Fluorescent motilin peptides

Details Fluorescent motilin peptides Fluorescent motilin peptides

2000-03-31

2004-01-13

Jones, Dwayne C. (Department: 1614)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

25 or more amino acid residues in defined sequence

C530S350000, C435S007100

Reexamination Certificate

active

06677430

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to peptide-based compounds having light-emitting moieties. Peptides may be chemically linked with detectable “labels” and used as probes, for example, to monitor peptide, cytokine, drug, and hormone receptors at the cellular level. Typically, the labeled peptide is placed in contact with a tissue or cell culture where it binds to an available receptor. Once bound, the label is detected, allowing properties such as receptor distribution or receptor binding kinetics to be monitored.
Peptides are typically labeled with radioactive elements such as
125
I or
3
H. In this case, emission of high-energy radioactive particles is monitored using standard &ggr;-ray detectors, thereby allowing detection of the label. While detection techniques for
125
I and
3
H are well-known, radioactive compounds by nature have limited half lives, and are often both toxic and expensive. Moreover, current detection technology makes it difficult or impossible to detect radioactive probes in real-time, thereby precluding study of kinetic processes.
Peptides labeled with non-radioactive molecules have also been disclosed. Macielag et al. (
Int. J. Pept. Protein Res
. (1994) 44(6):582-588)) discloses a biotin-labeled motilin peptide. However, detection of this biotin-labeled motilin peptide requires and multiple assay steps.
Motilin is a 22-amino acid polypeptide secreted from the endocrine cells of the mucosa of the upper part of the small intestine. Motilin binds to G-coupled receptors to stimulate peristaltic contractions of the stomach and small intestine during fasting (Lee et al.,
Am. J. Physiol
. (1983) 245G547-G553; Poitras , P.
Gastroenterology
(1984) 87:909-913; Scarpignato et al,
Can. J. Gastroenter
. (1999) 13(A): 50A-65A; Usellin et al.,
Histochemistry
(1984) 81:363-368). There is also evidence that suggests motilin and motilin receptors are present in the brain and have a role in the regulation of food intake (Asakawa et al.,
Peptides
(1998) 19(6):987-990).
Motilin's wide tissue distribution makes it a particularly desirable peptide to label and use to monitor cell receptors, as these peptides exhibit multiple biological roles and their receptors are located in a variety of tissues. Labeled motilin polypeptides are valuable in the identification of agonists and antagonist of motilin which may be useful as appetite stimulants or in the treatment of digestive disorders, for example, hypokinetic and hyperkinetic digestive disorders, such as irritable bowel syndrome associated constipation or diarrhea, or Chron's disease.
There exists the need for peptides that are chemically linked to detectable labels that are easily detected, yet do not decrease the biological activity of the peptide.
SUMMARY OF THE INVENTION
The present invention provides a compound containing a motilin peptide and a light-emitting moiety that is both biologically active and optically detectable. The peptide is chemically attached to the light-emitting moiety at an amino acid position that is not involved in binding to the peptide receptor. In this way, the peptide's affinity for the binding site is not significantly decreased and the compound retains high biological activity. Furthermore, the compound can be easily detected using standard optical means.
In general, in one aspect, the invention provides a biologically active compound of the formula:
where R
1
is a light-emitting moiety and R
2
is a motilin peptide, fragment, derivative or analog thereof The peptide is linked at a first amino acid position to (C—X) which, in turn, is selected from the group including C═O, C═S, CH(OH), C═C═O, C═NH, CH
2
, CH(OR), CH(NR), CH(R), CR
3
R
4
, and C(OR
3
)OR
4
where R, R
3
, and R
4
are alkyl moieties or substituted alkyl moieties. Optionally the compound may include a linker moiety between the peptide and the C—X binding group. Preferably, the compound exhibits substantial biological activity in the presence of receptors having affinities for motilin peptides. The compound may also be in the form of a pharmaceutically acceptable salt or complex thereof. Preferably, one of the C-terminal eight residues of the motilin peptide of SEQ ID NO:1(referred to herein is the C-terminal octapeptide) that is not required for motilin receptor binding or stability is attached to (C—X), either directly or through a linker moiety (Macielag et al., supra).
In preferred embodiments, the motilin peptide can be any peptide that shares sufficient homology with SEQ ID NO:1, Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln or a fragment, derivative, or analog thereof. In particularly preferred embodiments, motilin peptides homologs, such as the canine motilin SEQ ID NO:2, Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gly (MW 2685) and porcine motilin SEQ ID NO:3, Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln (MW 2697) may be used as motilin peptides in the present invention. Alternatively, the motilin peptide could be a modified motilin peptide that includes various substitutions or contains modified amino acids, such as beta-alanine or norleucine and the like. In preferred embodiments, any one of the eight residues of the C-terminal octapeptide of motilin is preferably chemically bound to the (C—X) moiety of the present invention. In one preferred embodiment, the motilin peptide is chemically bound to the (C—X) moiety through Lys
20
. In a particularly preferred embodiment, the peptide is attached to the (C—X) moiety through Lys
20
and the (C—X) bond is preferably either C═O or C═S. In yet another preferred embodiment, the peptide may be amidated at the C-terminus.
In other preferred embodiments, the light-emitting moiety (R
1
) is selected from the group including 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (e.g., 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino) hexanoic acid) (BODIPY® TMR) or 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY® 576/589)), fluorescein, fluorescein isothiocynate (FITC, Texas red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, indopyras dyes, Cascade blue, coumarins, nitrobenzo-2-oxa-diazole (NBD), Lucifer Yellow, propidium iodide, CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates, lanthanide chelates, non-fluorescent dialdehydes (OPA, NDA, ADA, ATTOTAG reagents from Molecular Probes) which react with primary amines (N-term lys) in the presence of a nucleophile (i.e. CN) to form fluorescent isoindoles, dansyl dyes fluorescamine and dabcyl chloride, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid, long lifetime dyes comprised of metal-ligand complexes (MLC) which consist of a metal center (Ru, Re, Os) and organic or inorganic ligands complexed to the metal such as [Ru(bpy)
3
]
2+
and [Ru(bpy)
2
(dcbpy)], and the like and derivatives thereof. The light-emitting moiety can be attached to the peptide by reaction of a reactive side group (of a light-emitting molecule) with any C-terminal eight residues of the motilin peptide. Suitable reactive side groups include, by way of example only, indoacetamide, maleimide, isothiocyanate, succinimidyl ester, sulfonyl halide, aldehydes, glyoxal, hydrazine and derivatives thereof.
The above-identified compound is useful in the labeling of cell receptor sites, cell sorting, flow cytometry and performing fluoroimmunoassays. In another aspect, the invention provides a method for labeling a receptor having an affinity for a motilin peptide by contacting the receptor with one or more of the compounds described above. Cell receptor sites, can be imaged by contacting candidate cell receptor sites with the compound of the invention, and then detecting the bound compounds as an indication of the cell receptor sites. Cell sorting can be performed by contacting a population of cells with compound and isolating cells bound to the c

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