Fluorescent CRF receptor-binding peptides

Details Fluorescent CRF receptor-binding peptides Fluorescent CRF receptor-binding peptides

1999-07-19

2004-01-20

Jones, Dwayne C. (Department: 1614)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S324000, C435S007100

Reexamination Certificate

active

06680367

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.
CRF is a hypothalamic peptide hormone that plays an important role in coordinating autonomic, endocrine and behavioral stress responses in both the brain and the periphery. Specifically, CRF stimulates the synthesis and release of adrenocorticotrophic hormone (ACTH) from the pituitary gland. Beta-endorphin and other proopiomelanocortic (POMC)-derived peptides are synthesized and released from the cortex, cerebellum and possibly other sites in the body. The subsequent ACTH-induced release of adrenal glucocorticoids represents the final stage in the hypothalamic-pituitary-adrenal axis (HPA), which mediates the endocrine reponse to stress. CRF may also affect a variety of peripheral functions, including cardiovascular activity, inflammation, reproduction and integration of the immune system's response to stress. Clinical studies have shown that CRF hypersecretion is associated with various diseases, such as major depression, anxiety-related illness, eating disorder, as well as inflammatory disorder. Low levels of CRF were found in Alzheimer's disease, dementias, obesity, and many endocrine diseases. Several peptides, for example sauvagine, urocortin and urotensin have sequence and biological activity similar to that of CRF. Urocortin is a neuropeptide that may play a role in some CRF-mediated actions as well as appetite suppression and inflammation. Sauvagine regulates blood pressure, plasma hormone concentrations, and neuronal and gastric activity.
Corticotropin releasing factor (CRF) and related peptides all bind to CRF receptors that belong to the superfamily of G protein-coupled receptors, which includes calcitonin and calcitonin-like receptors. CRF related peptides are particularly desirable peptides 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. For example, the CRF receptors fall into two distinct classes termed CRF1 and CRF2 receptors. The CRF2 receptor exists as three splice variants of the same gene that have been designated CRF2a, CRF2b and CRF2g. The pharmacology and localization of all CRF receptor proteins in the brain has been well established. The CRF1 receptor subtype is localized primarily to cortical and cerebellar regions of the brain, while the CRF2a receptor is localized to subcortical regions. The CRF2b receptor is primarily localized in the brain to cerebral arterioles and to choroid plexus heart skeletal muscle. The CRF2g receptor has most recently been identified in human amygdala.
There exists the need for CRF receptor-binding peptides that are chemically linked with detectable labels that are easily detected, yet do not decrease the biological activity of the peptide. Such labeled peptides are generally useful in the study of CRF related peptides and peptide binding receptors and may lead to the discovery of novel agents for treatment of depression, anxiety and other CRF related illnesses.
SUMMARY OF THE INVENTION
The present invention provides a compound containing a CRF related 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, R
2
is a CRF-related peptide and fragment, derivative or analog thereof and L is a linker moiety, which may be present or absent. 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 CRF-related peptides. The compound may also be in the form of a pharmaceutically acceptable salt or complex thereof. Preferably, the N-terminus of said CRF-related peptide is attached to (C—X), either directly or through a linker moiety.
In preferred embodiments, the CRF related peptide can be any peptide that shares sufficient homology or activity with CRF (SEQ ID NO:1). In particularly preferred embodiments, the CRF related peptide is any one of sauvagine, urocortin, urotensin or CRF. Sauvagine includes the amino acid sequence Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile (Sequence ID NO.2). Urocortin includes the amino acid sequence Asp-Asn-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Thr-Leu-Leu-Glu-Leu-Ala-Arg-Thr-Gln-Ser-Gln-Arg-Glu-Arg-Ala-Glu-Gln-Asn-Arg-Ile-Ile-Phe-Asp-Ser-Val (SEQ ID NO:3). Alternatively, the CRF related peptide could be a modified CRF or CRF related peptide that contains various substitutions, deletions or modified amino acids. The peptide [Nle 21,38]CRF contains norleucine at positions 21 and 38 and includes the amino acid sequence Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile (SEQ ID NO:4). It is preferred that the first residue of the peptide is attached to the (C—X) moiety and is preferably chemically bound to the (C—X) moiety through the N-terminal amino acid. In still other preferred embodiments, the (C—X) bond is either C═O or C═S. In 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, fluorescein, FITC, Texas red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4′6-diamidino-2-phenylindole, 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 &ls

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