Potassium channel modulators

This invention concerns peptides (I) having intracellular potassium channel modulating activity comprising the amino acid sequence shown in SEQ ID No: 2: (N terminal function) (I) Met Ile Ser Ser Val Cys Val Ser Ser 1 5} Tyr Arg Gly Arg Lys Ser Gly Asn Lys 10 15 Pro Pro Ser Lys Thr Cys Leu Lys Glu Glu 20 25 (C terminal function) in which the cysteines are optionally linked via a disulphide bridge and wherein ______________________________________ Met represents L-methionine Ile represents L-isoleucine Ser represents L-serine Val represents L-valine Cys represents L-cysteine Tyr represents L-tyrosine Arg represents L-arginine Gly represents glycine Lys represents L-lysine Asn represents L-asparagine Pro represents L-proline Thr represents L-threonine Leu represents L-leucine Glu represents L-glutamic acid ______________________________________ or a variant thereof, with the proviso that excluded is the 13-Lys variant (where 13-Arg is replaced by 13-Lys) in which the cysteines are not linked via a disulphide bridge, which are useful in a test method of screening for compounds having potassium channel modulating activity.

This invention relates to potassium channel modulators, in particular to 
linear and cyclic peptides which block potassium channels intracellularly 
and are useful in screening for potential potassium channel openers having 
therapeutic utility. 
Voltage gated potassium ion (K+) channels which produce outward currents 
are present in the cell membranes of neurones and serve to repolarise the 
cell following a depolarisation by opening and allowing potassium ions to 
flow from the inside of the cell to the outside. They are, therefore, one 
of the main regulating influences on the nerve cell firing and determine 
the amount of current reaching the terminal regions of the cells. This in 
turn regulates the amount of neurotransmitter substances released from the 
nerve terminals. In addition, they help to determine the refractory period 
of the nerve cell and hence the probability of the cell firing again 
within a certain time. This governs neuronal excitability and also the 
tendency of a cell to undergo repetitive firing. An ability to modify the 
functioning of these channels by chemical means is the aim of current 
research in the search for therapeutically useful agents. 
The present application is particularly concerned with the intracellular 
block of the K+ channels. 
Voltage-dependent potassium (K+) channels open in response to a positive 
shift in membrane potential. After some variable time these channels close 
again; certain types of K+ channels close fairly quickly ("inactivate" in 
several milliseconds) after opening, others remain open for seconds. It 
was suggested over twenty years ago that the rapid closing mechanism is 
due to a `molecular plug` or ball swinging into the open channel thereby 
blocking the further passage of ions. This simple mechanism has recently 
been shown to account for the rapid inactivation of several K channels. 
Initially Aldrich et al., Science 250 568-571 (1990), demonstrated that 
only 20 amino acids at the N terminal of Shaker B channels acts as the 
blocking particle. Similarly, Ruppersburg J P et al., Nature 353 657-660 
(1991), have recently shown for some mammalian (rat) potassium channels 
(called Raw3 and RCK4) that the N-terminal regions act as the natural 
channel closing particle. 
Robertson B., and Owen D., J. Physiol., 459, 92P, 1993 have also shown that 
non-inactivating K channels from a mammalian brain (MK-1) may be blocked 
by a peptide derived from the N-terminal sequence of the Shaker B channel. 
This has the overall effect of transforming this previously sustained 
channel into a rapidly inactivating one. Rudy, D., et al., J. Neuroscience 
Res., 29, 401-412 (1991), give the amino acid sequence for a human brain 
inactivating potassium channel (HKShIIIc), which is extremely homologous 
to the rat inactivating K channel Raw3. 
All of the above known peptide sequences are understood to be `linear` 
molecules in so far as there is no intramolecular chemical bonding. 
However physical intramolecular forces may give the molecule some degree 
of constraint. 
A 28-amino acid peptide derived from the N-terminal sequence of HKShIIIc 
was synthesised (`human` 28 mer peptide) and tested in the non-activating 
MK-1 channel to determine if this isolated peptide was capable of blocking 
the channel. This `human` 28 mer peptide had the sequence listed in SEQ ID 
No: 
Acetyl Nle Ile Ser Ser Val Cys Val Ser Ser Tyr Arg 
1 5 10 
Gly Arg Lys Ser Gly Asn Lys Pro Pro Ser Lys Thr 
15 20 
Cys Leu Lys Glu Glu NH.sub.2 
25 
An acetyl group was used to block the terminal NH.sub.2 -group. 
We have found that the `human` 28 mer peptide was active in transforming 
MK-1 into an inactivating channel. 
Most surprisingly we have found that the cyclic cysteine--cysteine bridged 
analogues were also active and apparently more potent than the `linear` 
form as K+ channel blockers. 
Accordingly this invention provides a peptide (I) having intracellular 
potassium channel blocking activity comprising the amino acid sequence 
listed in SEQ ID No: 2: 
(N terminal function) Met Ile Ser Ser Val Cys 
(I) 
1 5 
Val Ser Ser Tyr Arg Gly Arg Lys Ser Gly Asn Lys 
10 15 
Pro Pro Ser Lys Thr Cys Leu Lys Glu Glu 
20 25 
(C terminal function) 
in which the cysteines are optionally linked via a disulphide bridge and 
wherein 
______________________________________ 
Met represents L-methionine 
Ile represents L-isoleucine 
Ser represents L-serine 
Val represents L-valine 
Cys represents L-cysteine 
Tyr represents L-tyrosine 
Arg represents L-arginine 
Gly represents glycine 
Lys represents L-lysine 
Asn represents L-asparagine 
Pro represents L-proline 
Thr represents L-threonine 
Leu represents L-leucine 
Glu represents L-glutamic acid 
______________________________________ 
or a variant of said polypeptide having intracellular potassium channel 
modulating activity with the proviso that excluded is the 13-Lys variant 
(where 13-Arg is replaced by 13-Lys) in which the cysteines are not linked 
via a disulphide bridge. 
As an example of a variant mention is made of the 1-Nle (norleucine) 
analogue replacing 1-methionine. Similarly Glu residues may be replaced by 
aspartic acid (Asp), and Asn residue by Gln (L-glutamine). In addition Arg 
and Lys residues may be interchanged. The term variant means any analogue 
having one (or more) different amino acid residues providing that 
intracellular potassium channel modulating activity is retained. The term 
also covers omission or addition of amino acid residues where said 
intracellular activity is retained. 
The .alpha.-terminal group may be NH.sub.2 (i.e. the N-terminal function is 
H--) or a substituted amino group, e.g. mono- or di-alkyl amino or N-acyl 
such as N-alkanoyl, e.g. N-acetyl. The C-terminal group may be hydroxy 
(i.e. the peptide is an acid) or a derivative thereof, e.g. an ester 
function e.g. --O-alkyl, or an amide, e.g. --NH.sub.2, --NHalkyl or 
--N(alkyl).sub.2. 
As used herein `acyl` refers to carbonyl groups such as alkyl-, aryl- or 
aralkyl-carbonyl, e.g. having 2 to 15 carbon atoms, e.g. 2 to 7 for alkyl, 
7 to 11 for aryl and 8 to 12 carbon atoms for aralkyl. 
Examples of `alkyl` groups as used herein are straight or branched chain 
alkyl groups especially those having 1 to 6 carbon atoms e.g. methyl, 
ethyl, propyl, isopropyl and butyl. Examples of `aryl` are those of 6 to 
10 carbon atoms e.g. phenyl and naphthyl each optionally substituted. 
Examples of aralkyl are groups of 7 to 11 carbon atoms e.g. benzyl, 
phenethyl or naphthylmethyl each optionally substituted. The term 
`optionally substituted` means optional substitution by groups or radicals 
commonly used in pharmaceutical chemistry, such as alkyl, alkoxy, hydroxy, 
halo, nitro, amino, alkylamino, acylamino, carboxy, alkoxycarbonyl, 
mercapto, haloalkyl and aminocarbonyl. 
The term `aryl` also includes `heteroaryl`, i.e aromatic mono- or bi-cyclic 
groups having 5 to 10 ring atoms, at least one of which is a heteroatom, 
e.g. oxygen, nitrogen or sulphur. Examples are furanyl, thienyl, pyrrolyl, 
pyridyl, quinolyl and isoquinolyl. 
The peptides of this invention can be used with a potassium channel, e.g. 
the MK-1 channel to provide a useful screen for the development of novel 
drugs designed to interfere with the normal inactivation processes of 
potassium ion channels. Whole-cell recordings of MK-1 channels expressed 
in cell lines show the modification of MK-1 current into a rapidly 
inactivating current as the added peptide dialyses into the cell. Addition 
of test compound to the cell can be made from the extracellular or 
intracellular side to determine whether such molecules hinder the 
inactivation process, either by binding to the inactivation peptide 
itself, or its `receptor` on the ion channel. Compounds which impede the 
inactivation of potassium ion channels result in more effective braking of 
cellular activity, and lead to a decrease in both cell excitability and 
neurotransmitter release, and are potentially useful in preventing 
epileptiform activity. 
Accordingly this invention also provides a test method for screening a test 
compound for potassium channel modulating activity which comprises 
administering said test compound to a cell having a potassium channel, 
said cell containing a peptide of this invention, to determine whether the 
inactivation process is affected by said test compound. 
The cyclic peptides of this invention are prepared by a process comprising 
oxidising a corresponding linear peptide to form the disulphide bridge. 
Oxidation may be conveniently carried out by air (or gaseous O.sub.2) 
oxidation or by use of potassium ferricyanide. 
The corresponding linear peptide precursor can itself be prepared by 
deprotecting a fully or partially proctected precursor or resin supported 
precursor as described hereinafter. 
For example a fully or partially protected precursor peptide may be 
represented by the formula (II) shown in SEQ ID No: 3: 
X.sup.1 Met Ile Ser(R.sup.1) Ser(R.sup.1) Val Cys(R.sup.2) 
Val Ser(R.sup.1) Ser(R.sup.1) Tyr(R.sup.3) Arg(R.sup.4) 
(II) 
1 5 
10 
Gly Arg(R.sup.4) Lys(R.sup.5) Ser(R.sup.1) Gly Asn Lys(R.sup 
.5) Pro Pro Ser(R.sup.1) Lys(R.sup.5) 
15 20 
Thr(R.sup.1) Cys(R.sup.2) Leu Lys(R.sup.5) Glu(R.sup.6) 
Glu(R.sup.6) X.sup.2 
25 
or a variant thereof, 
where X.sup.1 is hydrogen or an .alpha.-amino protecting group, 
R.sup.1 is an hydroxy protecting group for the side chain of Ser or Thr or 
hydrogen, 
R.sup.2 is a mercapto protecting group or hydrogen, 
R.sup.3 is an hydroxy protecting group for the side chain of Tyr or 
hydrogen, 
R.sup.4 is an guanyl protecting group for the side chain of Arg or 
hydrogen, 
R.sup.5 is an amino protecting group for the side chain of Lys or hydrogen, 
R.sup.6 is a carboxy protecting group for the side chain of Glu or 
hydrogen, 
X.sup.2 is OH, a carboxy protecting group or bond to a solid phase support, 
e.g. X.sup.2 =--O--CH.sub.2 [polystyrene resin support] where the latter 
group represents one of the many functional groups present in the 
polystyrene resin; 
providing at least one protecting group is present when X.sup.2 is OH. 
When R.sup.2 is hydrogen the abovementioned precursor peptide may be 
cyclised by oxidation (e.g. gaseous O.sub.2) prior to removal of all 
protecting groups. 
Protecting groups for the .alpha.-amino group (X.sup.1) are illustrated by 
(1) acyl type protecting groups such as: formyl, trifluoroacetyl, phthalyl, 
p-toluenesulfonyl (tosyl), nitrophenylsulfenyl, etc; 
(2) aromatic urethane type protecting groups such as benzyloxycarbonyl and 
substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, 
p-nitrobenzyloxycarbonyl, 
(3) aliphatic urethane protecting groups such as tert-butyloxycarbonyl, 
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, allyloxycarbonyl, 
2,2,2-trichloroethoxycarbonyl, amyloxycarbonyl; 
(4) cycloalkyl urethane type protecting groups illustrated by 
cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl; 
(5) thiourethane type protecting groups such as phenylthiocarbonyl; 
(6) alkyl type protecting groups such as triphenylmethyl (trityl); 
(7) trialkylsilane groups such as trimethylsilane. 
The side chain nitrogen atoms of arginine, are protected by a group 
(R.sup.4) which may be nitro, tosyl, benzyloxycarbonyl, 
adamantyloxycarbonyl or tert-butyloxycarbonyl, preferably the tosyl group. 
Protection for the side chain amino group of lysine (R.sup.5) may be by 
tosyl, t-amyloxycarbonyl, t-butyloxycarbonyl, diisopropyloxycarbonyl, 
benzyloxycarbonyl, halobenzyloxycarbonyl, nitrobenzyloxycarbonyl, and the 
like, the 2-chlorobenzyloxycarbonyl group being preferred. Protection for 
the hydroxyl group of tyrosine, threonine and serine (R.sup.1, R.sup.3) 
may be with acetyl, benzoyl, tert-butyl, benzyl. The benzyl group is 
preferred for this purpose. 
The protecting group for the sulfydryl group of the cysteinyl amino acid 
residue (R.sup.2) can be a group selected from benzyl; substituted benzyl 
wherein the substituent is at least one of methyl, methoxy, nitro, or halo 
(e.g. 3,4-dimethylbenzyl, p-methoxybenzyl, p-chlorobenzyl, p-nitrobenzyl, 
etc.); trityl, benzyloxycarbonyl, benzhydryl, p-methoxybenzyloxycarbonyl, 
benzylthiomethyl, ethylcarbamoyl, thioethyl, tetrahydropyranyl, 
acetamidomethyl, benzoyl, s-sulfonate salt, etc.; p-methoxybenzyl group 
being preferred. 
The carboxy group of glutamic acid may be protected (R.sup.6) by a benzyl 
or substituted benzyl group. 
The corresponding linear protected peptide precursor may be prepared by 
known methods for building up an amino acid sequence as described in 
standard textbooks on peptide synthesis. For example solid phase 
methodology can be used where the peptide is bound to a polystyrene resin 
support or a benzhydrylamine resin support following techniques generally 
known in the art for building up amino acid sequences from an initial 
resin supported amino acid such as illustrated by Merrifield, JACS, 85 
2149 (1963). 
The resin support employed may be any suitable resin conventially employed 
in the art for the solid phase preparation of polypeptides, e.g. a 
copolymer of styrene and divinyl benzene in which the degree of 
crosslinking by the divinyl benzene is from 0.5 to 3%; which resin has 
been chloromethylated to provide sites for ester formation with the 
initially introduced protected amino acid. The projected C-terminal (amino 
protected) amino acid may be coupled to the chloromethylated resin 
according to the procedure of Gisin, Helv. Chim. Acta., 56 1476 (1973). 
Following the coupling of the first amino protected to the resin support, 
the amino protecing group may be removed by standard methods employing 
trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or 
HCl in dioxane. The deprotection may be carried out at a temperature 
between 0.degree. C. and room temperature. After removal of the amino 
protecting group the remaining a-amino protected and, if necessary, side 
chain protected amino acids are coupled, seriatim, in the desired order to 
obtain the product. Alternatively, multiple amino acid groups may be 
coupled by the solution method prior to coupling with the resin supported 
amino acid sequence. The selection of an appropriate coupling reagent is 
within the skill of the art. Particularly suitable coupling reagents are 
N,N'-diisopropylcarbodiimide and N,N'-dicyclohexylcarbodiimide. 
Each protected amino acid or amino acid sequence is introduced into the 
solid phase reactor in a two to six fold excess and the coupling is 
carried out in a medium of dimethylformamide: methylene chloride or in 
either dimethylformamide or methylene chloride alone. In cases where 
incomplete coupling occurs the coupling procedure is repeated before 
removal of the .alpha.-amino protecting group, prior to introduction of 
the next amino acid to the solid phase reactor. The success of the 
coupling reaction at each stage of the synthesis can be monitored by the 
ninhydrin reaction as described by E Kaiser et al., Analyt. Biochem., 34, 
595 (1970). 
The necessary .alpha.-amino protecting group employed for each amino acid 
introduced in the polypeptide is preferably tert-butyloxycarbonyl, 
although any such protecting group may be employed as long as it is not 
removed under coupling conditions and is readily removed selectively in 
relation to the other protecting groups present in the molecule under 
conditions which otherwise do not affect the formed molecule. Additional 
examples of such a-amino protecting groups from which selection may be 
made, after consideration of the rest of the polypeptide molecule, are 
trityl, phthalyl, tosyl, allyloxycarbonyl, cyclopentyloxycarbonyl, 
tert-amyloxycarbonyl, benzyloxycarbonyl and o- or 
p-nitrobenzyloxycarbonyl. 
The criteria for selecting protecting groups for R.sup.1 -R.sup.6 are: 
(a) the protecting group must be stable to the reagent and under the 
reaction conditions selected for removing the a-amino protecting group at 
each step of the synthesis, 
(b) the protecting group must retain its protecting properties (i.e not be 
split off under coupling conditions), and 
(c) the protecting group must be readily removable upon conclusion of the 
polypeptide synthesis, under conditions that do not otherwise affect the 
polypeptide structure. 
Standard methods of removing the protecting groups sequentially or 
simultaneously may be used. 
They may be removed before, after of simultaneously with cleavage from the 
resin support when present. Preferably cleaving and deprotection are 
carried out at the same time e.g. using hydrogen fluoride and anisole, to 
obtain the fully deprotected linear peptide. If a protected cyclic 
precursor peptide is prepared then deprotection gives the final cyclic 
peptide (I). 
Where a variant of the sequence above is desired to be prepared, the 
variant amino acid(s) protected if required is (are) incorporated at the 
appropriate stage(s) in the synthesis. 
The C-terminal group obtained by cleaving a compound of formula II where 
X.sup.2 is OCH[polystyrene resin support] using HF is the carboxy 
function, i.e X.sup.2 =COOH. Alternatively the peptide may be removed from 
the support by ammonolysis with ammonia to give a CONH.sub.2 terminal 
group, or by reaction with an amine such as alkyl NH.sub.2 to give a CONH 
alkyl group. Cleavage by transesterification gives an ester terminal group 
e.g. COOR where R is an organic radical, e.g. alkyl or aralkyl as 
illustrated hereinabove. 
The terminal amino acid function if not already modified prior to coupling 
may be modified after coupling by appropriate reaction of the precursor 
peptide, e.g. acylation using for example an acyl halide. 
The wet solution method for preparing the compounds of this invention 
comprises coupling the requisite amino acids protected, modified and/or 
activated as necessary in any order of succession to give the desired 
peptide sequence and thereafter, in any order, removing one or more 
protecting groups and oxidising if desired to give a disulphide bridge. 
The coupling of the amino acids in the above mentioned process may be 
carried out by the standard methods used in peptide chemistry. Such 
methods are described in the literature for example in standard textbooks 
on peptide synthesis--see for example Schroder and Lubke, "The Peptides", 
Academic Press 1965 and Greenstein and Winitz, "Chemistry of the Amino 
Acids" Vol. 2, John Wiley and Sons Inc. 1961. 
The following Example illustrates the invention: