Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-09-26
2004-03-23
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C436S086000, C530S300000
Reexamination Certificate
active
06710026
ABSTRACT:
The present invention relates to compounds useful in the treatment of disorders associated with abnormal blood glucose levels, particularly in the prevention of phosphorylase-a binding to the glycogen targeting subunit (G
L
) of protein phosphatase 1 (PP1). Such compounds are useful for increasing glycogen synthesis and thereby reducing blood glucose levels. The compounds find utility in the treatment of disorders, such as type I and type II diabetes, associated with higher than normal levels of blood glucose (hyperglycaemia).
Most of the adverse physiological consequences in type I and type II diabetes arise from the higher than normal levels of blood glucose. Although high blood glucose levels can be reduced by administration of insulin in type I diabetes and by dietary restrictions in the case of type II diabetes, a drug which aids reduction of blood glucose levels would be advantageous in the treatment of these disorders. The liver, which is the main organ regulating glucose homeostasis, is able to store glucose in the form of glycogen and the synthesis of hepatic glycogen from glucose is under the control of hepatic glycogen synthase.
Protein phosphatase 1 is major protein serine/threonine phosphatase in eukaryotic cells, which regulates numerous distinct cellular processes. This is achieved by the interaction of die catalytic subunit of PP1 (PP1C) with a diverse range of targeting subunits that localise PP1 C to specific sites within the cell, modulate its activity towards particular substrates and allow its activity to respond to extracellular signals.
The family of proteins that target PP1 to glycogen and regulate its activity towards the enzymes of glycogen metabolism consists of four members, G
M
/PPP1R3, G
L
/PPP1R4, PPP1R5 and PPP1R6/PTG. The liver specific glycogen-targeting subunit, G
L
is a 33 kDa protein that, when bound to PP1, enhances the rate at which the latter dephosphorylates and activates the rate determining enzyme in glycogen synthesis, glycogen synthase, whilst suppressing the rate at which it inactivates glycogen phosphorylase. The stimulation of hepatic glycogenolysis by glucagon (acting via cyclic AMP and PKA (Protein Kinase A)) and &agr;-adrenergic agonsists (acting via Ca
2+
) is achieved by activation of phosphorylase kinase, which increases the levels of the active phosphorylated form of glycogen phosphorylase (phosphorylase a). In addition, phosphorylase a binds to G
L
and potently inhibits its glycogen synthase phosphatase activity thereby inhibiting glycogen synthesis. Insulin lowers hepatic cAMP levels, causing a reduction in the level of phosphorylase a and alleviation of the phosphorylase a-mediated inhibition of the PP1G
L
complex, while the binding of glucose to phosphorylase a, increases the rate at which phosphorylase is inactivated. These mechanisms contribute to the stimulation of glycogen synthesis by insulin and high blood glucose. The inhibition of the PP1G
L
complex by phosphorylase a occurs at nanomolar concentrations and is thought to be via an allosteric mechanism since the K
m
for phosphorylase a as a substrate is in the micromolar range. This view is strengthened by the finding that phosphorylase a (but not phosphorylase b) binds directly to G
L
in protein blotting experiments.
Recent studies identified conserved regions between the glycogen targeting subunits G
M
/PPP1R3 and G
L
/PPP1R4. A peptide corresponding to one of these regions. G
M
63-75 (amino acids 63 to 75 of G
M
) was shown to bind PP1 and the amino terminal 38 residues of the myofibrillar targeting subunit of PP1 were also demonstrated to interact with PP1 . The G
M
63-75 peptide, which contains a small motif common to the myofibrillar binding subunit and many other of the PP1 targeting subunits has been crystallised as a complex with PP1 and its structure solved to 2.8 Å resolution. This motif, Lys/Arg-Val/Ile-Xaa-Phe/Trp, which has also been identified by a random peptide library approach, is found in all the four glycogen targeting subunit and is located at residues 60-64 of G
L
. However, incubation of the PP1G
L
complex purified from hepatic glycogen-protein particles with a PP1-binding peptide from G
M
failed to dissociate the PP1-G
L
complex, even though the peptide abolished the suppression of phosphorylase phosphatase activity conferred on PP1 by association with G
L
.
The present invention seeks to provide biological materials and methods which may be useful in the treatment of disorders, especially those such as diabetes type I and type II, associated with higher than normal levels of blood glucose.
According to a first aspect of the invention there is provided the use in medicine of a compound which is capable of blocking the interaction of phosphorylase a with the glycogen-targeting subunit (G
L
) of protein phosphatase 1.
Preferably, the compound is for use in the manufacture of a medicament for use in treating disorders associated with higher than normal levels of blood glucose. Preferably the medicament is for use in the treatment of a disorder selected from type I and/or type II diabetes.
Preferably, the compound is a polypeptide comprising the sequence of the C-terminal 16 amino acids of human G
L
sequence, or a fragment or variant thereof of which is capable of binding phosphorylase a.
Thus, the sequence may be PEWPSYLGYEKLGPYY (SEQ ID. NO: 1), which may be the sequence of the C-terminal 16 amino acids of rat liver G
L
.
By “variant” we include the meaning of polypeptides comprising an amino acid sequence which, although not identical to the 16 amino acid sequence, are capable of binding phosphorylase a.
By “fragment” we include the meaning that the polypeptide comprises less than the 16 amino acid sequence mentioned above, but is capable of binding phosphorylase a.
The identification of variants and fragments within the scope of the invention can be carried out using the methods described herein.
Preferably the polypeptide increases the activity of hepatic glycogen synthase.
Polypeptides in which one or more of the amino acid residues are chemically modified, before or after the polypeptide peptide is synthesised, may be used in accordance with the invention, providing that the function of the peptide, namely the blocking of the interaction between G
L
and phosphorylase a, remains substantially unchanged. Such modifications include forming salts which acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, and attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such modifications may protect the peptide from in vivo metabolism.
The peptides may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may increase the activity of the polypeptide in blocking the binding of G
L
and phosphorylase a.
In a second aspect, the invention provides a pharmaceutical composition comprising an inhibitor compound which is capable of blocking the interaction of phosphorylase a with the glycogen targeting subunit (G
L
) of protein phosphatase (PP1), together with a pharmaceutically acceptable excipient or carrier. Preferably the inhibitor compound comprises a polypeptide having the sequence of the C-terminal 16 amino acids of human G
L
sequence, or a fragment or variant thereof of which is capable of binding phosphorylase a, for example the 16 amino acid sequence PEWPSYLGYEKLGPYY (SEQ ID. NO: 1) or a fragment or variant thereof of which is capable of binding phosphorylase a.
In a third aspect, the invention provides a method of identifying an inhibitor compound that is capable of blocking the interaction of phosphorylase a with the glycogen-targeting subunit of PP1 comprising: providing a polypeptide comprising the sequence of the C-terminal 16 amino acids of human G
L
sequence, or a fragment or variant thereof of which is capable of binding phosphorylase a, for example the 16 amino acid sequence PEWPSYLGYEKLGPYY (SEQ ID. NO: 1), or a fragment or variant thereof which binds ph
Armstrong Christopher George
Cohen Patricia Townsend Wade
Doherty Martin John
Medical Research Counsel
Nashed Nashaat T.
Rogalsky, I Weyand, LLP
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