Alkyl peptide amides and applications

Details Alkyl peptide amides and applications Alkyl peptide amides and applications

2000-08-01

2003-08-26

Borin, Michael (Department: 1631)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C514S013800, C530S326000, C530S327000

Reexamination Certificate

active

06610657

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
This invention pertains to compositions capable of regulating protein activity, particularly activity dependent upon binding between two or more proteins. Specifically, the invention pertains to compositions and methods for delivering a peptide inhibitor moiety into a living cell including moieties, capable of inhibiting binding between a protein kinase and at least one other protein in the cell such as anchoring proteins.
Signaling molecules, such as hormones and neurotransmitters, elicit cellular responses through interrelated biochemical reactions. A variety of specific and selective reagents have been developed, which make it possible to study how such signaling molecules mediate such cellular responses in vitro. The study of such responses is an important first step in the discovery and development of new drugs. The development of specific and selective reagents capable of being used, in similar studies in either cell culture medium or in a whole animal is of equal importance. The most preferred such reagents are, traditionally, ones which enable the study of specific processes in living cells without breaking the cell or introducing artifacts therein.
Synthetic molecules such as synthetic peptides or peptidomimetics capable of interacting with, or competing with signaling protein molecules, such as protein kinases or the insulin receptor, have been reported in the literature. Various strategies have been reported for introducing such synthetic molecules into living cells, including electroporation, osmotic shock, and permeabilization of cells using agents such as saponins, streptolysin O, or liposomes. However, those delivery systems tend to cause cellular damage. Microinjection, another known method for introducing such synthetic molecules into living cells, is less damaging, but suffers the drawback of being labor intensive.
A recent publication by Liotta et al. indicates that synthetic peptides can be modified so that they passively migrate into the membranes of living cells, where the modified peptides can interact with trans-membrane proteins (Liotta et al.,
J. Biol. Chem
. 269:22996-23001, 1994). The Liotta et al. publication demonstrates that a synthetic Tris-sulfotyrosyl dodecapeptide analogue of the insulin receptor 1146-kinase domain is a good inhibitor of tyrosine dephosphorylation of the insulin receptor in situ. The most significant observation made by Liotta et al. was the fact that a particular synthetic tris-sulfotyrosyl dodecapeptide (TRDIYETDYYRK-amide) (SEQ ID NO:1), a stearyl peptide amide, caused an 4.5-fold increase in insulin-stimulated receptor autophosphorylation in intact CHO/HIRc cells. That stearyl peptide amide displayed specificity toward tyrosine-class phosphatases only, as it did not have an effect on the activities of the serine/threonine phosphatases PP-1, PP-2A, or alkaline phosphatase. Approaches similar to that of Liotta et al. but with fatty acid-peptide conjugates had been used to inhibit protein kinase C (PKC) and tyrosine kinase activities in intact cells (Eichholtz et al.
J. Biol. Chem
., 268: 1982-1986, 1993; Liotta et al.,
J. Biol. Chem
. 269(37):22996-23001, 1994; and O'Brian et al.,
Biochem. Pharmacol
. 39:49-57, 1990).
One type of signaling protein molecule, signal transduction enzymes (e.g. protein kinases and phosphatases), play pivotal roles in mediating cellular responses to a wide variety of stimuli. Such enzymes are often targeted to specific substrates or cellular compartments through their interaction with cellular “anchoring proteins” (Hubbard and Cohen,
Trends Biochem. Sci
. 18:172-177, 1993). The anchoring or compartmentalization of such proteins is thought to be critical in determining the specificity of response for a particular stimulus (Scott and Carr,
News Physiol. Sci
. 7:143-148, 1992; Rubin,
Biochem. Biophys
. Acta 1224:467-479, 1994; Mochly-Rosen,
Science
268:247-251, 1995). Anchoring of cyclic AMP-dependent protein kinase A (PKA or A-kinase) is accomplished by the binding of its regulatory subunit (R) to an amphipathic helix-binding motif located within A-kinase anchoring proteins (AKAPs) (Carr et al.,
J. Biol. Chem
. 266:14188-14192, 1991).
Many different peptides have been developed and synthesized which are known to have functional activities in vitro, including peptides designed to interact with anchor proteins or other proteins known to have binding regions in the intracellular space of living cells. The protein kinase anchoring inhibitory peptides, such as Ht31, (DLIEEAASRIVDAVIEQVKAAGAY) (SEQ ID NO:2), are included in the category of peptides designed to interact with anchor proteins (Carr et al.,
J. Biol. Chem
. 267(19):13376, 1992). Ht31 has been shown to inhibit the binding of the regulatory subunit of PKA to anchoring protein using cellular extracts in vitro. The PKA regulatory subunit from which the sequence of Ht31 was derived was used to synthesize the peptide moieties of the new reagents used in several of the examples of this application. Peptides such as Ht31, which are known to inhibit PKA binding to anchor proteins, are referred to herein as “anchor inhibiting peptides” or (AIPs).
Peptides known to be functionally active in vitro have also been used to develop and synthesize inactive peptides for use as control peptides. The alterations made in the amino acid sequence of an active peptide to produce an inactive peptide are expected to have a disruptive effect on the predicted &agr;-helix conformation of the active parent peptide, a conformation considered essential to ensure the maintenance of functional activity. Altered inactive peptides of this type have been used as controls in assaying the functional activity and other properties of the parent peptides from which they were derived. The sequence of any such control peptide is derived from the sequence of its functionally active counterpart by substituting a few amino acid residues in the parent amino acid sequence with other amino acid residues likely to produce a significantly different conformation (See, e.g. Carr et al., supra; and Rosenmund et al.,
Nature
368:853-856, 1994).
Microinjection of AIPs into neurons or skeletal muscle cells has been shown to disrupt PKA anchoring and PKA modulation of glutamate receptor channels (Rosenmund et al., supra) and voltage-gated calcium channels (Johnson et al.,
Proc. Natl. Acad. Sci. USA
91:11492-11496, 1994). However, microinjection is impractical for normal pharmaceutical applications. If a more practical method were found for introducing AIPs and other functionally active peptides intracellularly, it would lead to many possible applications, particularly those applications involving the control and regulation of cyclic adenosine monophosphate (cAMP) mediated responses in living cells.
Cyclic AMP (cAMP) is known to mediate the motility of sperm and a variety of other ciliated cells (Satir,
Modern Cell Biol
. 4:1-46, 1985; Tash,
Cell Motil. Cytoskel
. 14:332-339, 1989; Bedford and Hoskins, in
Marshall's Physiology of Reproduction
, Lamming, ed., pp. 379, Churchill Livingstone, N.Y. 1990). Increases in the level of this nucleotide are associated with development of motility in the epididymis (Bedford and Hoskins, supra; Hoskins et al.,
J. Reprod. Fertil
. 37:131-133, 1974). Cell-permeant cAMP analogs, e.g., cAMP phosphodiesterase inhibitors, and adenyl cyclase activators, all stimulate motility of sperm from several species (Garbers et al.,
Biol. Reprod
. 7:132, 1972; Garbers et al.,
Adv. Cyclic Nucleotide Res
. 9:583-595, 1978; Hoskins,
Journal of Biological Chemistry
248:1135-1140, 1973; Hoskins et al.,
Biol. Reprod
. 13:168-176, 1975; Vijayaraghavan and Hoskins,
J. Cyclic Nucleotide Protein Phosphoryl. Res
. 10:499-510, 1985). The kinetic and metabolic responses to cAMP elevation have been discovered to occur within 5 to 10 minutes (Garbers et al.,
Biol. Reprod
. 7:132, 1972; Garbers et al.,
Adv. Cyclic Nucleotide Res
. 9:583-595, 1978).
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