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.

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. Coapplication experiments reveal competitive-like functional interactions between the effects of (-)-CM-TPMF and (-)-B-TPMF. Despite belonging to a different chemical class than GW542573X, the K(Ca)2.1 selectivity of (-)-CM-TPMF and (-)-B-TPMF depend critically on Ser293 as revealed by loss- and gain-of-function mutations. We conclude that compounds occupying the TPMF site may either positively or negatively influence the gating process depending on their substitution patterns. It is noteworthy that (-)-CM-TPMF is 10 times more potent on K(Ca)2.1 than NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime), an unselective but hitherto the most potent K(Ca)3/K(Ca)2 channel activator. (-)-B-TPMF is the first small-molecule inhibitor with significant selectivity among the K(Ca)2 channel subtypes. In contrast to peptide blockers such as apamin and scyllatoxin, which preferentially affect K(Ca)2.2, (-)-B-TPMF exhibits K(Ca)2.1 selectivity. These high-affinity compounds, which exert opposite effects on K(Ca)2.1 gating, may help define physiological or pathophysiological roles of this channel.

Selective activation of the SK1 subtype of human small-conductance Ca2+-activated K+ channels by 4-(2-methoxyphenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (GW542573X) is dependent on serine 293 in the S5 segment.

A new small molecule, 4-(2-methoxy-phenylcarbamoyloxymethyl)-piperidine-1-carboxylic acid tert-butyl ester (GW542573X), is presented as an activator of small-conductance Ca(2+)-activated K(+) (SK, K(Ca)2) channels and distinguished from previously published positive modulators of SK channels, such as 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethylpyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA), in several aspects. GW542573X is the first SK1-selective compound described: an EC(50) value of 8.2 +/- 0.8 microM (n = 6, [Ca(2+)](i) = 200 nM) was obtained from inside-out patches excised from hSK1-expressing HEK293 cells. Whole-cell experiments showed that hSK2 and hSK3 channels were more than 10 times, and hIK channels even more than 100 times, less sensitive to GW542573X. The Ca(2+)-response curve of hSK1 was left-shifted from an EC(50)(Ca(2+)) value of 410 +/- 20 nM (n = 9) to 240 +/- 10 nM (n = 5) in the presence of 10 microM GW542573X. In addition to this positive modulation, GW542573X activated SK1 in the absence of Ca(2+) and furthermore induced a 15% increase in the maximal current at saturating Ca(2+). Thus, GW542573X also acts as a genuine opener of the hSK1 channels, a mechanism of action (MOA) not previously obtained with SK channels. The differential potency on hSK1 and hSK3 enabled a chimera approach to elucidate site(s) important for this new MOA and selectivity property. A single amino acid (Ser293) located in S5 of hSK1 was essential, and substituting the corresponding Leu476 in hSK3 with serine conferred hSK1-like potency (EC(50) = 9.3 +/- 1.4 microM, n = 5). GW542573X may activate SK channels via interaction with "deep-pore" gating structures at the inner pore vestibule or the selectivity filter in contrast to 1-EBIO and CyPPA that exert positive modulation via the intracellular calmodulin binding domain.

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.

Selective positive modulator of calcium-activated potassium channels exerts beneficial effects in a mouse model of spinocerebellar ataxia type 2.

Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder caused by a polyglutamine expansion within the Ataxin-2 (Atxn2) protein. Purkinje cells (PC) of the cerebellum fire irregularly and eventually die in SCA2. We show here that the type 2 small conductance calcium-activated potassium channel (SK2) play a key role in control of normal PC activity. Using cerebellar slices from transgenic SCA2 mice we demonstrate that SK channel modulators restore regular pacemaker activity of SCA2 PCs. Furthermore, we also show that oral delivery of a more selective positive modulator of SK2/3 channels (NS13001) alleviates behavioral and neuropathological phenotypes of aging SCA2 transgenic mice. We conclude that SK2 channels constitute a therapeutic target for SCA2 treatment and that the developed selective SK2/3 modulator NS13001 holds promise as a potential therapeutic agent for treatment of SCA2 and possibly other cerebellar ataxias.