Biologically active peptide of ob protein

Details Biologically active peptide of ob protein Biologically active peptide of ob protein

1999-09-09

2001-02-13

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C435S069400, C514S013800, C514S014800, C530S324000, C530S326000, C530S327000, C530S345000, C530S410000

Reexamination Certificate

active

06187751

ABSTRACT:

The invention relates to novel compounds, in particular novel proteins, to compositions containing such compounds and to the use of such compounds in medicine.
The mechanism of the physiological regulation of energy balance in the body—food intake verses energy output—has been the subject of debate for many years. In a recent publication in Nature (Y. Zhang et al, Nature, 372, 425-431, 1994) suggest that one of the molecules which plays a key role in energy balance regulation is the ob protein. Zhang et al also report the cloning and sequencing of both mouse and human ob gene protein.
To date there is no indication in the art as to the nature of the secondary or tertiary structural characteristics of the human or mouse ob protein. Zhang et al also report that the ob protein amino acid sequence has no significant homology with any sequence in Genbank.
It has now been indicated that the tertiary structure of the mouse and human ob proteins comprises a modified form of a four-helix bundle structure with an ‘up-up, down-down’ topology. In addition, novel molecules with the potential to mimic the physiological activity of the ob protein have been identified during investigations into the regions of the ob protein sequence which are indicated to interact with a putative receptor for this protein.
Such molecules have potential use in the treatment of nutritional and metabolic disorders, particularly obesity and diabetes in the case of agonists and anorexia and cachexia in the case of antagonists.
Accordingly, in a first aspect, the present invention provides a protein fragment of the ob protein, being an active site of said protein.
The active site is suitably provided by the ob protein when it is in the form of a four helix bundle structure, particularly that having an up-up down-down topology.
In particular, the active site is formed from one or more amino acids selected from one or more of the four helices forming the secondary structure of the ob protein, especially a protein fragment consisting of amino acid residues 26 to 39, 74 to 88, 93 to 113 or 142 to 161.
A suitable active site is formed from one or more amino acids selected from: R41, D30, T37, K26, T33, T40, R149, Q155, V145, Q151, S148,L158, W159, S73, Q84, Q77, V81, K74,S88,A80,T87, R105, S98, E102, I95, D106,N99 and H109 on the ob protein.
Hereinafter protein fragments of the ob protein will be referred to using an analogous abbreviation to the following: ‘the protein fragment consisting of amino acid residues 26 to 39’ is abbreviated to ‘ob26-39’.
A suitable active site is formed from one or more amino acids selected from ob protein fragments ob26-39 and ob93-113.
A suitable active site is formed from one or more amino acids selected from ob protein fragments ob 74 -88 and ob142-161.
A suitable active site is formed from amino acids:
R41, D30, T37, K26, T33, T40, R149, Q155, V145, Q151, L158 and W159 selected from the amino acid sequence of the human ob protein.
A suitable active site is formed from amino acids:
S73, Q84, Q77, V81, K74, R105, S98, E102, I92, D106 and H109 selected from the amino acid sequence of the human ob protein.
A particular ob gene is the human ob gene.
Particular compounds with the potential to mimic the physiological activity of the ob protein are those formed by linking together amino acids selected from the above mentioned active sites. Accordingly, in a further aspect the invention provides a compound of formula (I):
wherein:
T
1
and T
2
are optional N-terminator groups such as aliphatic acyl, &ohgr;-methoxy, &agr;-oxycarbonyl or polyethylene glycol which may as further options be linked covalently to each other or incorporate moieties capable of targeting the construct to the blood-brain barrier such as fatty acid esters or phospholipids;
S
1
, S
2
, S
3
and S
4
are each linear peptide sequences of 10-30 aminoacids (preferably 12-20) which amino acids are selected from a sequentially contiguous residue from the amino acid sequence of a helix of the ob protein;
H
1
, H
2
, H
3
and H
4
each independently represents a bond or a linear sequence of 3-12 aminoacids;
(L)M and (L)N each independently comprise small hydrophilic aminoacid repeats, preferably of glycine or alanine. n and m being 1-8, preferably 2-4;
X
1
and X
2
are each either a bond, the aminoacid cysteine (in which case a disulphide bond forms the link between the sequence pairs), a homo- or heterobifunctional cross-linking agent (such as N,N′bis-maleimido diaminoalkane) so as to extend the length of the bridge within the range 2-20 atoms, preferably 2-8, or X represents glycine.
A particular helix of the ob protein is a protein fragment selected from ob26-39, ob74-88, ob93-113 or ob142-61.
Suitably, S
1
, S
2
, S
3
and S
4
represents a linear peptide sequences of amino acids selected from the sequentially contiguous residues of a helix of the ob protein, example include AKVQDDTKTLIKTIVTRI (SEQ ID NO: 1), SKMDTLAVYQQILT (SEQ ID NO: 2), NVIQISNDLENLRDLLHVLAFSK (SEQ ID NO: 3), and TEVVALSRLLQGSLQDMLWQL (SEQ ID NO: 4).
Examples of H sequences include AK,AQ and LQ repeats. These sequences will be prepared as fusions such A-(Linker)-D and B-(Linker)-C and may be combined as (A-D)-(B-C) heterodimers or as homodimers such as (A-D)
2
. Optionally, three helical sequences may be employed (e.g (A-B) - D).
In particular the invention includes the proteins having the sequence ID Nos set out below.
The invention also extends to the functional analogues, variants and derivatives of the proteins mentioned herein, including functional analogues, variants and derivatives of the peptide fragments and compound of formula (I).
Functional analogues includes functionally analogous proteins wherein one or more amino acids of the proteins mentioned herein are replaced with alternative amino acids.
Functional analogues also include small molecule agonists or antagonists of the compounds of formula (I) or the proteins mentioned herein, which may be identified and prepared by methods such as those disclosed in International patent application, publication number WO9605309.
Functional derivatives includes the proteins of the invention chemically modified by the attachment of groups or moieties so as to improve the physical properties, such as stability, or the therapeutic properties, for example the pharmaco kinetic properties, of the protein.
The peptides of the present invention may be prepared by conventional methods using liquid or solid-phase peptide peptide synthesis or by expression of DNA encoding such sequences in a suitable host. Preferably, that host will be bacterial, more preferably
Echerichia coli
K12.
Following synthesis or expression and purification by standard methods, the isolated peptides are coupled either directly molecular oxygen or other oxidising agents (homodimerisation) or by conversion on one component to a thiol-reactive form (for example a 2-pyridyldthio- derivative) for reaction with the free thiol form of the second component (heterodimerisation).
‘Amino acid’ is understood herein to mean naturally occurring L-amino acid. All representations of peptides herein follows the normal convention of N-terminal at the left and C-terminal at the right.
Peptide bonded units of the proteins associated with the present invention can be prepared by standard peptide synthesis techniques using a peptide synthesiser (Atherton, E. and Sheppard, R. C. (eds.) (1989) Solid Phase Peptide Synthesis: A practical approach, LRL Press, Oxford) followed by procedures appropriate to direct disulphide or amide bond formation.
Methods of well-known peptide synthesis are set forth by Ali et al.,
J. Med. Chem.,
29:984 (1986) and
J. Med. Chem.,
30:2291 (1987) and are incorporated by reference herein. Preferably, the peptides are prepared by the solid phase technique of Merrifield (
J. Am. Chem. Soc.,
85:2149 (1964)). However, a combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, tetra-, or penta-peptide fragments may be prepared by solid phase synthesis and either coupled or

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