Peptide binding the KLVFF-sequence of amyloid-&bgr;

Details Peptide binding the KLVFF-sequence of amyloid-&bgr; Peptide binding the KLVFF-sequence of amyloid-&bgr;

1998-06-10

2001-12-18

Borin, Michael (Department: 1631)

Chemistry: analytical and immunological testing

Biospecific ligand binding assay

C435S007100, C514S002600, C514S017400, C514S018700

Reexamination Certificate

active

06331440

ABSTRACT:

INTRODUCTION
The present invention relates to compounds, which are of special interest by their ability to bind to the KLVFF-sequence in the peptide amyloid &bgr; and to inhibit polymerization of the amyloid &bgr; peptide. The compounds according to the invention are e.g. useful as medicaments and as tools for identification of substances to be used in the treatment or prevention of amyloidosis.
BACKGROUND OF THE INVENTION
Amyloidosis is a condition which is characterized by the deposition of amyloid in organs or tissues of the human or animal body, either as a primary disease or unknown cause or secondary to chronic disease, such as tuberculosis or osteomyelitis. In addition, it has also be found that the pre-eminent neuropathological feature of Alzheimer's disease (AD), a chronic condition of brain atrophy, is the deposition of amyloid in the brain parenchyma and cerebrovasculature (D. J. Selkoe,
Neuron
6, 487-498 (1991); D. J. Selkoe,
Annu. Rev. Cell Biol.
10, 373-403 (1994)).
The basic component of such amyloid is a peptide termed amyloid &bgr;, or A&bgr; (G. C. Glenner, C. W. Wong,
Biochem. Biophys. Res. Commun.
120, 885-890 (1984)). It is a 40 to 42 amino acids long proteolytic fragment of the Alzheimer amyloid precursor protein (APP), a protein expressed in most tissues (J. Kang, et al.,
Nature
325, 733-736 (1987)). Genetic and neuropathological studies provide strong evidence for a central role of A&bgr; in the pathogenesis of AD, but the pathophysiological consequences of the amyloid deposition are still unclear. However, it has been suggested that A&bgr; polymers and amyloid are toxic to neurons, either directly or indirectly, and hence cause neurodegeneration (C. Behl, J. B. Davis, R. Lesley, D. Schubert,
Cell
77, 817-827 (1994); D. T. Loo, et al., ibid 90, 7951-7955 (1995)).
The amyloid associated with Alzheimer's disease (AD) consists of thin fibrils of polymerized A&bgr;. A rational pharmacological approach for the prevention of amyloidogenesis would therefore be to use drugs that specifically interfere with A&bgr;—A&bgr; interaction and polymerization. Previous studies showed that A&bgr; polymerization in vivo and in vitro is a highly specific process, which probably involves an interaction between binding sequences in the A&bgr; peptide (J. Näslund, et al.,
Proc. Natl. Acad. Sci. USA
91, 8378-8382 (1994); J. Näslund, et al.,
Biochem. Biophys. Res. Commun.
204, 780-787 (1994)).
Wood et al (S. J. Wood, R. Wetzel, J. D. Martin, M. R. Hurle,
Biochemistry
34, 724-730 (1995)) suggest that amino acid residues within or close to A&bgr;-16-20 are important for the adoption of the correct &bgr;-pleated sheet structure of A&bgr; and show that amino acids 17-23 in the amyloid &bgr; peptide (A&bgr;) are essential for fibril formation and probably make up the &bgr;-sheet core of the fibrils. In addition, Wood et al. have investigated the ability of their peptides to form amyloid fibrils in a solution containing solely the mutated or the wild-type peptide. However, no method or principle which makes it possible to inhibit A&bgr; of wild type from forming amyloid fibrils is devised and no use of the peptides as medicaments is suggested.
WO 95/08999 relates to amelioration of amnesia in Alzheimer's disease caused by deposition of amyloid &bgr; protein. Three peptides are disclosed, which overcome the amnestic effects of &bgr;-12-28, a peptide homologous to A&bgr;. In addition, WO 95/08999 describes the screening of several other peptides, which were neither significantly amnestic nor memory enhancing, of which one is KLVFF, SEQ. NO. 15 of the sequence listing therein.
In EP 0 584 452, novel amyloid precursor proteins and the sequences thereof are disclosed. Peptide sequences that comprise KLVFF are revealed. However, neither binding to amyloid &bgr; peptide nor any inhibition of the polymerization thereof is suggested.
SUMMARY OF THE INVENTION
Thus, the polymerization of the amyloid &bgr; peptide (A&bgr;) into amyloid fibrils is a critical step in the pathogenesis of Alzheimer's disease.
In vitro and in vivo studies of A&bgr; have shown that the A&bgr; molecules interact with a high degree of specificity during polymerization and fibril formation. It was assumed that ligands which bind to recognition sequences would be capable of inhibiting A&bgr; polymerization and possibly also dissolve preformed A&bgr; polymers in situ. The strategy in finding such A&bgr; ligands was to identify critical binding regions in A&bgr; and, based on their sequences, develop a compound capable of blocking the A&bgr;—A&bgr; binding.
According to the invention, it was hypothesized that compounds capable of binding to regions in the A&bgr;-molecule critical for its polymerization might inhibit amyloid fibril formation, as described in more detail below.
According to the invention, it has now been found that the Lys-Leu-Val-Phe-Phe (KLVFF) (SEQ ID NO:1) sequence in A&bgr; is necessary for polymerization to occur. Peptides incorporating this sequence bind to A&bgr; and are capable of blocking the fibril formation of A&bgr;-1-40 and are therefore potentially useful as drugs.
In addition, compounds have been found, which
1) are capable of binding to full-length A&bgr;,
2) are capable of blocking A&bgr; fibril formation and
3) do not form fibrils by themselves.
In addition, it has also been found that alanine-substituted A&bgr;-1-28 (Ala at position 16,17,20), in contrast to wild-type A&bgr;-1-28, does not form fibrils.
Thus, it was concluded that the Lys-Leu-Val-Phe-Phe (16-20) motif serves as a structural basis for the development of peptide and non-peptide agents aimed at inhibiting amyloidogenesis in vivo. This is a novel finding and the compounds are of utmost interest as being useful as drugs for Alzheimer's disease.
Further, the findings according to the invention are even more surprising on the basis of what was concluded from WO 95/08999 mentioned above. In WO 95/08999, it was concluded that KLVFF is not a potential candidate for the development of substances that can antagonize binding of A&bgr; and thus attenuate symptoms and progression of AD. Even though the teaching of said WO publication indicates the opposite, according to the present invention, it has now been found that KLVFF on the contrary is most useful for the development of new compounds defined by Formula (I) and (II) below.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds which are able to bind to the Lys-Leu-Val-Phe-Phe-sequence, or KLVFF-sequence, in the peptide amyloid &bgr;. More specifically, the compounds according to the invention are defined by their formula (I):
R
1
—A′—Y′—Leu—X′—Z′—B′—R
2
  (I)
in which
X′ means any group or amino acid imparting to the compound of formula (I) the ability to bind to the KLVFF-sequence in amyloid &bgr; peptide, or two amino acids imparting the same ability, but with the proviso that one is not proline;
Y′ means any amino acid;
Z′ means any non-acidic amino acid;
A′ means a direct bond or an &agr;-amino acid bonded at the carboxyl terminal of the &agr;-carboxy group or a di-, tri-, tetra- or pentapeptide bonded at the carboxyl terminal of the &agr;-carboxy group;
B′ means a direct bond or an &agr;-amino acid bonded at the &agr;-nitrogen or a di-, tri-, tetra- or pentapeptide bonded at the &agr;-nitrogen or the N-terminal &agr;-amino acid;
R
1
is H or —CO—R
3
bonded at the &agr;-amino group of A′;
R
2
is H, —OR
4
or NR
5
R
6
, all bonded to the &agr;-carboxyl group of the &agr;-carboxyterminal of B′;
R
3
is a straight or branched carbon chain of 1-4 carbon atoms;
R
4
is a straight or branched carbon chain of 1-4 carbon atoms;
R
5
and R
6
independently are H, alkyl, cycloalkyl, aryl or substituted aryl or together are —(CH
2
)
n
—, where n is 4-5;
R
1
and R
2
together can form a hydrocarbon ring or heterocyclic ring; and
all the &agr;-amino acids can be wither D- or L-isomers;
with the proviso that (I) is not Lys-Leu-Val-Phe-Phe

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