The present invention relates to novel peptides and derivatives thereof useful as inhibitors of neuronal amine transporters of neurotransmitters such as noradrenaline, serotonin, dopamine, glutamic acid and glycine. The invention also relates to pharmaceutical compositions comprising these peptides, nucleic acid probes useful in finding active analogues of these peptides, assays for finding compounds having neuronal noradrenaline transporter inhibitory activity and the use of these peptides in the prophylaxis or treatment of conditions such as but not limited to incontinence, cardiovascular conditions and mood disorders.
The marine snails of the genus Conus (cone snails) use a sophisticated biochemical strategy to capture their prey. As predators of either fish, worms or other mollusks, the cone snails inject their prey with venom containing a cocktail of small bioactive peptides. These toxin molecules, which are referred to as conotoxins, interfere with neurotransmission by targeting a variety of receptors and ion-channels. The venom from any, single Conus species may contain more than 100 different peptides. The conotoxins are divided into classes on the basis of their physiological targets. To date, ten classes have been described. The ω-conotoxin class of peptides target and block voltage-sensitive Ca2+-channels inhibiting neurotransmitter release. The α-conotoxins and ψ-conotoxins target and block nicotinic ACh receptors, causing ganglionic and neuromuscular blockade. Peptides of the μ-conotoxin class act to block voltage-sensitive Na+-channels inhibiting muscle and nerve action potentials. The δ-conotoxins target and delay the inactivation of voltage-sensitive Na+-channels, enhancing neuronal excitability. The κ-conotoxin class of peptides target and block voltage-sensitive K+-channels, and these also cause enhanced neuronal excitability. The conopressins are vasopressin receptor antagonists and the conantokins are NMDA receptor antagonists. More recently, the prototype of a new γ-conotoxin class, which targets a voltage-sensitive nonspecific cation channel, and of a new σ-conotoxin class, which antagonizes the 5HT3 receptor, have been described.
It has now been found that a new class of conotoxin exists, hereinafter referred to as the χ-conotoxin class, which are characterised by having the ability to inhibit neuronal amine transporters.
Compounds which inhibit neurotransmitter reuptake have been found to be useful in the treatment of lower urinary tract disorders, such as urinary incontinence, detrusor instability and interstitial cystitis. One such compound is “imipramine” which, in addition to inhibiting noradrenaline reuptake, has been shown to affect calcium channel blockade, and to exhibit anticholinergic, local anaesthetic activity and a number of other effects. Other compounds capable of inhibiting noradrenaline reuptake are described in U.S. Pat. No. 5,441,985. These compounds are said to have a reduced anticholinergic effect relative to imipramine.
In the case of the peptides of the present invention this inhibition of neurotransmitter reuptake is achieved by selectively inhibiting the neuronal neurotransmitter transporter, such as the noradrenaline transporter, which functions to rapidly clear released noradrenaline from the synapse back into neurons.
The peptides of the present invention are the first peptides to have activity in inhibiting an amine transporter. All other conotoxin peptides characterised to date target ion channels or receptors on cell surfaces.
According to one aspect of the present invention there is provided an isolated, synthetic or recombinant χ-conotoxin peptide having the ability to inhibit a neuronal amine transporter.
Preferably, the neuronal amine transporter is the neuronal noradrenaline transporter.
The χ-conotoxin peptide may be a naturally occurring peptide isolated from a cone snail, or a derivative thereof.
Preferably the χ-conotoxin peptide is χ-MrIA or χ-MrIB, or a derivative thereof. χ-MrIA and χ-MrIB may be isolated from the venom of the mollusk hunting cone snail, Conus marmoreus.
They are both peptides of 13 amino acid residues in length, and contain 2-disulphide bonds; the peptides show most homology to members in the α-conotoxin class, which act as nicotinic ACh receptor antagonists.
The amino acid sequences of χ-MrIA and χ-MrIB are as follows:
SEQ ID NO. 1
SEQ ID NO. 2
The C-terminus may be a free acid or amidated.
In the sequences above the “O” refers to 4-hydroxy proline (Hyp). This amino acid residue results from post translational modification of the encoded peptide and is not directly encoded by the nucleotide sequence.
Preferably, the χ-conotoxin peptide is a selective inhibitor of the neuronal noradrenaline transporter. The terms “selective” and “selectively” as used herein mean that the activity of the peptide as an inhibitor of neuronal noradrenaline transporter is considerably greater than any activity at the α1-adrenoceptors.
U.S. Pat. No. 5,441,985 indicates that inhibitors of noradrenaline reuptake which have a negligible anticholinergic effect are particularly useful in the treatment of lower urinary tract disorders. It has been found that χ-MrIA also has no detectable anticholinergic effect.
Accordingly in a preferred embodiment of the invention there is provided an isolated, synthetic or recombinant χ-conotoxin peptide having the ability to selectively inhibit neuronal noradrenaline transporter, and having negligible or no anticholinergic effect.
χ-MrIA has also been found to have no activity as a sodium channel blocker or as an inhibitor of dopamine transporter. The absence in χ-MrIA of these additional pharmacological activities commonly associated with other noradrenaline transporter inhibitors and in preferred peptides according to the invention, makes these peptides useful pharmacological tools.
The χ-conotoxin peptides according to the invention may be naturally occurring peptides, such as χ-MrIA and χ-MrIA or may be derivatives of naturally occurring peptides.
The term “derivative” as used herein in connection with naturally occurring χ-conotoxin peptides, such as χ-MrIA and χ-MrIB, refers to a peptide which differs from the naturally occurring peptides by one or more amino acid deletions, additions, substitutions, or side-chain modifications. Such derivatives which do not have the ability to inhibit neuronal noradrenaline transporter do not fall within the scope of the present invention.
Tyr. It is to be understood that some non-conventional amino acids may also be suitable replacements for the naturally occurring amino acids. For example ornithine, homoarginine and dimethyllysine are related to His, Arg and Lys.
Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g. substituting a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.
Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.
Preferably, amino acid substitutions are conservative.
Additions encompass the addition of one or more naturally occurring or non-conventional amino acid residues. Deletion encompasses the deletion of one or more amino acid residues.
As stated above the present invention includes peptides in which one or more of the amino acids has undergone sidechain modifications. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH. Any modification of cysteine residues must not affect the ability of the peptide to form the necessary disulphide bonds. It is also possible to replace the sulfhydryl groups of cysteine with selenium equivalents such that the peptide forms a diselenium bond in place of one or more of the disulphide bonds.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with dimethylpyrocarbonate.
Praline residue may be modified by, for example, hydroxylation in the 4-position.
A list of some amino acids having modified side chains and other unnatural amino acids is shown in Table 1.