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl-N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tert-butyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[125I]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent.
