U.S. patent application number 10/647711 was filed with the patent office on 2005-06-30 for alkyl peptide amides and applications.
This patent application is currently assigned to Promega Corporation. Invention is credited to Goueli, Said A..
Application Number | 20050143309 10/647711 |
Document ID | / |
Family ID | 21859400 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050143309 |
Kind Code |
A1 |
Goueli, Said A. |
June 30, 2005 |
Alkyl peptide amides and applications
Abstract
Disclosed are alkyl peptide amides capable of inhibiting or
reducing sperm cell motility. Methods of inhibiting or reducing
sperm motility using certain alkyl peptide amides are also
disclosed.
Inventors: |
Goueli, Said A.; (Madison,
WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
|
Assignee: |
Promega Corporation
Madison
WI
|
Family ID: |
21859400 |
Appl. No.: |
10/647711 |
Filed: |
August 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10647711 |
Aug 25, 2003 |
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09630115 |
Aug 1, 2000 |
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6610657 |
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09630115 |
Aug 1, 2000 |
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09308507 |
May 19, 1999 |
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09308507 |
May 19, 1999 |
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PCT/US97/21509 |
Nov 20, 1997 |
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60031428 |
Nov 21, 1996 |
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Current U.S.
Class: |
514/44R ;
514/7.5; 514/9.8; 530/326; 530/327; 530/328 |
Current CPC
Class: |
C07K 14/47 20130101;
A61P 15/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/013 ;
514/014; 514/015; 530/326; 530/327; 530/328 |
International
Class: |
A61K 038/10; C07K
007/08 |
Claims
I claim:
1. An alkyl peptide amide, comprising: an alkyl moiety having at
least twelve (12) carbon atoms, the alkyl moiety including a
carbonyl terminus; a peptide moiety, the peptide moiety having an
inhibitor region and an N-terminus, the inhibitor region comprising
a sequence of amino acids substantially homologous to a binding
domain within a first protein, wherein the binding domain is
capable of binding a second protein in the intracellular space of a
living cell; and an amide linkage between the carbonyl terminus of
the alkyl moiety, and the N-terminus of the peptide moiety.
2. An alkyl peptide amide according to claim 1, wherein the alkyl
moiety comprises at least eighteen (18) carbon atoms.
3. An alkyl peptide amide according to claim 1, wherein the peptide
moiety comprises at least about four (4) and up to about forty (40)
amino acid residues.
4. An alkyl peptide amide according to claim 1, wherein the
inhibitor region of the peptide moiety comprises a sequence of
amino acids substantially homologous to the region of an A-kinase
anchoring protein first protein capable of binding a cyclic AMP
dependent protein kinase A second protein.
5. An alkyl peptide amide according to claim 4, wherein the
inhibitor region of the peptide moiety comprises a sequence of
amino acids selected from the group consisting of: LIEEAASRIVDAVI;
TAEEVSARIVQVVT; LIETASSLVKNAIQ; LIETASSLVKNAIE; EVAAEVLAEVITAAVKAV;
IIDMASTALKSKSQ; LAEKIVAEAIEKAE; and VISEATEQVLATTVGKVAGRVC.
6. An alkyl peptide amide, comprising: an alkyl moiety of at least
twelve (12) carbon atoms, the alkyl moiety having a carbonyl
terminus; a peptide moiety of at least about four (4) and up to
about forty (40) amino acid residues, having an inhibitor region
and an N-terminus, the inhibitor region comprising a sequence of
amino acids selected from the group consisting of: LIEEAASRIVDAVI;
TAEEVSARIVQVVT; LIETASSLVKNAIQ; LIETASSLVKNAIE, EVAAEVLAEVITAAVKAV;
IIDMASTALKSKSQ; LAEKIVAEAIEKAE; and VISEATEQVLATTVGKVAGRVC; and an
amide linkage between the carbonyl terminus of the alkyl moiety and
the N-terminus of the peptide moiety.
7. A pharmaceutical composition of an alkyl peptide amide
comprising: a pharmaceutically acceptable table carrier; and an
alkyl peptide amide, comprising: an alkyl moiety of at least twelve
(12) carbon atoms, the alkyl moiety having a carbonyl terminus; a
peptide moiety, the peptide moiety having an inhibitor region and
an N-terminus, the inhibitor region comprising a sequence of amino
acids substantially homologous to a binding domain of a first
protein, wherein the binding domain is capable of binding a second
protein in the intracellular space of a living cell; and an amide
linkage between the carbonyl terminus of the alkyl moiety, and the
N-terminus of the peptide moiety;
8. A pharmaceutical composition according to claim 7, the inhibitor
region comprising the sequence of amino acids is substantially
homologous to the binding domain of the first protein which is an
anchor portion, wherein the binding domain is capable of binding
the second protein which is a protein kinase.
9. A pharmaceutical composition according to claim 7, wherein the
amount of alkyl peptide amide in the composition is sufficient to
substantially inhibit binding of the first protein to the second
protein, when administered to a living cell containing the second
protein and the first protein.
10. A pharmaceutical composition according to claim 9, wherein the
amount of alkyl peptide amide is sufficient to cause at least a 50%
reduction of activity of the second protein when administered to
the living cell.
11. A method of using an alkyl peptide amide to inhibit the binding
of a first protein to a second protein in a living cell, comprising
the steps of: (a) selecting a living cell having a first protein, a
second protein, and a cell membrane surrounding an intracellular
space, wherein the first protein has at least one binding domain
capable of binding the second protein in the intracellular space;
(b) providing an alkyl peptide amide, comprising: an alkyl moiety
of at least twelve (12) carbon atoms, the alkyl moiety having a
carbonyl terminus; a peptide moiety, the peptide moiety having an
inhibitor region and an N-terminus, the inhibitor region comprising
a sequence of amino acids substantially homologous to the binding
domain of the first protein for the second protein; and an amide
linkage between the carbonyl terminus of the alkyl moiety, and the
N-terminus of the peptide moiety; and (c) exposing the alkyl
peptide amide to the living cell so that the peptide moiety
permeates the cell membrane and inhibits the binding of the first
protein to the second protein.
12. A method according to claim 11, wherein the selection step (a)
is accomplished by selecting a first protein which is an anchor
protein having a binding domain; and wherein the providing step (b)
is accomplished by providing the alkyl peptide amide having the
inhibitor region of the peptide moiety which consists of a sequence
of amino acids substantially homologous to the binding domain of
the anchor protein.
13. The method of claim 12, further comprising: selecting the
living cell in step (a) having an anchor protein with a binding
domain capable of binding the second protein, wherein the second
protein is a protein kinase A; and providing the alkyl peptide
amide in step (b), wherein the inhibitor region of the peptide
moiety comprises a sequence of amino acids substantially homologous
to the protein kinase A binding domain of the anchor protein first
protein.
14. A method according to claim 10, wherein a sufficient amount of
the alkyl peptide amide is combined with the living cell to disrupt
the binding of the protein kinase second protein to the anchor
protein first protein in step (c) to result in an inhibition of
biological responses in the living cell which are mediated by
protein kinase A.
15. A method according to claim 10, further comprising: selecting
the living cell in step (a) having a first protein and a second
protein, wherein the second protein is protein kinase A, and
wherein the first protein is present in the intracellular space of
the cell wherein the first protein has a binding domain which binds
to protein kinase A; and providing the alkyl peptide amide in step
(b), wherein the inhibitor region of the alkyl peptide amide
comprises a sequence of amino acids substantially homologous to the
protein kinase A binding domain of the first protein.
16. An alkyl peptide, comprising: an alkyl moiety having at least
twelve (12) carbon atoms; a peptide moiety, the peptide moiety
having an inhibitor region comprising a sequence of amino acids
substantially homologous to a binding domain of a first protein,
wherein the binding domain is capable of binding a second protein
in the intracellular space of a living cell; and a linkage between
the alkyl moiety, and the peptide moiety.
17. An alkyl peptide according to claim 15 wherein the linkage is
an amide linkage.
18. An alkyl inhibitor amide comprising: an alkyl moiety having at
least twelve (12) carbon atoms; an inhibitor moiety, the inhibitor
moiety having an inhibitor region capable of inhibiting binding
between a first protein and a second protein in the intracellular
space of a living cell; and an amide linkage between the alkyl
moiety and the inhibitor moiety.
19. The alkyl inhibitor amide according to claim 18, wherein the
inhibitor moiety is a peptide moiety.
20. The alkyl inhibitor amide according to claim 18, wherein the
inhibitor moiety is a peptidomimetic moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the priority date,
under 35 U.S.C. .sctn.119, of U.S. Provisional Application No.
60/031,428, filed 21 Nov. 1996.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
BACKGROUND OF THE INVENTION
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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), 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).
[0007] 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).
[0008] 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), 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).
[0009] Peptides known to be functionally active in vftro 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 predict d .alpha.-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).
[0010] 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.
[0011] 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, New York, 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).
[0012] Sperm are known to lack nucleic-acid and protein-synthetic
activity, thereby considerably reducing the possible range of
targets of cAMP action. Sperm have been found to have distinct
subcellular structures easily distinguished with the aid of a light
microscope. Immunogold staining of sperm, followed by examination
under a light microscope, has been used to demonstrate a
predominant localization of the type II subunit (RII) of a PKA
anchoring protein at the outer membrane of the mitochondria that
spiral around the proximal flagella (Lieberman et al., J. Cell
Biol. 107:1809-1816, 1988). A developmentally regulated sperm AKAP
(i.e., AKAP-84) has also been localized to sperm mitochondria (Lin
et al., J. Biol. Chem. 270:27804-27811, 1995).
[0013] Cyclic AMP and the proteins which regulate the production of
cAMP in living cells, such as the various protein kinase regulatory
anchoring proteins (e.g. AKAPs), are important in mediating many
different responses in a wide variety of cells, other than the
motility response in sperm cells. For example, AKAPs have been
found to play a critical role in the regulation of synaptic
function in cultured hippocampal neurons (Rosenmund et al.,
supra).
[0014] Therefore, what is needed is a product or process for
introducing peptides known to be functionally active into the
intracellular space of living cells. As used herein, the term
"intracellular space" refers to the region of a cell bounded by the
cell membrane or cell wall. In the case of a eukaryotic cell, the
term intracellular space refers to everything contained within the
cytoplasm of the cell, including all organelles and regions of
proteins protruding into the cytoplasm from the inner surface of
the cell membrane. Particularly needed is a product or process for
introducing AIPs into the intracellular space of living cells and,
more particularly, AIPs capable of inhibiting the binding of
specific protein kinases to specific protein kinase anchor
proteins. Disclosed herein is such a product, e.g., an alkyl
peptide amide, and a process for the introduction of at least the
amide portion of the product into the intracellular space of living
cells.
[0015] Also disclosed herein are examples of the synthesis and use
of such alkyl peptide amides to inhibit the activity of protein
kinase A in HeLa cells and to inhibit binding between the RII
subunit of protein kinase A and the anchor binding protein in sperm
from various different organisms.
BRIEF SUMMARY OF THE INVENTION
[0016] The principal aspects of the invention are as follows. In
one aspect the present invention is an alkyl peptide,
comprising:
[0017] an alkyl moiety having at least twelve (12) carbon
atoms;
[0018] a peptide moiety, the peptide moiety having an inhibitor
region comprising a sequence of amino acids substantially
homologous to a binding domain within a first protein, wherein the
binding domain is capable of binding a second protein in the
intracellular space of a living cell; and
[0019] a linkage between the alkyl moiety and the peptide
moiety.
[0020] In another aspect, the present invention is an alkyl peptide
amide, comprising:
[0021] an alkyl moiety having at least twelve (12) carbon atoms,
the alkyl moiety including a carbonyl terminus;
[0022] a peptide moiety, the peptide moiety having an inhibitor
region and an N-terminus, the inhibitor region comprising a
sequence of amino acids substantially homologous to a binding
domain within a first protein, wherein the binding domain is
capable of binding a second protein in the intracellular space of a
living cell; and
[0023] an amide linkage between the carbonyl terminus of the alkyl
moiety, and the N-terminus of the peptide moiety.
[0024] In a further aspect, the present invention is a
pharmaceutical composition comprising an alkyl peptide amide
described above in a pharmaceutically acceptable carrier.
[0025] In yet a further aspect, the present invention is a method
of using an alkyl peptide amide to inhibit the binding of a first
protein to a second protein in a living cell, comprising the steps
of:
[0026] (a) selecting a living cell having a first protein, a second
protein, and a cell membrane surrounding an intracellular space,
wherein the first protein has at least one binding domain capable
of binding the second protein in the intracellular space;
[0027] (b) providing an alkyl peptide amide, comprising:
[0028] an alkyl moiety of at least twelve (12) carbon atoms, the
alkyl moiety having a carbonyl terminus;
[0029] a peptide moiety, the peptide moiety having an inhibitor
region and an N-terminus, the inhibitor region comprising a
sequence of amino acids substantially homologous to the binding
domain within the first protein for the second protein; and
[0030] an amide linkage between the carbonyl terminus of the alkyl
moiety, and the N-terminus of the peptide moiety; and
[0031] (c) exposing the alkyl peptide amide to the living cell so
that the peptide moiety permeates the cell membrane and inhibits
the binding of the first protein to the second protein.
[0032] The alkyl peptide amides of this invention are efficient
delivery systems for introducing peptide moieties into the
intracellular space of living cells, including sperm cells and HeLa
cells. The motility of sperm cells from a wide variety of different
species can be inhibited after such cells are exposed to the alkyl
peptide amide form of AIPs (i.e. alkyl AIP amides). This last form
of the alkyl peptide amides of this invention (alkyl AIP amides)
can be included in pharmaceutical compositions and used as
contraceptives, either alone or in combination with other
contraceptive substances, e.g., a spermicidal agent such as
nonoxynol-9.
[0033] The pharmaceutical compositions of the alkyl peptide amides
of this invention include pharmaceutical contraceptive
compositions, particularly such compositions comprising an alkyl
peptide amide known to inhibit sperm motility, such an alkyl AIP
amide. The pharmaceutical contraceptive compositions of this
invention can be used in the form of a topical ointment or lotion
e.g., to lubricate the vagina before intercourse. Other modes of
application of the pharmaceutical composition of the invention are
likely to occur to one skilled in the contraceptive art. By
inhibiting sperm motility, the contraceptive compositions of this
invention could prevent fertilization of the ovum (alone or in
combination with other contraception techniques), with minimal side
effects.
[0034] The pharmaceutical contraceptive compositions of this
invention are likely to produce few side effects because: (1) the
peptide moiety of the alkyl peptide amide has a substantially
similar sequence to a natural cellular component; (2) only very low
concentrations of the alkyl peptide amide component are needed for
a composition to be effective as an inhibitor of sperm motility;
and (3) the effect, i.e., sperm motility inhibition, has been found
to be temporary, i.e., to be reversible once sperm cells are no
longer in contact with a composition containing the alkyl peptide
amide.
[0035] The detailed disclosure below demonstrates that the
compositions and methods of the present invention can be used to
introduce inhibitor moieties, such as peptide or peptidomimetic
moieties into the intracellular space of a variety of different
types of cells, including sperm cells obtained from various
different species, and HeLa cells (a continuously cultured cell
line, originally obtained from human cancerous cervical tissue.)
Aspects of this invention may also have utility in other
applications which will occur to one skilled in this art in light
of the disclosure herein. One such aspect is the inhibition of
transformation in certain tumorous cells. All such other
applications are included within the scope of this invention.
BRIEF DESCRIPTION OF THE DRAWING(S)
[0036] FIG. 1 Schematic diagram showing the disruption of binding
between a pair of cAMP dependent regulatory subunit proteins and a
membrane bound anchoring protein by an alkyl AIP amide in the
intracellular space of a living cell.
[0037] FIG. 2 Plot of PKA Kinase activity versus inhibitor
concentration obtained from processing and assaying HeLa cells
after treatment with PKI or with s-PKI (N-ST-PKI).
[0038] FIG. 3 Autoradiograms of Western blots of sodium D-lauroyl
sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of
.sup.32P-labeled cAMP-dependent protein kinase A subunits,
RI.alpha., RI.beta., RII.alpha., and RII.beta. of sperm extracts
from bovine (B), human (H), and monkey (M).
[0039] FIG. 4 Autoradiograms of Western blot/SDS-PAGE of bovine
caudal epididymal sperm lysed and separated into supernatant (S)
and pellet (P) fractions by centrifugation, and probed using
anti-RII.alpha., anti-RII.beta., and anti-RI.beta. antibodies.
[0040] FIG. 5 Autoradiograms of Western blot/SDS-PAGE of sperm
extract from bovine, human, and monkey probed with .sup.32P-labeled
RII.alpha. or RII.beta..
[0041] FIG. 6 Autoradiograms of Western blot/SDS-PAGE showing the
disruption of RII binding to bovine sperm AKAP in the presence or
absence of 20 .mu.M of the anchor inhibiting peptide Ht31, 20 .mu.M
of a stearyl peptide amide form of Ht31 (s-Ht31), or 20 .mu.M of a
stearyl peptide amide form of an Ht31 control peptide
(s-Ht31-P).
[0042] FIG. 7 Plot of forward motility as a percentage of control
versus incubation time, obtained from testing bovine sperm after
incubation for varying amounts of time in a buffer containing 50
.mu.M s-Ht31-P (control) (x), or 5 .mu.M (.circle-solid.), 10 .mu.M
(.box-solid.), or 50 .mu.M (.diamond-solid.) of s-Ht31.
[0043] FIG. 8 Plot of forward motility as a percentage of control
versus concentration of stearyl peptide amides (derivatives of
AIPs), obtained from testing bovine sperm after 5 minutes of
incubation in a buffer, after adding varying amounts of s-Ht31
(.diamond-solid.) or s-Ht31-P control (.tangle-solidup.).
[0044] FIG. 9 Histogram of forward motility index obtained from
bovine caudal epididymal sperm after treatment with water (control)
or 10 .mu.M s-Ht31 and recovery for varying amounts of time in a
buffer containing 2 mM calcium (+Calcium) or 2 mM EGTA
(-Calcium).
[0045] FIG. 10 Histogram of forward motility index of bovine caudal
epididymal sperm after treatment with water (control) or 10 .mu.M
s-Ht31 and recovery for varying amounts of time in a buffer with or
without 2 mM calcium and 50 mM bicarbonate.
[0046] FIG. 11 Histogram of forward motility index of monkey
ejaculated sperm after incubation for 5 or 30 minutes in a buffer
containing dimethyl sulfoxide (DMSO) (control) and varying amounts
of the stearyl AIP amide s-AKAP79.
[0047] FIG. 12 Histogram of forward motility index of monkey sperm
from neat semen or semen collected after swim-up analysis, after
incubation for 15 minutes after no addition (control), or after
addition of 100 .mu.M s-Ht31, 2.5 mM EGTA, or 2.5 mM EGTA plus 50
.mu.M s-Ht31.
[0048] FIG. 13 Histogram of forward motility index of human
ejaculated sperm after swim-up analysis, after incubation for 15
minutes after no addition (control), or after addition of 100 .mu.M
s-Ht31 or s-Ht31-P.
[0049] FIG. 14 Histogram of forward motility index of bovine caudal
and caput sperm, after incubation for 15 minutes in the presence or
absence of 50 .mu.M 2-chloro-2'-deoxyadenosine (CDA) and/or 50
.mu.M of the protein kinase inhibitor H-89.
[0050] FIG. 15 Histogram of PKA kinase activity of bovine caudal
and caput sperm, after incubation as described in FIG. 12 followed
by an assay for PKA activity.
[0051] FIG. 16 Plot of forward motility as a percentage of control,
obtained from bovine sperm incubated for 5 minutes in a buffer
containing increasing amounts of s-Ht31 (.diamond-solid.), s-AKAP79
(.quadrature.), or s-Ht31-P (control) (.DELTA.).
DETAILED DESCRIPTION OF THE INVENTION
[0052] The alkyl peptide of this invention comprises an alkyl
moiety linked to a peptide moiety or to a peptidometric moiety. A
peptidomimetic moiety consists of a moiety produced from at least
one organic molecule other than a peptide, which mimics the
biological activity of a peptide moiety of an alkyl peptide. The
alkyl peptide of this invention is preferably an alkyl peptide
amide, more preferably an alkyl peptide amide comprising an alkyl
moiety and a peptide moiety linked by an amide bond between a
terminal nitrogen residue of the peptide moiety (an N-terminus) and
the terminal carbonyl residue of the alkyl moiety (carbonyl
terminus) . The alkyl moiety comprises a saturated, straight chain
of at least 12 carbon atoms (including the carbonyl terminus), or a
chain of at least 12 carbon atoms optionally substituted by at
least one other component such as a halogen, or a lower alkyl group
having from one to six carbon atoms and especially a very short
alkyl group such as a methyl or ethyl group. The alkyl moiety is
preferably a saturated, straight chain of at least 14 carbon atoms
and most preferably a stearyl group (i.e. a straight 17 carbon atom
chain having a carbonyl terminus).
[0053] Herein below is a detailed description of the alkyl peptide
amide aspect of the present application. However, the detailed
description below is not intended to limit the invention to that
particular embodiment of the invention.
[0054] In addition to an N-terminus, the peptide moiety also
includes an inhibitor region, comprising a sequence of amino acids
substantially homologous to a sequence of amino acids in a binding
domain of a specific protein (a first protein), a binding domain
capable of binding a particular protein of interest (a second
protein) in the intracellular space of a living cell. The sequence
of amino acids in the inhibitor region of the peptide moiety is
preferably substantially homologous to a sequence of amino acids in
the binding domain of a first protein capable of binding a protein
kinase in the intracellular space of a living cell, more preferably
a binding domain capable of binding PKA. In one preferred aspect of
the alkyl peptide amide of this invention the inhibitor region of
the peptide moiety consists of an amino acid sequence substantially
homologous to the sequence of amino acids in a binding domain of a
first protein, wherein the first protein is an anchoring protein,
more preferably a binding domain of AKAP which is known to, or
suspected of, binding to the regulatory subunit of PKA.
[0055] The inhibitor region of the peptide moiety must be of a
sufficient length to inhibit the binding of a second protein to the
binding domain of a first protein, wherein the sequence of amino
acids in the inhibitor region of the moiety is substantially
homologous to the binding domain of the first protein. The length
of the entire peptide moiety is most preferably sufficient for the
moiety to pass into the intracellular space of a living cell with
the inhibitor region of the moiety sufficiently intact to disrupt
binding between a second protein and a first protein. The minimum
length of the entire peptide moiety component of the alkyl peptide
amides of this invention is preferably about 4 amino acid residues,
more preferably about 10 amino acid residues, and most preferably
about 20 amino acid residues. The maximum length of the entire
peptide moiety is preferably less than about 40 amino acid
residues, more preferably less than about 30 amino acid residues,
and most preferably less than about 25 amino acid residues.
[0056] The peptide used to produce the alkyl peptide moiety can be
synthesized using any standard peptide synthesis method, preferably
using an automated peptide synthesizer, more preferably using an
automated peptide synthesizer using 9-fluorenylmethoxycarbonyl
(FMOC) chemistry employing base-mediated coupling. The polypeptide
can also be purified before use in making the alkyl peptide amide
of the invention, using any suitable means for purifying a
polypeptide known in the art (Shaw, C., Methods in Molecular
Biology 32:275-287 1994).
[0057] The carbonyl terminus of the alkyl moiety is linked to the
N-terminus of the peptide moiety to form the alkyl peptide amide of
the invention. The link is preferably formed, through a
base-catalyzed nucleophilic addition, by reacting the carboxylic
acid terminus of a fatty acid, such as stearic acid, with the
N-terminus of a peptide having an inhibitor region, under reaction
conditions designed to protect the peptide from degradation.
Formally, the reaction may be written as follows: 1
[0058] wherein R'is a saturated chain of at least 11 carbon atoms,
optionally substituted by at least one component such as a halogen
or a lower alkyl group, as described above; wherein R.sup.2 is an
amino acid R group (see e.g. description of amino acid R groups in
A. L. Lehninger, Principals of Biochemistry, Worth Publishers,
Inc., 1982, p. 101); and wherein R.sup.3 is a peptide chain of at
least four amino acid residues, terminating in a carboxyl group
(i.e. the C-terminus of the peptide moiety).
[0059] The peptide is preferably protected during the reaction
described above by being covalently linked to at least one
substantially inert protection group prior to being exposed to the
fatty acid. The fatty acid is preferably activated before being
exposed to the peptide. The link between the fatty acid and the
peptide is preferably formed in the presence of an activator using
diiosopropylethylamine, an activator such as benzotriazole-1
-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosph- ate (BOP)
or 0-(7-azabenzotriazole- 1 -yl)-1,1,3,3,-tetramethyluroniumhexa-
fluorophosphate (HATU).
[0060] Formation of the alkyl peptide amide in the reaction
described above can be monitored using any one of a number of
different monitoring methods, including staining samples of the
reaction mixture with ninhydrin (an amino acid selective blue
stain), or cleaving the resulting products with trifluoroacetic
acid (TFA). Monitoring is preferably done using a combination of
mass spectrometry and high performance (or high pressure) liquid
chromatography (HPLC).
[0061] The alkyl peptide amide product of the peptidelfatty acid
linkage reaction described above is preferably isolated from other
components of the reaction solution, preferably by HPLC, and more
preferably by reverse phase HPLC using a C8 column employing a
TFA/acetonitrile buffer system. If HPLC is used to purify the alkyl
peptide amide, the eluent is preferably analyzed to identify the
fractions of eluent containing the alkyl peptide amide, preferably
using mass spectrometry, more preferably using a time of flight
(TOF) mass spectrometry analyzer. Fractions containing the largest
amounts of alkyl peptide amide are then preferably pooled, and
analyzed again to confirm the purity of the final alkyl peptide
amide product.
[0062] Alkyl peptide amides synthesized and isolated according to
the preferred procedures described above can be stored in a
stabilizing buffer at room temperature or colder, but are more
preferably stored in lyophilized form, even more preferably in
lyophilized form under nitrogen.
[0063] The alkyl peptide amides of this invention are designed to
introduce the peptide moiety into the intracellular space of a
living cell, where it can disrupt or inhibit binding between one or
more first proteins and one or more second proteins. The capacity
of a given alkyl peptide amide to disrupt or inhibit binding
between a first protein and a second protein often depends upon how
similar the sequence of the inhibitor region of the peptide moiety
is to the sequence of amino acids in the binding domain of the
first protein, wherein the binding domain-is capable of binding the
second protein in the intracellular space of a living cell. ln a
preferred aspect, the sequence of amino acids in the inhibitor
region of the peptide moiety of the alkyl peptide amide is
preferably substantially homologous to the amino acid sequence of
the binding domain of the first protein, more preferably
substantially homologous to a binding domain capable of binding to
a protein kinase in the intracellular space of a cell, most
preferably substantially homologous to a sequence of amino acids in
the PKA binding domain of an anchoring protein, such as in the PKA
binding domain of AKAP. Peptides containing sequences of this last
most preferred type are commonly known as AIPs. This particular
preferred embodiment of the alkyl peptide amides of the present
invention is referred to herein as alkyl AIP amides.
[0064] It is contemplated that nonhomologous peptides or other
organic molecules such as peptidomimetics that have structural
identity that is sufficient to disrupt the interaction between
AKAPS and PKA could be substituted for the peptide moiety of alkyl
AIP amides described herein, and similarly be used to inhibit sperm
motility and other AKAP-anchored, PKA-medicated responses.
[0065] FIG. 1 is a schematic diagram showing the interaction of an
alkyl AIP amide with a membrane-bound anchoring protein to inhibit
binding between the anchoring protein and a regulatory subunit
homodimer of PKA. In the absence of such a binding inhibitor, cAMP
generating enzymes such as adenylate cyclase (a membrane associated
enzyme) can be activated by ligands (hormones, neurotransmitters,
etc.) or by activators (forskolin or cAMP analogues). The activated
adenylate cyclase converts ATP to the second messenger cAMP. The
second messenger cAMP, in turn, interacts with PKA, causing the
regulatory subunit homodimer to dissociate from the catalytic
subunit of PKA. The regulatory subunits are then translocated to
the membrane where they attach to the anchoring protein. By
inhibiting or disrupting binding between the regulatory subunits of
PKA and the anchoring protein, the alkyl AIP amides of this
invention inhibit the physiological functions mediated by the
interaction of an anchoring protein with the regulatory
subunits.
[0066] FIG. 1 is merely illustrative of one possible mode of action
of one embodiment of the alkyl peptide amides of this invention.
This invention is not limited by any theory or any postulated mode
of action. Thus neither the schematic diagram of FIG. 1 nor its
description immediately above are intended to limit the scope of
the invention.
[0067] The present invention is not limited to the alkyl peptide or
alkyl peptide amide aspects described in general terms above, and
in greater detail through the definitions and examples below. The
present invention encompasses any alkyl inhibitor amide comprising
an alkyl moiety linked to an inhibitor moiety through an amide
bond, wherein the inhibitor moiety comprises an inhibitor region
capable of inhibiting or disrupting binding between a first protein
and a second protein in the intracellular space of a living cell.
The inhibitor moiety is preferably a peptide moiety or a
peptidomimetic moiety, wherein the peptidomimetic moiety consists
of a moiety produced from at least one organic molecule other than
a peptide, which mimics the biological activity of a peptide
inhibitor moiety.
[0068] The inhibitor region of a peptide inhibitor moiety consists
of a sequence of amino acid residues which is capable of inhibiting
or disrupting binding between a first protein and a second protein
in the intracellular space of a living cell. The sequence of amino
acid residues in the inhibitory region of such a peptide moiety can
be selected by sythesizing and testing a series of peptides with
random sequences until a sequence is identified which produces the
targeted inhibitory effect. When such a screening method is used,
the resulting inhibitory region sequence will not necessarily be
substantially homologous to the binding domain of either the first
or the second protein. In the most preferred aspect, however, the
sequence of amino acids in the inhibitor region of the alkyl
peptide amide of the present invention is substantially homologous
to at least one sequence of amino acid residues in the binding
domain of a first protein which is capable of binding a second
protein in the intracellular space of a living cell.
[0069] The alkyl peptidomimetic amides of the present invention are
preferably produced by forming an amide bond linking the carboxyl
residue at the carboxylic acid terminus of a fatty acid molecule to
the amine residue at a terminus of a peptidomimetic moiety
precurser. The composition of the inhibitor region of the
peptidomimetic moiety is selected to mimic the inhibitory or
disruptive functionality of the inhibitory region of an peptide
moiety of an alkyl peptide amide of this invention. The inhibitory
functionality of an alkyl peptidomimetic amide of the present
invention is preferably similar to that of an alkyl peptide amide,
more preferably at least as high as that of an alkyl peptide amide,
and most preferably greater than that of an alkyl peptide amide.
Methods for producing peptidomimetic molecules with such inhibitory
potencies are known. (See, e.g. Eichler et al., Med. Res. Rev., 15:
481-496, 1995, incorporated by reference herein.).
[0070] The peptide moiety of the alkyl inhibitor amide can be
produced by forming an amide bond with an amine group at either
terminus of a peptide molecule. Normally, a peptide molecule
contains only one N-terminus. However, the carboxylic acid residue
(i.e. the C-terminus) of a peptide molecule can be modified by
substituting an amine group for the hydroxyl group at that
terminus, thereby producing a second N-terminus.
[0071] Definitions and Details of Preferred Embodiments
[0072] The following definitions and additional details of the
preferred embodiments of the invention are provided to guide those
of ordinary skill in the art in understanding and making and using
the compositions of this invention. Definitions of common terms
used here may also be found in Rieger et al., Glossary of Genetics:
Classical and Molecular, 5th edition, Springer-Verlag: New York,
1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
The standard one-letter nomenclature for amino acid residues is
used herein. "Anchoring Inhibiting Peptides". An anchoring
inhibitiag peptide or AIP is defined as a peptide that (1) is at
least 4 amino acids in length; (2) possesses an amphipathic
.alpha.-helical structure; (3) binds to PKA; and (4) interferes
with binding of PKA to an AKAP.
[0073] Subcellular localization of PKA is directed through the
regulatory (R) subunit. There are two R subunit classes, RI and
RII, which form the type I and type II holoenzymes, respectively.
Type II PKA is present in all eukaryotic cells, whereas the tissue
distribution of type I PKA is more restricted. Type II PKA
localization is dictated by the association of RII with AKAPs.
Tissue-specific AKAP localization has been detected by
protein-blotting techniques or by fractionation on RII-Sepharose
affinity columns.
[0074] All AKAPs identified to date contain an amphipathic helix
domain that is responsible for RII binding (Carr et al., J. Biol.
Chem. 266:14188-14192, 1991; Carr et al., J. Biol. Chem.
267:13376-13382, 1992; Carr et al., J. Biol. Chem. 267:16816-16823,
1992; Coghlan et al., J. Biol. Chem. 269:7658-7665, 1994; McCartney
et al., J. Biol. Chem. 270:9327-9333, 1995). "Amphipathic Helix."
An amphipathic helix is an .alpha.-helix with opposing hydrophilic
and hydrophobic faces oriented down the long axis of the helix. As
discussed in Example 3 below, substantial disruption of the
.alpha.-helical structure of a functionally active peptide, such as
an AIP, significantly reduces or eliminates functional activity,
such as the binding of an AIP to RII.
[0075] The RII-binding amphipathic helix of an AKAP can be
identified by a computer-aided secondary structure prediction
method (Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:140-144,
1984). A characteristic of the amphipathic helix motif of about 14
amino-acid residues is the ordered placement of alternating pairs
of hydrophobic and hydrophilic amino acids within the linear
sequence of a protein. In addition, each RII-anchoring protein
contains acidic amino acids distributed over the hydrophilic face
of the helix. In particular, a glutamic acid residue at position 3
is located within the first turn of the amphipathic helix of most
wild-type AKAPS.
[0076] The same computer-aided secondary structure prediction
method (or a combination of that method and a library screening
approach), could also be employed to identify amphipathic helix
motifs in the regions of AKAPs and other first proteins suspected
to bind specific second proteins. The sequence of amino acids in
the identified motifs could then be employed to obtain peptides
with equivalent disrupting potency to the specific AIP sequences
identified herein below.
[0077] Preferably, the AIP includes an amino acid sequence that is
substantially identical to, or more preferably identical to that of
the RII-binding portion of a "native" (naturally-occurring or
wild-type) AKAP. Additional amino-acid residues may be included,
preferably at the amino-terminus or carboxyl-terminus of the AIP,
so as not to interfere with binding of the AIP to PKA.
[0078] Amphipathic helix sequences predicted to bind RII include,
but are not limited to the sequences listed on Table 1, below:
1 TABLE 1 AKAP Amino Name Acid Residues Amino Acid Sequence Ht 31
494-507 LIEEAASRIVDAVI MAP2 87-100 TAEEVSARIVQVVT AKAP 150 429-442
LIETASSLVKNAIE AKAP 79 392-405 LIETASSLVKNAIQ AKAP 95 642-659
EVAAEVLAEVITAAVKAV AKAP 100 396-411 IIDMASTALKSKSQ AKAP 220 905-918
LAEKIVAEAIEKAE AKAP 84 355-376 VISEATEQVLATTVGKVAGRVC
[0079] (Carr et al., J. Biol. Chem. 266:14188-14192, 1991; Carr et
al., J. Biol. Chem. 267:13376-13382, 1992; Carr et al., J. Biol.
Chem. 267:16816-16823, 1992; Coghlan et al., J. Biol. Chem.
269:7658-7665, 1994; McCartney et al., J. Biol. Chem.
270:9327-9333, 1995; Carr et al., J. Biol. Chem. 267:13376-13382,
1992). Any of the sequences listed immediately above are suitable
for incorporation into the alkyl moiety of an alkyl peptide amide
of this invention.
[0080] The sequence of the RII-binding domain of a wild-type AKAP
polypeptide, i.e., a "native" AIP sequence, can be modified by
substituting a hydrophobic amino-acid residue with another
hydrophobic residue or substituting a hydrophilic residue with
another hydrophilic residue, for example. Preferably acidic
residues are replaced with other acidic residues. Residues that lie
outside the predicted amphipathic helix region of an RII-binding
region of an AKAP may enhance RII binding by stabilizing the
overall conformation of the region and are preferably included in
the AIP sequence. As a result, AIPs and alkyl AIP amides that
include additional sequences from the RII-binding region of an AKAP
flanking the helix-forming sequence are preferred. AIPs shorter
than 14 amino-acid residues may also bind RII.
[0081] Suitable AIP sequences can also be identified by screening
peptides for binding to RII. Random peptide sequences of at least
about 14-amino acid residues that would be expected to form
amphipathic helices can be identified by computer analysis.
Alternatively, such sequences can be identified by screening for
binding to PKA regulatory subunits using conventional methods
including, but not limited to, combinatorial chemistry and
expression library approaches (e.g., phage expression libraries,
including those in which a peptide is joined in reading frame to an
outer structural protein of the phage). The Examples below and Carr
et al. (J. Biol. Chem. 266:14188-14192, 1991), discuss various
assays, e.g., an RII gel overlay procedure, that are useful for
screening phage expression libraries for polypeptides that bind to
RII.
[0082] The AIPs used to make the preferred alkyl AIP amides of this
invention are produced by standard synthesis techniques, preferably
by chemical synthesis (see, e.g., Merrifield, J. Amer. Chem. Soc.
85:2149-2156, 1963). Alternatively, the AIPs can also be produced
by standard genetic engineering techniques, i.e., by the expression
of an AlP-encoding nucleic acid sequence in an appropriate host
cell. The AIP may be expressed as a fusion polypeptide if binding
to PKA is not significantly diminished by the fusion partner. For
guidance regarding expression of polypeptides in various host
cells, see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed.,
vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, New York, 1989, and Current Protocols in
Molecular Biology, ed. Ausubel et al., Greene Publishing and
Wiley-lnterscience, New York, 1987 (with periodic updates).
[0083] Alkyl AIP amides are capable of permeating cell membranes,
and of introducing the AIP peptide moieties into the intracellular
space of a living cells, where they can competitively disrupt the
binding of PKA to AKAPs and cause loss of PKA modulation of
cellular responses. Some of the alkyl AIP amides of this invention
also inhibit sperm motility in a time and concentration-dependent
manner, when introduced to sperm in the form of an alkyl AIP amide.
The inhibition of sperm motility by such an alkyl AIP amide (e.g.,
s-Ht31) is reversible, but only if calcium is present in the
suspension buffer. Most spermicidal agents in current use employ
non-specific toxic compounds (e.g., nonoxynol-9) that have adverse
effects on cells lining the vaginal tract. Compositions of the
alkyl AIP peptides of this invention are expected to have minimal
side effects at concentrations that fully inhibit sperm
motility.
[0084] Peptide Sequence Homology. Preferably, the inhibitor region
of the peptide moiety of each of the alkyl AIP amides of this
invention is at least about 70% homologous to a native AKAP-derived
polypeptide, preferably at least about 80% homologous, and more
preferably at least about 95% homologous. The inhibitor region of
any of the alkyl peptide amides of this invention is said to be
"substantially homologous" to the region of a first protein capable
of binding a second protein if the alkyl peptide amide has an
equivalent capacity to disrupt such binding, compared to an alkyl
peptide amide having an inhibitor region which is homologous to the
same region of the same first protein. The most preferred
embodiments of the alkyl peptide amides of this invention,
including the alkyl AIP arnides, have such "substantial homology"
or otherwise exhibit sufficient disruption or binding inhibition
capability.
[0085] Peptide homology is typically analyzed using sequence
analysis software such as the Sequence Analysis Software Package
available from the Genetics Computer Group, University of Wisconsin
Biotechnology Center, Madison, Wis.).
[0086] "Isolated," "Purified," "Homogeneous" Peptides or
Polypeptides. A peptide or polypeptide is "isolated" if it has been
substantially separated from contaminants, e.g., cellular
components (nucleic acids, lipids, carbohydrates, and other peptide
species) that may accompany it during purification. Such a peptide
or polypeptide can also be referred to as "pure" or "homogeneous"
or "substantially pure or homogeneous." For example an AIP is
isolated when at least 60-90% by weight of a sample is composed of
the peptide,-preferably 95% or more, and more preferably more than
99%. Purity or homogeneity is determined using conventional assay
techniques, such as polyacrylamide gel electrophoresis (PAGE) of a
protein sample, followed by visualization of a single polypeptide
band upon staining the polyacrylamide gel with a suitable reagent,
or by high pressure liquid chromatography (HPLC).
[0087] AIP Moieties Having a Non-native Sequence. Preferably, a
modification to a natural AKAP sequence consists of a
"conservative" amino-acid substitution. "Conservative" amino acid
substitutions include those listed in Table 2, below.
2 TABLE 2 Original Residue Conservative Substitutions Alanine (A) G
Arginine (R) K Asparagine (N) Q, H Aspartic Acid (D) E Cysteine (C)
S Glutamic Acid (E) D Glycine (G) A Glutamine (Q) N, H Histidine
(H) N, Q Isoleucine (I) L, V Leucine (L) I, V Lysine (K) R
Methionine (M) L, I Phenylalanine (F) L, Y Proline (P) G Serine (S)
T Threonine (T) S Tryptophan (W) F, Y Tyrosine (Y) W, F Valine (V)
L, I
[0088] "Non-conservative substitutions" are amino acid
substitutions which produce a peptide which reduces binding of a
particular second protein (such as PKA) to a first protein (such as
an AKAP) by at least about 50%, compared to a peptide (such as an
AIP) having a sequence homologous to that of the binding domain of
the first protein. Preferably, such substitutions reduce binding by
25% or less, more preferably have no effect on binding, and most
preferably improve binding. Nonconservative substitutions can
result from changes in: (a) the tertiary structure of the peptide;
(b) the charge or hydrophobicity of the peptide; or (c) the bulk of
an amino acid side chain. Substitutions generally expected to
produce the greatest changes in protein properties are those in
which: (a) a generally hydrophilic amino acid residue is
substituted for, or by, a generally hydrophobic amino acid residue;
(b) a proline residue is substituted for, or by, any other residue;
or (c) an amino acid residue having a bulky side chain, e.g., a
phenylalanine residue, is substituted for, or by, a residue not
having such a side chain, e.g., a glycine residue. Preferably, an
amino acid residue having a generally electropositive side chain,
e.g., a lysine, arginine, or histidine residue, is not substituted
for, or by, a residue having a generally electronegative side
chain, e.g., a glutamine or aspartic acid residue.
[0089] Alkyl AIP Amides. An alkyl AIP amide is a form of the alkyl
peptide amides of this invention produced by linking an alkyl group
to the N-terminus of an AIP as described above. The most preferred
form of the alkyl AIP amide is a stearyl AIP amide, wherein the
alkyl moiety most preferably comprises a straight chain, saturated,
alkyl group of 18 carbon atoms in length. In their most preferred
form, the alkyl AIP amides of this invention are able to permeate
cell membranes (e.g., sperm-cell membranes) and affect binding of
PKA to an AKAP within the intracellular spaces of the cell.
[0090] The most preferred form of the alkyl AIP amides of the
present invention are preferably delivered in such a way that PKA
activity is affected only or primarily in target cells, e.g., by
topical application, by injection (e.g., into a joint to treat
arthritis) or by cell- or tissue-specific delivery of the AIP by
methods well known in the art.
[0091] Pharmaceutical Compositions. A description of pharmaceutical
compositions of this invention is provided below. Particular
emphasis is placed on describing compositions of a particularly
preferred form of the alkyl peptide amides of this invention, viz.,
alkyl AIP amides capable of inhibiting sperm motility and their use
in contraceptive compositions. However, most of the teachings
below, even those specifically directed to specialized
contraceptive compositions, can be applied equally to the
formulation of pharmaceutical compositions of other alkyl peptide
amides of this invention. It is contemplated that the compositions
and methods of the present invention will suggest many variations,
both human and veterinary, to one skilled in this art.
[0092] The present invention encompasses pharmaceutical
compositions that include an "effective amount" of an alkyl AIP
amide, or mixture of alkyl AIP amides, and one or more non-toxic
pharmaceutically acceptable carriers, excipients, diluents, and/or
adjuvants. An "effective amount" of an alkyl AIP amide is, e.g., an
amount effective to interfere substantially with PKA binding to an
AKAP in a target cell, thereby causing at least about a 50%,
preferably at least about a 75%, and most preferably at least about
a 90% reduction of PKA activity in the cell. For contraceptive
compositions, an "effective amount" of an alkyl AIP amide is an
amount effective to diminish sperm motility by at least about 50%,
preferably at least about 70%, more preferably at least about 80%,
and most preferably at least about 90%.
[0093] The dosage rate, e.g., 0.05 to about 20 mg/kg of body
weight, is a function of the nature and body weight of the human or
animal subject to be treated. The dosage unit of any biologically
active substance delivered to a subject depends, for example, on
the condition treated, nature of the formulation, nature of the
condition, embodiment of the claimed pharmaceutical compositions,
mode of administration, and condition and weight of the patient,
and the activity of the particular substance used. See, e.g., The
Pharmacological Basis of Therapeutics, Goodman, Gilman, et al.,
eds., Macmillan, New York, 1994, and Principles of Pharmacology,
Munson et al., ed., Chapman & Hall, New York, 1995. Standard
pharmaceutical formulation techniques are preferably used in
testing the potency of a given pharmaceutical composition, such as
the techniques disclosed in Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa., latest edition.
[0094] The pharmaceutical compositions or medicaments of the alkyl
peptide amides of the present invention can be formulated for
administration by any of various routes. The compositions can be in
the form of, for example, tablets, capsules, powders, granules,
lozenges, dragees, pills, ampoules, suppositories, or liquid or gel
preparations, such as oral, topical, or sterile parenteral
solutions or suspensions. In the instance of contraceptives, the
medicament is preferably formulated in a suppository, foam, gel, or
cream, for example, or applied to a condom, diaphragm, cervical
cap, sponge, or other conventional contraceptive barrier prior to
intercourse.
[0095] Tablets and capsules for oral administration can be in a
form suitable for unit-dose presentation and can contain
conventional diluents and excipients. The pharmaceutical
compositions will generally contain from 0.5 to 90% of the alkyl
peptide amide by weight of the total composition. In addition to an
alkyl peptide amide or combination of alkyl peptide amides, the
pharmaceutical compositions and medicaments can also contain other
pharmaceutically active compounds.
[0096] For contraceptive use, an alkyl AIP amide or combination of
alkyl AIP amides may be combined with one or more other
contraceptive substances, e.g., nonylphenoxypolyethoxyethanol
(nonoxynol-9), p-diisobutylphenoxy-polyethoxyethanol-(octoxynol-9),
gossypol, gramicidin, neem seed extracts (Praneernl, reetha
saponins, quinine hydrochloride, etc. Nonoxynol-9 and octoxynol-9
are surface-acting agents that disrupt the cell membrane, thereby
killing the sperm cells with which the substances come into
contact. The alkyl AIP amide can act in concert with such
surface-acting agents by inhibiting sperm motility long enough for
the surface-acting agents to come into contact with the sperm cells
to kill the cells. Due to the motility inhibition effect of the
alkyl AIP amide, contraceptive compositions combining an alkyl AIP
amide and, for example, nonoxynol-9, may employ a lower level of
nonoxynol-9 than if nonoxynol-9 is used alone. In addition to
topical (female) contraceptives, systemic (male) contraceptives are
also contemplated in which delivery of an alkyl AIP amide is
targeted to an appropriate site to affect sperm motility.
[0097] Calcium at sufficient concentrations can overcome the sperm
motility inhibition by an alkyl AIP amide. For this reason, a
non-toxic chelating agent (such as EDTA or EGTA), or anion that
combines with calcium ion to form an insoluble product, such as
phosphate, is preferably included in the contraceptive compositions
of this invention.
[0098] It is further contemplated that the pharmaceutical
compositions of this invention will include diluents and/or
excipients. Diluents and excipients suitable for use in the
pharmaceutical compositions of this invention include: binding
agents such as syrup, acacia, gelatins, sorbitol, tragacanth,
carboxymethyl cellulose and other cellulose derivatives, alginates,
and polyvinylpyrrolidone; fillers and extenders such as sugars,
starches, calcium phosphate, sorbitol, mannitol, or glycine;
tableting lubricants, such as magnesium stearate, talc,
polyethylene glycol or silica; disintegrants, such as potato
starch; or acceptable wetting agents, such as sodium lauryl
sulfates; and diluents such as water, ethyl alcohol, isopropyl
alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide,
oils (e.g., ground nut oil), glycerol, tetrahydrofurfuryl alcohol,
polyethylene glycols, and fatty acid esters of sorbitol or mixtures
thereof. Tablets can be coated by conventional methods.
[0099] Pharmaceutical compositions of the present invention may
include oral liquid preparations, or dry preparations designed to
be reconstituted with water or another suitable vehicle before use.
Suitable oral liquid preparations contemplated for inclusion in the
pharmaceutical compositions of this invention include preparations
in the form of: suspensions, solutions, emulsions, syrups or
elixirs. Suitable liquid preparations can also contain conventional
additives such as suspending agents, e.g., sorbitol, methyl
cellulose, glucose, gelatin, or hydrogenated edible fats;
emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia;
non-aqueous vehicles, e.g., almond oil, fractionated coconut oil,
oily esters such as glycerine, propylene glycol, or ethyl alcohol;
preservatives such as methyl or propyl p-hydroxybenzoate or sorbic
acid.
[0100] The pharmaceutical compositions of this invention may also
include additives, e.g., buffers such as sodium metabisulphite or
phosphate buffers; preservatives such as bactericidal and
fungicidal agents, including phenyl mercuric acetate or nitrate,
benzalkonium chloride or chlorhexidine; thickening agents, such as
hypromellose; or flavoring or coloring agents.
[0101] The pharmaceutical compositions of this invention,
particularly the contraceptive compositions of alkyl AIP amides,
can be applied topically. For topical application to the skin, the
alkyl peptide amide(s) can be made up into a cream, lotion, or
ointment using conventional formulations. For topical applications
to the eye, the alkyl peptide amide(s) of this invention can be
made up into a solution or suspension in a suitable sterile aqueous
or non-aqueous vehicle.
[0102] The pharmaceutical compositions of this invention can also
be administered parenterally in a sterile medium. The drug can be
dissolved or suspended in the vehicle, depending on the vehicle or
concentration used. Adjuvants such as local anesthetics,
preservatives, and buffering agents can also be dissolved in the
vehicle. Commonly used excipients for injectable forms of the
pharmaceutical compositions of the present invention include
physiological saline, Hank's solution, Ringer's solution, and the
like. Injection can be, e.g., intravenous, intramuscular,
intraperitoneal, or subcutaneous.
[0103] The pharmaceutical compositions of this invention can also
be administered by transdermal or transmucosal delivery by
including agents which effect penetration of these tissues, such as
bile salts, fusidic acid derivatives, cholic acid, and the
like.
[0104] The invention will be better understood by reference to the
following examples, which are intended merely to illustrate the
synthesis, purification, and use of alkyl peptide amides of the
present invention in two different types of living cells, sperm
cells and HeLa cells. However, the scope of this invention is not
to be considered limited to the specific species, compositions, and
uses of alkyl peptide amides used in the examples below.
EXAMPLE 1
Stearyl Peptide Amide Synthesis and Purification
[0105] The peptides described in the examples herein below were all
synthesized by Quality Controlled Biochemicals, Inc. (of Hopkinton,
Mass., USA), on an automated synthesizer using
9-fluorenylmethoxycarbonyl (FMOC) chemistry employing base-mediated
coupling. The activator of choice was either BOP or HATU, using
diisopropylethylamine as a solvent. Stearic acid was added together
with an activator of attachment to the free amino-terminus of the
protected peptide. The progress of the stearation reaction was
monitored by ninhydrin, a TFA test cleave, or by mass spectral or
HPLC analysis.
[0106] The final stearated peptide product was purified by reverse
phase HPLC using a C8 column employing a TFA/acetonitrile buffer
system. To identify the correct peak and facilitate recovery of
pure material, the molecular weight confirmation of the stearated
material was performed using a TOF mass spectrometry analyzer.
Analytical HPLC traces of the pooled fractions confirmed the
expected purity. Pooled fractions were lyophilized to a dry powder
under nitrogen. The stearyl peptide amides constructed and used in
the Examples below were as follows:
3 s-PKI: Stearyl-TTYADFIASGRTGRRNAIHD amide s-Ht31:
Stearyl-DLIEEAASRIVDAVIEQVKAAGAY amide s-Ht31-P:
Stearyl-DLIEEAASRPVDAVPEQVKAAGAY amide s-AKAP79:
Stearyl-YETLLIETASSLVKNAIQLSIE amide
EXAMPLE 2
Inhibition of Hela Cell PKA by a Stearyl PKI Amide
[0107] A. Characterization of a cAMP-Dependent Protein Kinase
Inhibitor Peptide:
[0108] The PKI peptide described immediately above is known to be a
very potent inhibitor, having an lC.sub.50 for purified PKA in the
nanomolar range, and having an IC.sub.50 for PKA supplied in the
form of a cellular extract in the micromolar range. The term
"IC.sub.50" as used herein refers to the concentration of inhibitor
required to produce a 50% reductionin enzyme activity of PKA. The
results showed that the stearyl PKI amide, s-PKI, inhibited the
enzymatic activity of the catalytic subunit of PKA in cellular
extracts in a concentration-dependent manner, and with lC.sub.50
similar to that for the non-stearated PKI. That indicated the
modification of PKI by the addition of stearyl moiety to produce
s-PKI as described in Example 1, above, did not alter the inhibitor
potency towards PKA (see FIG. 2).
[0109] The results above led to experiments designed to test the
efficacy of s-PKI on the activity of PKA in vivo. HeLa cells were
chosen as the model for these studies. However, any other cell
could have been used to test the inhibitory potency of this
particular stearyl peptide amide PKI, since PKA is a ubiquitous
enzyme. Activation of PKA in any cell is dependent on the
availability of the second messenger cAMP. Therefore, any agent
that activates or inhibits the enzyme adenylate cyclase will
accordingly cause an increase or decrease in the endogenous level
of cAMP and thus alter the activity of PKA.
[0110] Forskolin, a plant alkaloid, is known to be a potent
activator of adenylate cyclase in HeLa cells, resulting in a
significant increase in the endogenous level of cAMP. This agent
was employed in the studies reported herein, by adding it to the
medium at a concentration known to be sufficient to significantly
increase the amount of cAMP in HeLa cells. The HeLa cells used in
this study were grown in a mixture of Dulbecco's Modified Eagle
Medium (DMEM) and fetal bovine serum (FBS), containing either 100
.mu.M of forskolin, or an equal volume of DMSO as a vehicle
control.
[0111] When the unmodified PKI was tested against PKA and compared
with that of s-PKI, it was found that s-PKI inhibited the enzymic
activity of-PKA in vivo in HeLa cells within sixty minutes after
addition of the inhibitor to the medium and the inhibition was
observed for both basal (no forskolin) and forskolin-stimulated
cells. In contrast, the unmodified non-stearated PKI did not have
any significant inhibitory effect on the enzyme activity of
PKA.
[0112] B. Effect of Concentration of s-PKI Used to Treat HeLa
Cells:
[0113] Cultures of HeLa Cells were treated with differing amounts
of s-PKI, in the presence or absence of forskolin, as follows.
About 10.sup.6 HeLa cells were incubated in a 75 centimeter-squared
(cm.sup.2) flask containing 5 milliliters (ml) DMEM/10% FBS medium
in the presence of 0, 10, 25, 50, and 100 micromolar (pM) s-PKI for
thirty (30) minutes. The cells were then transferred to a medium
solution (DMEM/10% FBS) supplemented with either forskolin (100
.mu.M) or an equal volume of vehicle control (0.1% DMSO), and
incubated for an additional 30 minutes.
[0114] At the end of the second 30 minutes, the cells were
harvested and processed, as follows: The cells were first washed to
remove any adhering inhibitor or other adherent material, and then
scraped into serum-free DMEM. The resulting scraped cells were then
lysed in lysis buffer, thereby forming a lysate. The lysate was
centrifuged and the supernatant was used to assay the PKA kinase
activity as described by Goueli, et al (Anal. Biochem. 225: 10-17,
1995). Protein concentration was determined using the assay
protocol described in the aforementioned reference, which is
incorporated by reference herein.
[0115] The results of this assay are displayed in Table 3,
below.
4TABLE 3 s-PKI PKA activity Concentration (pmoles
.sup.32P/min/.mu.g Percent (.mu.M) protein) Inhibition 0 3.258 0 10
2.536 22 25 2.456 25 50 1.775 45 100 0.80 75
[0116] C. Effect of Incubation Time in Presence of s-PKI on HeLa
Cells:
[0117] Table 4, below, presents the results of a specific study
done to determine the effect of incubation time of s-PKI on the
kinase activity of PKA in HeLa cells. HeLa cells were grown in
medium supplemented with s-PKI for varying amounts of time, and the
inhibitory effect on PKA kinase activity was measured. HeLa cells
(10.sup.6 cells per dish) were grown in DMEM medium supplied with
10% FCS and fresh medium was added with and without inhibitor
(s-PKI) for various periods (0, 1, 2, and 3 hours) with and without
the addition of 100 .mu.M of forskolin 30 minutes before harvesting
the cells.
[0118] The harvested cells were then processed, and the PKA kinase
activity was assayed as follows. The medium was removed and the
cells were washed in fresh medium several times and then lysed
using a lysis buffer. The lysate was centrifuged and the
supernatant was used to assay the PKA kinase activity as described
by Goueli et al. (1995) Anal. Biochemistry 225: 10-17. Protein
concentration was determined using published protocols (See
Id.)
5TABLE 4 Incubation PKA Activity (pmoles Percent of Activity Time
(hr.) .sup.32P/min/.mu.g protein) Remaining 0 2.84 0.0 1 1.432 50.4
2 1.791 63.0 3 1.996 70.3
[0119] The data in Table 4 demonstrate that s-PKI lost its
effectiveness as an inhibitor after a few hours in the medium. The
presence of forskolin was found to have no effect upon activity.
These results could indicate that s-PKI was degraded in the medium
by an exogenous or secreted protease (or esterase), or that it was
degraded intracellularly.
[0120] The apparent loss in effectiveness of s-PKI as an inhibitor
of PKA activity over time could have been accounted for and
counteracted in any one of a number of ways. For example, s-PKI
could have been added to the medium every few hours, and the medium
replenished more frequently than it was in this Example.
Alternatively, a new stearyl peptide amide could have been designed
with a subset of the sequence of PKI, so the resulting modified
form of s-PKI could have survived degradation, thus remaining
intact in the medium and in the cells for longer periods of
time.
EXAMPLE 3
Sperm Motility Assays
[0121] A. Materials and Methods
[0122] Sperm Preparation. Testes from mature bulls with intact
tunica were obtained from a local slaughterhouse and sperm from
caput or caudal epididymis were isolated and washed as previously
described (Vijayaraghavan et al., Biol Reprod. 32:489-500, 1985).
The sperm were resuspended in buffer A (120 mM NaCl, 10 mM KCl, 10
mM Tris[hydroxymethyl]aminornhethane (TRIS), pH 7.4) supplemented
with 10 mM glucose and 10 mg/ml bovine serum albumin (BSA) for
motility measurements. Monkey semen was obtained by
electro-ejaculation and processed by procedures previously reported
(Smith et al.,Biol. Reprod. 54:719-727, 1996). Human semen samples
were obtained from a fertility clinic at the Oregon Health Sciences
University.
[0123] Sperm Motility Measurement. Head motility parameters were
determined as previously described (Vijayaraghavan et al.,Biol.
Reprod. 54:709-718, 1996; Stephens et al.,Biol. Reprod. 38:577-586,
1988). A 3-4 .mu.L aliquot of sperm suspension (5
.times.10.sup.7/ml) was loaded onto a counting chamber at
37.degree. C. After bulk fluid movement had subsided, six different
locations on the slide were recorded. The videotaped segments were
analyzed by a computerized image-analysis system (CASMA) as
previously described (Vijayaraghavan et al.,Biol. Reprod.
54:709-718, 1996; Stephens et al.,Biol. Reprod. 38:577-586, 1988).
This computer system measured several parameters of head motion,
including forward motility. The forward Motility Index (FMI) was
used as the measure of motility. FMI is a product of percent motile
(% M, percent of sperm moving at velocity greater than 20 mm/sec)
and average velocity (Va, the five-point smoothed average of the
head positions through arnalysis of at least twenty frames). In
most cases both components of FMI were found to increase
together.
[0124] PKA Activity. PKA activity was assayed as previously
described (Carr et al., J. Biol. Chem. 268:20729-20732, 1993) with
minor changes. Whole caput or caudal sperm were treated with
2-chloro-2'-deoxyadenosine (50 .mu.M), H-89 (50 .mu.M), or both for
30 min. at 37.degree. C. The sperm were then washed twice in ice
cold homogenization buffer supplemented with protease inhibitors,
benzamidine (10 mM), leupeptin (4 .mu.g/ml), and
N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (100 .mu.M), and
sonicated for one min. Reaction mixtures (20 microliters (.mu.l )
total) contained 250 .mu.M Kemptide (i.e., LRRASLG), 250 .mu.M
(y-.sup.32P) ATP, 25 mM Na.sub.3VO.sub.4, 50 mM 3-[N-morpholino]
propanesulfonic acid (MOPS) (pH=7.0), 10 mM MgCl.sub.2, 0.25 mg/ml
BSA and where indicated, 10.mu.M cAMP. Assays were initiated by
addition of labeled ATP, incubated for 2 min at 30.degree. C., and
stopped by addition of 30 .mu.l of 1 N HCl. Twenty .mu.l of the
reaction was then spotted on phosphocellulose paper followed by
three washes in 75 mM phosphoric acid. The papers were then
analyzed by Cerenkov counting. All determinations were made in
quadruplicate.
[0125] Western blotting and overlay assays. The overlay procedure
is a modified Western blot procedure. Proteins were separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to a nylon membrane (Immobilon). After treatment with Blotto
(Protein Methods, Bollag and Edelstein, eds., Wiley-Liss, New York,
1991) to prevent non-specific binding, radiolabelled RII.alpha. or
RII.beta. probes were applied (Carr and Scott, Trends Biochem. Sci.
17:246-249, 1992). Recombinant RII.alpha. was produced as
previously described (Carr et al., J. Biol. Chem. 266:14188-14192,
1991; Carr et al., J. Biol Chem. 267:13376-13382, 1992). RII.alpha.
and RII.beta. were gifts from Dr. John Scott, Oregon Health
Sciences University, Portland, Oreg. Two different variations of
the overlay assay were used. In the first variation, recombinant
RII radiolabeled by incubation with the catalytic subunit of PKA
and y.sup.32P-ATP was used. After separation from free
y.sup.32P-ATP, the .sup.32P-labeled RII (500,000 CPM/10 ml blotto)
was incubated with the blocked blot for four hours followed by
washing and autoradiography. In the second variation, the blot was
incubated with cold RI.beta. (1 .mu.g/10 ml Blotto), washed, then
incubated with anti-RI.beta. antiserum. After the blot was again
washed, it was incubated with secondary antibody conjugated to
horseradish peroxidase. A final wash was followed by development
with an enhanced chemiluminescence kit (Renaissance.TM., New
England Nuclear). The PKA isoform-specific antibodies were
affinity-purified antibodies obtained from Triple Point Biologics,
Forest Grove, Oreg.
[0126] B. Results
[0127] Identification of AKAPs and PKA Isoforms in Bovine, Human
and Monkey Sperm.
[0128] Cyclic AMP has been known to stimulate sperm motility in a
variety of species. To determine if PKA anchoring is involved in
regulating motility, the PKA isoforms and AKAPs present in
mammalian sperm were identified. Immunoblot analysis of sperm
proteins with affinity-purified, isoform-specific antibodies
detected three (RII.alpha., RII.beta., and RI.beta.) of the four
known PKA isoforms in bovine, human and monkey sperm (FIG. 3).
[0129] FIG. 3 shows the results of identifying PKA subunits in
various mammalian sperm. Sperm from bovine (B), human (H) and
monkey (M), were lysed and the proteins were separated by SDS-PAGE
and analyzed by Western blotting for regulatory subunits of PKA
(RI.alpha., RI.beta., RII.alpha., or RII.beta.) using isoform
specific antibodies.
[0130] No RI.alpha. was detected at the 50 to 55 kDa range in sperm
from any of the species, even though RI.alpha. was detectable in
bovine testis. The lower Mr bands detected in bovine and human
sperm with anti-RI.alpha. antibody might be breakdown products of
RI.alpha.. However, the other isoforms showed very little apparent
proteolysis. The fuzziness of the bands detected with the
RII.alpha. and RI.beta. antibodies suggest that these proteins may
be at least partially phosphorylated.
[0131] Identification of PKA Isoforms in Three Different Species of
Sperm Using Four Different Antibodies.
[0132] To determine if the PKA isoforms are associated with the
soluble or insoluble fractions of sperm sonicates, bovine sperm
were homogenized, centrifuged at 16,000.times.g for 30 min and
subjected to western blot analysis (FIG. 4). Bovine caudal
epididymal sperm were lysed by sonication, separated into
supernatant (S) or pellet (P) fractions by centrifugation at
16,000.times.g, and probed using anti-RII.alpha., anti-RII.beta.
and anti-RI.beta. antibodies.
[0133] Greater than 50% of all three R subunit isoforms are present
in the pellet fraction of sperm sonicates, and RI.beta. is found
almost exclusively in this fraction. These results suggest that all
of these isoforms are associated with structural or cytoskeletal
elements of the sperm.
[0134] Overlay analysis of bovine, human and monkey sperm using
.sup.32P-labeled RII.alpha. or RII.beta. as probes detected a
single dominant AKAP in each species (FIG. 5). A single predominant
AKAP was detected in bovine, human, and monkey sperm using either
RII.alpha. or RII.beta. as a probe. The bovine and human AKAPs had
relative molecular weights of 110 kDa, while the monkey AKAP was
slightly larger, at a relative molecular weight of 115 kDa. The
overlay assay also detects the endogenous RII isoforms accounting
for the bands observed at 55 kDa.
[0135] The bands detected at approximately 55 kDa by the RII.beta.
probe are probably due to dimerization of the probe with endogenous
RII, although it is not clear why these proteins are preferentially
binding to RII.alpha. compared to RII.beta.. Overlay analysis using
RI.beta. did not detect any binding proteins. These data suggest
that a single AKAP in sperrn may be responsible for the
localization of both RI.alpha. and RII.beta.. The fact that
RI.beta. is clearly present in the particulate fraction, but does
not interact with denatured proteins on the blot, suggests that the
overlay method may not be appropriate for detecting AKAPs that
interact with RI.beta.. Instead, a non-denaturing binding assay,
such as a band-shift assay (Carr and Scott, Trends Biochem. Sci.
17:246-249, 1992) may be used for detecting RI-binding
proteins.
[0136] All AKAPs identified to date contain an amphipathic helix
domain responsible for RII binding (Carr et al., J. Biol. Chem.
266:14188-14192, 1991; Carr et al., J. Biol. Chem. 267:13376-13382,
1992; Carr et al., J. Biol. Chem. 267:16816-16823, 1992; Coghlan et
al., J. Biol. Chem. 269:7658-7665, 1994; McCartney et al., J. Biol.
Chem. 270:9327-9333, 1995; Carr et al., J. Biol. Chem.
267:13376-13382, 1992). A peptide, Ht31, containing an amphipathic
helix domain, binds to RII and competitively inhibits the
interaction of RII with other AKAPs. Addition of this AIP to the
overlay assay blocked RII binding with AKAP 110 (FIG. 6),
suggesting that this sperm AKAP also contains an amphipathic
helix-binding domain. A cell-permeable stearated Ht31 counterpart,
N-St Ht31, also inhibited in vitro binding of RII to AKAP 110,
albeit at a reduced potency (85% inhibition s compared with 100% by
the non-stearated Ht31). The control stearated peptide, s-Ht31-P,
which has a proline substitution preventing amphipathic helix
formation, had no effect on RII binding.
[0137] FIG. 6 shows the results of disrupting the binding of RII to
sperm AKAPs by an AlP, or by a control alkyl peptide amide. RII
overlays were performed in the absence (lane 1) or presence of 20
.mu.M Ht31, s-Ht31, or s-Ht31-P (lanes 2, 3 or 4, respectively).
Addition of the three peptide species inhibited RII binding to
AKAP110 by 100%, 85% and 0%, respectively, as determined by
densitometric scanning analysis.
[0138] Effect of Stearyl AIP Amides on Sperm Motility. Stearyl AIP
amides, such as s-Ht31 or s-AKAP79 were added to vigorously motile
sperm under a variety of conditions to determine whether PKA
anchoring would be sufficiently inhibited to produce a reduction in
sperm motility. The results, shown below, indicated that each
stearyl AIP amide tested inhibited basal motility in a
concentration- and time-dependent manner.
[0139] In the first such experiment, the vital dyes SYBR-green and
rhodamine 123 were added to sperm before and after treatment with
s-Ht31 in order to determine whether s-Ht31 affects sperm viability
or structural integrity. Only viable, intact cells normally take up
such dyes. Both treated and control sperm were found to accumulate
these dyes to the same extent, suggesting that the stearyl peptide
amide (s-Ht31) did not decrease viability or disrupt the integrity
of the sperm plasma membrane. In experiments using sperm loaded
with the chromophores BCECF or Fura 2, addition of digitonin, but
not s-Ht31, caused release of these dyes, confirming that peptide
treatment did not compromise the integrity of the plasma
membrane.
[0140] FIG. 7 summarizes the results of the second such experiment.
This second experiment was conducted as follows. Sperm were
incubated in buffer A containing s-Ht31 at 5 .mu.M
(.circle-solid.), 10 .mu.M (.box-solid.) or 50 .mu.M
(.diamond-solid.) or a control peptide, s-Ht31-P at 50 .mu.M
(.times.). Motility was assayed at the times indicated by
computer-automated sperm motility analysis (CASMA) as described
previously. Complete arrest of motility was observed at
concentrations from 5 to 50 .mu.M within three to five minutes
after treatment (FIG. 7).
[0141] FIG. 8 is a plot of the results of experiments in which
sperm were incubated for 5 min in buffer A containing increasing
concentrations of s-Ht31 (.diamond-solid.) or control peptide
s-Ht31-P (.tangle-solidup.) and the motility index was determined.
When motility is measured five minutes post-treatment, the
concentration of s-Ht31 needed to produce 50% inhibition is
approximately 1 .mu.M (FIG. 8). A control peptide, s-Ht31-P, which
is ineffective in disrupting PKA anchoring to AKAPs (see FIG. 6),
had no effect on sperm motility at concentrations up to 20 .mu.M,
suggesting that motility inhibition by s-Ht31 was due to disruption
of PKA anchoring.
[0142] The next experiments in this set demonstrated that sperm
with inhibited motility after exposure to s-Ht31 could regain full
motility after contact with s-Ht31 ceased, at least under certain
conditions. In each such experiment, sperm motility was monitored
5, 30, and 60 minutes after exposure to s-Ht31 in the presence of
buffers with various salt compositions. FIG. 9 shows sperm motility
recovery results from bovine caudal epididymal sperm suspended in
buffer A supplemented with 2 mM calcium (+Calcium) or 2 mM EGTA
(-Calcium), after treatment with water (control) or with 10 .mu.M
s-Ht31. The results of this first experiment (shown in FIG. 9)
demonstrated the motility of sperm treated with s-HT31 only
recovered after treatment when the sperm were suspended in
calcium-containing media. When the sperm were suspended in a medium
depleted of external calcium, by the addition of EGTA, treatment
with s-Ht31 caused irreversible motility arrest. The presence or
absence of external calcium did not affect the motility of
untreated sperm.
[0143] Sperm motility and motility inhibition by s-Ht31 was also
assayed in the presence and absence of bicarbonate. Bicarbonate was
used in this experiment because of its demonstrated tendencies to
stimulate sperm adenyl cyclase (Okamura et al., J. Biol. Chem.
260:9699-9705, 1985), and to increase intracellular pH
(Vijayaraghavan et al.,Biol. Reprod. 32:489-500, 1985). Bicarbonate
has also been described as an essential component of suspension
buffers required for optimal sperm function in vitro (Lee and
Storey, Biol Reprod. 34:349-356, 1986; Kopf and Gerton, in Elements
of Mammalian Fertilization, Wassermann, ed., pp. 153-203, CRC
Press, Boca Raton, Fla., 1991).
[0144] FIG. 10 shows the results of this last experiment. In the
experiment, sperm were incubated in buffer A containing 2 mM
calcium and 50 mM bicarbonate (where indicated). Motility was
assessed at 5, 30 and 60 min following addition of either water
(control) or 10 .mu.M s-Ht31. Sperm motility in the presence of
bicarbonate is significantly enhanced compared to untreated sperm
(FIG. 10). To determine the effect of AIPs on optimal motile sperm,
s-Ht31 was added to sperm in both basal and
bicarbonate-supplemented media (FIG. 10). The peptide was equally
effective in inhibiting motility in both media. Similar results
were obtained when sperm were pretreated with other activators of
motility, dibutyryl cyclic 3', 5'-adenosine monophosphate
(db-cAMP), isobutylmethylxanthine (IBMX), and CDA, all thought to
increase cAMP content.
[0145] In similar experiments, addition of the same stearyl AIP
amide (s-Ht31) to undiluted rhesus monkey (FIGS. 11 and 12) and
human ejaculated semen (FIG. 13) was likewise found to result in a
reduction in sperm motility. The experiments and figures displaying
this last set of results are summarized further below.
[0146] FIG. 11 shows the effect of s-AKAP79 on the motility of
monkey-ejaculate sperm at 5 min (light gray bar) and 30 min (dark
gray bar). Increasing concentrations of s-AKAP79 were added to neat
monkey semen and incubated at 37.degree. C. Motility was assayed at
5 and 30 min. As was observed in the s-Ht31 studies described
above, the results of this experiment showed s-AKAP inhibited sperm
motility in a concentration--and time-dependent manner.
[0147] FIG. 12 shows the effect of s-Ht31 on the motility of monkey
semen and swim-up sperm. Monkey sperm, either neat semen or semen
collected after swim-up analysis, were incubated with no addition
(Control) (open bar), 50 .mu.M s-Ht31 (solid bar), 2.5 mM EGTA
(hatched bar) or 2.5 mM EGTA plus 50 .mu.M s-Ht31 (dotted bar).
Motility was assayed after 15 min.
[0148] FIG. 13 shows the effect of s-Ht31 on the motility of
human-ejaculate swim-up sperm. Human sperm collected after swim-up
analysis were incubated for 15 min with no addition (Control), 100
.mu.M s-Ht31, or 100 .mu.M s-Ht31-P and assessed for motility.
[0149] Role of PKA in sperm motility and s-Ht31 action. If the
effect of anchoring inhibitor peptides on sperm motility is due to
the dissociation of the catalytic subunit of PKA from its preferred
substrates, then this effect should be mimicked by inhibitors of
PKA activity. To test this hypothesis, the effect of a potent
cell-permeable PKA inhibitor,
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide
(H-89), on sperm motility was studied.
[0150] FIG. 14 shows the effect of H-89 on sperm motility and PKA
activity from control sperm and sperm activated by
2-chloro-2'-deoxyadenosine (CDA). Bovine caudal and caput sperm
were incubated in buffer A in the presence or absence of 50 .mu.M
CDA and/or 50 .mu.M H-89. When sperm were treated with both CDA and
H-89, the H-89 was added 15 min prior to CDA. Motility was assessed
by CASMA at 15 min following treatment. Addition of high levels of
H-89 (50 .mu.M) inhibited basal sperm motility approximately 50% or
less, and had no effect on sperm motility stimulated by the
adenosine analog CDA (FIG. 14).
[0151] Essentially identical data to the data shown in FIG. 14 were
obtained when sperm were stimulated with isobutylmethylxanthine
(IBMX) or 8-bromo-cAMP (8-Br-cAMP) instead of CDA (FIG. 15). This
unexpectedly weak action of H-89 apparently results primarily from
a decrease in motility of a subpopulation of sperm, because a
considerable proportion of sperm maintain vigorous motility. This
contrasts sharply with the complete arrest of motility seen in
S-Ht31 treated sperm. Also, unlike the action of the alkyl AIP
amides, the effects of H-89 were strongly suppressed by CDA. The
effectiveness of H-89 was also observed with caput sperm, which are
immotile unless stimulated with agents such as CDA or IBMX.
Motility induction by CDA is unaffected by preincubating sperm with
H-89 before treatment (FIGS. 14 & 15). Together these
observations suggest that AIPs and PKA kinase inhibitors, have
different mechanisms of action.
[0152] In previous reports, initiation or stimulation of sperm
motility by CDA was associated with an elevation of cAMP and an
increase in PKA activity was assumed (Vijayaraghavan and
Hoskins,Biol. Reprod. 34:468-77, 1986). Because H-89 failed to
suppress stimulation of motility by CDA, PKA activity from caudal
and caput sperm which had been treated with CDA, H-89 or CDA plus
H-89 was measured. All assays were performed in the absence or
presence of cAMP to determine the basal and maximal PKA activities.
Bovine sperm were incubated as described for FIGS. 14 and 15 and
then assessed for PKA activity. Where indicated, 10 .mu.M cAMP was
added to the PKA assay. The results of this assay are shown in FIG.
15.
[0153] Treatment with CDA increased basal PKA activity by
approximately 60% in both caudal and caput sperm. H89 treatment, in
the presence or absence of CDA, strongly inhibited PKA activity.
Addition of cAMP to the assay was not able to overcome this
inhibition. To ensure that H-89 was affecting PKA activity in the
cells and not just its activity in the in vitro assay, the sperm
were washed several times before lysis to remove all exogenous
H-89. PKI (50 .mu.M), a non-permeable PKA inhibitor, was added as a
control to sperm. The sperm were then washed, lysed and assayed for
PKA activity in a manner identical to the H-89 treated sperm. PKI
had no effect on cellular PKA activity, although a similar
concentration, when added to the PKA assay, will inhibit virtually
100% of activity. These data suggest that motility stimulation
associated with treatments that elevate cAMP does not require
increases in the catalytic activity of PKA and can occur even if
PKA activity is substantially inhibited.
[0154] Finally, FIG. 16 shows the effect of s-Ht31, s-AKAP79, and
s-Ht31-P on the motility of bovine sperm. Sperm were incubated for
5 min in buffer A containing increasing concentrations of s-Ht31
(diamonds), s-AKAP79 (squares) or control peptide s-Ht31-P
(triangles) and motility was assessed.
[0155] C. Discussion
[0156] As an initial step in studying the role of PKA anchoring in
regulation of sperm function, PKA isoforms and AKAPs that are
present in mammalian sperm were identified. Three of the four
isoforms of the regulatory subunit of PKA were detected in bovine,
human and monkey sperm. RI.alpha., though abundant in testis, were
not detected in sperm. Consistent with previous reports (Horowitz
et al., J. Biol. Chem. 263:2098-2104, 1988), the major proportion
of all isoforms remained in the particulate fraction, presumably
due to their interaction with AKAPs. The Examples described above
also demonstrated bovine, human and monkey sperm each contain one
predominant AKAP with an apparent molecular weight between 110 and
115 kDa. The Examples also demonstrated that AKAPs of each such
organism studied predominately localized to the particulate
fraction and bound to both RII.alpha. and RII.beta., suggesting
that a single AKAP is responsible for the localization of both
these RII isoforms. In the overlay assays described above, in which
RI.beta. was used as a probe, no binding protein was deleted in any
of the sperm studied. The fact that RI.beta. was clearly in the
particulate fraction in Example, but does not interact with
denatured proteins on the blot, suggests that the overlay procedure
may not be appropriate for detecting AKAPs that interact with
RI.beta.. Preliminary analysis by electron microscopy detects Roll
in both the head and tail, while RII.alpha. is almost exclusively
associated with the axoneme.
[0157] In accord with these observations, Orr and colleagues
reported one predominant AKAP at molecular weight of approximately
120 kDa in bovine sperm (Horowitz et al., J. Biol Chem.
263:2098-2104, 1988), and Rubin and colleagues found a single
RII.beta. AKAP ({tilde over ()}120 kDa) that binds RII.beta. in
mature mouse sperm (Lin et al., J. Biol. Chem. 270:27804-27811,
(1995)). Others, however, have reported RII.alpha.-binding AKAPs of
82 kDa in mouse that bind RII.alpha. (Carrera et al., Dev. Biol.
165:272-284, 1994), 80 kDa in rat (Horowitz et al., J. Biol. Chem.
263:2098-2104, 1988) and 72 kDa in human (Pariset and Weinman, Mol.
Reprod. Develop. 39:415-422, 1994). The reason for these
differences is not clear, although proteolysis is one possibility.
Prolonged storage of SDS-solubilized extracts was found to result
in loss of the 110-kDa band and the appearance of an 80-kDa band.
The 72-kDa RII.alpha.-binding band reported in human sperm (Pariset
and Weinman, Mol. Reprod. Develop. 39:415-422, 1994) is distinct
from all other known AKAPs due to the fact that it was detected
using a peptide from RII.alpha. (amino-acid residues 45-75) which
does not contain the AKAP-binding domain (Luo et al., J. Biol.
Chem. 265:21804-21810, 1990; Scott et al., J. Biol. Chem.
265:21561-21566, 1990; Hausken et al., J. Biol. Chem.
269:24245-24251, 1994; Li and Rubin, J. Biol. Chem. 270:1935-1944,
1995). Finally, the cloning of a sperm AKAP of 84 kDa that is
uniquely expressed during spermatogenesis has been reported (Lin et
al., J. Biol. Chem. 270:27804 27811, 1995). This AKAP is not found
in mature sperm and thus its relationship, if any, to AKAP 110 in
mature sperm is unknown. To date, only sperm cells have been shown
to contain an AKAP of 110 kDa.
[0158] Several reports have suggested that flagellar activity in
sperm is regulated by cAMP (Tash, Cell Motil. Cytoskel. 14:332-339,
1989; Garbers and Kopf, in Advances in Cyclic Nucleotide Research,
Greengard and Robison, eds., Vol. 13, pp. 252-306, Raven Press, New
York, 1980; Lindemann and Kanous, Arch. Androl. 23:1-22, 1989; Tash
and Means, Prog. Clin. Biol. Res. 267:335-355, 1988). Both PKA and
AKAPs have been shown to be located at the same subcellular site in
sperm, the outer mitochondrial membrane located on the proximal
flagella (i.e., the sperm "midpiece") (Lieberman et al., J. Cell
Biol. 107:1809-1816, 1988; Lin et al., J. Biol. Chem.
270:27804-27811, 1995). Addition of a cell-permeable AIP
dramatically inhibits sperm motility in a dose- and time-dependent
manner. This inhibition is reversible, but only in the presence of
external calcium, suggesting that the regulatory mechanism being
disrupted may involve maintenance of calcium homeostasis or a
calcium-regulated function that recovers only in the presence of
exogenous calcium. Since mitochondria play an important role in
sperm calcium homeostasis (Babcock et al., J. Biol. Chem.
251:3881-3886, 1976; Vijayaraghavan and Hoskins, Cell Calcium
10:241-253, 1989; Vijayaraghavan et al.,Biol. Reprod. 40:744-751,
1989; Vijayaraghavan and Hoskins, Mol. Reprod. Develop. 25:186-194,
1990; Vijayaraghavan et al., Mol. Reprod. Dev. 38:326-333, 1994),
it is reasonable to propose that disruption of RII anchoring in
this region could be responsible for the changes in sperm
Ca.sup.2+regulation. The control peptide, s-Ht31-P, had no effect
on motility, suggesting that motility inhibition by s-Ht31 was not
due to the stearate moiety but due to disruption of PKA anchoring.
Reversibility of the motility inhibition and the observations that
s-Ht3 1-treated sperm exclude vital dyes provide evidence that the
motility inhibition is not due to disruption of sperm plasma
membrane integrity. The simplest model consistent with these data
is that the interaction of the regulatory subunit of PKA with AKAP
110 is essential for sperm movement.
[0159] It is usually assumed that the main function of this
interaction is to translocate the catalytic subunit to a
preferential subcellular site for specific phosphorylation of
protein substrates in the vicinity. This model is supported by
studies showing that microinjection of AIPs into neuronal or muscle
cells mimics the effect of PKA inhibitors causing loss of cAMP
modulation of the glutamate receptor and voltage-gated calcium
channels (Rosenmund et al., Nature 368:853-856, 1994; Johnson et
al., Proc. Natl. Acad. Sci. USA 91:11492-11496t 1994). FIGS. 14 and
15 demonstrate that in sperm inhibition of the kinase activity of
the catalytic subunit of PKA does not mimic the effect of AIPs,
suggesting that the interaction of the regulatory subunit with
sperm AKAPs has regulatory actions independent of the catalytic
subunit. Others have also suggested that the regulatory subunit
acts independently of the catalytic subunit (De Camilli et al., J.
Cell Biol. 103:189-203, 1986). An independent role of RII in the
regulation of sperm motility has also been suggested by reports
showing that the addition of axokinin (later shown to be RII)
[Noland et al.,Biol. Reprod. 37:171-180, 1987; Paupard et al., J.
Cell. Biochem. 37:161-175, 1988] to demembraned sperm was
sufficient to induce motility (Tash et al., Cell 38:551-559, 1984;
Tash et al., J. Cell Biol. 103:649-655, 1986; Tash and Bracho, J
Androl. 15:505-509, 1994).
[0160] Even though the activation of motility agents which increase
sperm cAMP is well substantiated, the role of PKA has never been
clear. For instance, PKA inhibitors reportedly do not alter the
stimulatory actions of cAMP on motility of intact or demembranated
sperm (Carr and Acott, Biology of Reproduction 43:795-805, 1990;
San Agustin and Witman, Cell Motility & the Cytoskeleton
27:206-18, 1994). These reports, however, did not conclusively
establish that the PKA inhibitors were indeed inhibiting sperm PKA.
Assay results reported herein demonstrate that under conditions
where sperm PKA is clearly inhibited, compounds that elevate cAMP
such as CDA, IBMX, and 8-Br-cAMP still induce or stimulate sperm
motility. The lack of effect of H-89, even at concentrations as
high as 50 .mu.M, on cAMP-mediated induction and stimulation of
motility is quite unequivocal. These data suggest that the
association of RII with AKAPs, but not PKA catalytic activity, is
required for sperm motility. Therefore, RII, independent of PKA
activity, may regulate other biochemical events such as phosphatase
activity and intracellular ion concentration.
[0161] This regulation may involve protein-protein interaction
between the RII/sperm AKAP complex and other sperm proteins. The
demonstration that the phosphatase PP2B, PKA, and PKC are all
anchored to the same AKAP in neurons (Klauck et al., Science
271:1589-1592, 1996) opens up several possible, previously
unidentified, roles for the individual members of this multimeric
complex. The interaction between RII and other sperm proteins could
also be cAMP-dependent, since cAMP is known to produce a
conformational change in the regulatory subunit. The data presented
herein show that RII anchoring, independent of PKA catalytic
activity, is essential for sperm motility and that cell-permeable
AIPs disrupt PKA anchoring and cellular function.
[0162] All publications and published patent documents cited in
this specification are incorporated herein by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0163] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. All modifications that are
within the spirit and scope of the invention are to be included
within the scope of the invention as described herein.
Sequence CWU 1
1
13 1 12 PRT Homo sapiens 1 Thr Arg Asp Ile Tyr Glu Thr Asp Tyr Tyr
Arg Lys 1 5 10 2 24 PRT Homo sapiens 2 Asp Leu Ile Glu Glu Ala Ala
Ser Arg Ile Val Asp Ala Val Ile Glu 1 5 10 15 Gln Val Lys Ala Ala
Gly Ala Tyr 20 3 14 PRT Homo sapiens 3 Leu Ile Glu Glu Ala Ala Ser
Arg Ile Val Asp Ala Val Ile 1 5 10 4 14 PRT Homo sapiens 4 Thr Ala
Glu Glu Val Ser Ala Arg Ile Val Gln Val Val Thr 1 5 10 5 14 PRT
Homo sapiens 5 Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile
Glu 1 5 10 6 14 PRT Homo sapiens 6 Leu Ile Glu Thr Ala Ser Ser Leu
Val Lys Asn Ala Ile Gln 1 5 10 7 18 PRT Homo sapiens 7 Glu Val Ala
Ala Glu Val Leu Ala Glu Val Ile Thr Ala Ala Val Lys 1 5 10 15 Ala
Val 8 14 PRT Homo sapiens 8 Ile Ile Asp Met Ala Ser Thr Ala Leu Lys
Ser Lys Ser Gln 1 5 10 9 14 PRT Homo sapiens 9 Leu Ala Glu Lys Ile
Val Ala Glu Ala Ile Glu Lys Ala Glu 1 5 10 10 22 PRT Homo sapiens
10 Val Ile Ser Glu Ala Thr Glu Gln Val Leu Ala Thr Thr Val Gly Lys
1 5 10 15 Val Ala Gly Arg Val Cys 20 11 20 PRT Homo sapiens 11 Thr
Thr Tyr Ala Asp Phe Ile Ala Ser Gly Arg Thr Gly Arg Arg Asn 1 5 10
15 Ala Ile His Asp 20 12 24 PRT Homo sapiens 12 Asp Leu Ile Glu Glu
Ala Ala Ser Arg Pro Val Asp Ala Val Pro Glu 1 5 10 15 Gln Val Lys
Ala Ala Gly Ala Tyr 20 13 22 PRT Homo sapiens 13 Tyr Glu Thr Leu
Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala 1 5 10 15 Ile Gln
Leu Ser Ile Glu 20
* * * * *