Note: Descriptions are shown in the official language in which they were submitted.
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Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Allosteric Binding
Ligands
To Modulate Serum Low Density Lipoprotein (LDL) Levels
Field Of Invention
This invention is related to the field of hypercholesterolemia. In particular,
the
invention provides compositions and methods to modulate circulating levels of
low density
lipoproteins by altering the conformation of the protein PCSK9 using a
synthetic ligand and/or
a synthetic ligand derivative having sequences of 3-8 amino acids ranging
between 350 ¨ 2,000
Da. Altering the conformation of PCSK9 affects the interaction between PCSK9
and an
endogenous low density lipoprotein receptor, and can lead to reduced or
increased levels of
circulating LDL-cholesterol. High LDL-cholesterol levels are associated with
increased risk for
heart disease. Low LDL-cholesterol levels may be problematic in other
conditions, such as
liver dysfunction; thus, there is also utility for ligands which can raise LDL
levels.
Background
Elevated plasma levels of low density lipoprotein cholesterol (LDL-C)
represent the
greatest risk factor for the development of coronary heart disease. Clearance
of LDL-C from
the plasma occurs primarily by the liver through the action of LDL receptors
(LDLRs), which
are cell surface glycoproteins that bind to apolipoprotein B100 (apoB100) on
LDL particles
with high affinity and mediate their endocytic uptake. Goldstein et al., Annu.
Rev. Cell Biol.
1:1-39 (1985). Autosomal dominant hypercholesterolemia (ADH) is associated
with
mutations that reduce plasma LDL clearance that are found in genes encoding
the LDLR
(familial hypercholesterolemia (FH)) or apoB100 (familial defective apoB100).
Hobbs et al.,
Annu. Rev. Genet. 24, 133-170 (1990); and Innerarity et al., J. Lipid Res.
31:1337-1349
(1990), respectively.
The low density lipoprotein (LDL) receptor (LDLR) mediates efficient
endocytosis of
VLDL, VLDL remnants, and LDL. As part of the endocytic process, the LDLR
releases
lipoproteins into hepatic endosomes.
One approach to modulating LDL-cholesterol levels would be to identify
peptides
which bind to PCSK9 and alter the kinetics of the interact between PCSK9 and
the LDLR such
that the rate of lipoprotein clearance by LDLR endocytosis is increased or
decreased, as
desired.
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Summary Of The Invention
This invention is related to the field of hypercholesterolemia. In particular,
the
invention provides compositions and methods to modulate circulating levels of
low density
lipoproteins by altering the conformation of the protein PCSK9 using a
synthetic ligand and/or
a synthetic ligand derivative having sequences of 3-8 amino acids ranging
between 350 ¨ 2,000
Da. Altering the conformation of PCSK9 affects the interaction between PCSK9
and an
endogenous low density lipoprotein receptor, and can lead to reduced or
increased levels of
circulating LDL-cholesterol. High LDL-cholesterol levels are associated with
increased risk for
heart disease. Low LDL-cholesterol levels may be problematic in other
conditions, such as
liver dysfunction; thus, there is also utility for ligands which can raise LDL
levels.
In one embodiment, the present invention contemplates a method, comprising: a)
providing; i) a PCSK9 protein, wherein said protein comprises a binding site
that induces
allosteric modulation and a low density lipoprotein receptor binding site; ii)
a ligand capable of
binding said binding site; iii) a plurality of hepatocyte cells comprising a
low density
lipoprotein receptor and low density lipoproteins; b) binding said synthetic
ligand to said
binding site, wherein said synthetic ligand induces a conformation shift of
said protein; and c)
modulating the affinity of said low density lipoprotein receptor binding site
for said low
density lipoprotein receptor by said conformational shift. In one embodiment,
the binding site
comprises His417, Lys421, pr0446, Trp453, G1n454, Giu628, Giy629, Asn652, and
Thr653
of the PCSK9
protein. In one embodiment, the synthetic ligand is an allosteric inhibitor
ligand wherein said
modulating decreases the affinity of said low density lipoprotein receptor
binding site for said
low density lipoprotein receptor such that internalization of said low density
lipoprotein by said
plurality of hepatocytes is increased. In one embodiment, the synthetic ligand
is an allosteric
enhancer ligand said modulating increases the affinity of said low density
lipoprotein receptor
binding site for said low density lipoprotein receptor such that
internalization of said low
density lipoprotein by said plurality of hepatocytes is decreased. In one
embodiment, the
conformational shift of said protein is selected from the group consisting of
an induced fit shift
and a biomechanical shift. In one embodiment, the synthetic ligand is a
synthetic peptide
selected from the group consisting of VYVRFW, VLELYW and ISDLSY. In one
embodiment, the allosteric inhibitor is a peptide is selected from the group
consisting of
SRX55, SRX56, SRX60, SRX61, SRX62, SRX63, SRX64, SRX65 and SRX66. In one
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embodiment, the allosteric enhancer is a peptide is selected from the group
consisting of
SRX64, SRX67, SRX68, SRX69, SRX72 and SRX73. In one embodiment, the synthetic
peptide comprises between approximately 3 ¨ 8 amino acids. In one embodiment,
the
synthetic peptide is six amino acids. In one embodiment, the synthetic peptide
is less than
1,300 Da. In one embodiment, the synthetic peptide ranges between
approximately 466-1067
Da. In one embodiment, the synthetic peptide ranges between approximately 175-
1,000 Da.
In one embodiment, the present invention contemplates, a method, comprising:
a)
providing; i) a PCSK9 protein, wherein said protein comprises a binding site
that induces
allosteric modulation and a low density lipoprotein receptor binding site; ii)
a synthetic ligand
capable of binding said binding site; iii) a plurality of hepatocyte cells
comprising a population
of low density lipoprotein receptors; b) binding said synthetic ligand to said
binding site,
wherein said synthetic ligand induces a conformation shift of said protein; c)
modulating said
population of said low density lipoprotein receptors by said conformational
shift. In one
42i, pro446, Trp453,
embodiment, the binding site comprises His417, Lys Gln454, ou628, 037629,
Asn652, and Thr653 of the PCSK9 protein. In one embodiment, the synthetic
ligand is an
allosteric inhibitor ligand wherein said modulating increases said population
of said low
density lipoprotein receptors measurable on the cell surface of said plurality
of hepatocytes. In
one embodiment, the synthetic ligand is an allosteric enhancer ligand wherein
said modulating
decreases said population of said low density lipoprotein receptors measurable
on the cell
surface of said plurality of hepatocytes. In one embodiment, the
conformational shift of said
protein is selected from the group consisting of an induced fit shift and a
biomechanical shift.
In one embodiment, the ligand is a synthetic peptide selected from the group
consisting of
VYVRFW, VLELYW and ISDLSY. In one embodiment, the allosteric inhibitor peptide
is
selected from the group consisting of SRX55, SRX56, SRX60, SRX61, SRX62,
SRX63,
SRX64, SRX65 and SRX66. In one embodiment, the allosteric enhancer peptide is
selected
from the group consisting of SRX64, SRX67, SRX68, SRX69, SRX72 and SRX73. In
one
embodiment, the synthetic peptide comprises between approximately 3 ¨ 8 amino
acids. In
one embodiment, the synthetic peptide is six amino acids. In one embodiment,
the synthetic
peptide is less than 1,300 Da. In one embodiment, the synthetic peptide ranges
between
approximately 466-1067 Da. In one embodiment, the synthetic peptide ranges
between
approximately 50-1,000 Da.
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In one embodiment, the present invention contemplates a compound of the
formula:
inNire[
0
HI II
Ri
R2 S2 0
n-2
wherein: i) n, the number of amino acid residues, is an integer in the range 3-
8; ii) the
constituent amino acids are single enantiomers of independently selected
natural or unnatural
amino acids; iii) R2 and R3, are independently selected from the group
consisting of hydrogen,
a lower alkyl, a branched alkyl, a hydroxyalkyl, a cycloalkyl, a heterocycle,
aryl, heteroaryl,
acyl, substituted or unsubstituted benzoyl, alkyl or aryl sulfonyl (e.g. mesyl
or tosyl) and
carbamoyl (e.g. BOC); iv) R1 is selected from the group consisting of ¨OH and -
NR4-R5; v)
R4 and R5, independently, are selected from the group consisting of hydrogen;
a lower alkyl,
an aryl, a cycloalkyl, an aromatic heterocycle, pyridine, tetrazole, alkoxy,
cycloalkoxy;
alternatively, R4 and R5 are joined as a heterocyle, such as piperidine;
pyrrolidine;
morpholine; piperazine; a substituted heterocycle, such as 4-methylpiperazine;
or a fused
heterocycle, such as dihydroquinoline or indoline and Si, S2 and S3 are side
chains. In one
embodiment, the compound further comprises a negatively charged polar group.
In one
embodiment, the negatively charged polar group is selected from at least one
of the group
consisting of 0-phosphate, 0-sulfate, 5-0-, and a 5-N- tetrazole incorporated
in said side-
chains Si, S2, or Sn. In one embodiment, the side chain selected from the
group consisting of
Sl, S2 and Sn comprises a phosphoserine. In one embodiment, the side chain 51
comprises -
CH2-NH-tetrazole. In one embodiment, the compound further comprises a glycine
C-
terminus. In one embodiment, the compound is selected from the group
consisting of Val-Tyr-
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Val-Arg-Phe-Trp, 3-Ala-Phe(3-CH2NH2)-Va1-D-Ser(p)-Phe-Trp, Thr-Leu-Cys(CH2-Ph)-
Thr-
Trp-Ser-Ser-Ser(p), Thr-Leu-Asp(NHCH2Ph)-Thr-Trp-Ser-Ser-Ser(p), Thr-Leu-
Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p), Thr-Leu-Hph-Thr-Trp-Ser-Ser-
Ser(p), Thr-
Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ser-Ser(p), Val-Leu-Glu-Leu-Tyr-Trp, Leu-Asp-
Leu-
Phe-Phe-Ser, Ile-Leu-Asp-Leu-Ser-Tyr, Ac-Trp-Ser-Ser(p), Ac-Trp-Ala-Ser(p), Ac-
Trp(5-F)-
Ala-Ser(p)-morpholine, Thr-Leu-Thr-Trp-Ser-Ser-Ser(p), Ac-Tyr-Trp-Gly, Phe(4-
Ph)-Ala-
Ser(p)-morpholine. In one embodiment, the compound comprises between
approximately 3 ¨ 8
amino acids. In one embodiment, the compound is six amino acids. In one
embodiment, the
compound is less than 1,300 Da. In one embodiment, the compound ranges between
approximately 466-1067 Da. In one embodiment, the compound ranges between
approximately 175-1,000 Da. In one embodiment, the compound comprises a
synthetic
peptide. In one embodiment, the compound is formulated as a pharmaceutical
composition. In
one embodiment, the pharmaceutical composition further comprises a
pharmaceutical drug. In
one embodiment, the pharmaceutical drug is selected from the group consisting
of a statin, a
cardiovascular drug, a metabolic drug, and an antihypertensive drug. In one
embodiment, the
pharmaceutical drug is selected from the group consisting of ezetimibe,
amlodipine besylate,
sitagliptin, metformin, atorvastatin, rosuvastatin and simvastatin. In one
embodiment, the
pharmaceutical composition is formulated as selected from the group consisting
of a tablet, a
liquid, a gel, a capsule, a sachet, a microparticle, a liposome, a
nanoparticle, a salt, a
transdermal patch, an ointment, a lotion, a cream, a gel, a drop, a strip, a
suppository, a spray
and a powder.
In one embodiment, the present invention contemplates a composition comprising
a
PCSK9 allosteric ligand ranging between approximately 350-1,500 Da. In one
embodiment,
the PCSK9 allosteric ligand is less than 1,300 Da. In one embodiment, the
PCSK9 allosteric
ligand comprises between approximately 3 ¨ 6 amino acids. In one embodiment,
the PCSK9
allosteric ligand ranges between approximately 550¨ 1,000 Da. In one
embodiment, the
composition is a pharmaceutical composition. In one embodiment, the
composition is a
pharmaceutical composition. In one embodiment, said administering further
comprises a
delivery system selected from the group including, but not limited to,
liposomes, microparticles
and nanoparticles. In one embodiment, the pharmaceutical composition comprises
an effective
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dose of said ligand. In one embodiment, the pharmaceutical composition
comprises salts. In
one embodiment, the pharmaceutical composition is formulated for oral
administration.
In one embodiment, the present invention contemplates a method, comprising: a)
administering a PCSK9 allosteric inhibitor peptide to a subject, wherein said
subject has at
least one symptom of a cardiovascular disease; and b) reducing said at least
one symptom of
cardiovascular disease by said PCSK9 allosteric inhibitor peptide
administration. In one
embodiment, said at least one symptom is reduced between 10% - 85%. In one
embodiment,
said at least one symptom is reduced between 20% - 65%. In one embodiment,
said at least
one symptom is reduced between 30% - 55%. In one embodiment, the
cardiovascular disease
comprises a coronary disease. In one embodiment, the cardiovascular disease
comprises
hypertension. In one embodiment, the cardiovascular disease comprises
hypercholesterolemia.
In one embodiment, the cardiovascular disease comprises atherosclerosis. In
one embodiment,
the at least one symptom comprises reduced circulating high density
lipoprotein. In one
embodiment, the at least one symptom comprises elevated circulating
cholesterol. In one
embodiment, the at least one symptom comprises elevated circulating low
density lipoprotein.
In one embodiment, the at least one symptom comprises high blood pressure. In
one
embodiment, the administering comprises an effective dose of said PCSK9
allosteric inhibitor
peptide. In one embodiment, said administering further comprises a delivery
system selected
from the group including, but not limited to, liposomes, microparticles and
nanoparticles. In
one embodiment, the effective dose comprises a pharmaceutical composition. In
one
embodiment, the pharmaceutical composition comprises salts. In one embodiment,
the
phan-naceutical composition is formulated for oral administration. In one
embodiment, the
allosteric inhibitor peptide comprises between approximately 3 ¨ 8 amino
acids. In one
embodiment, the allosteric inhibitor peptide is six amino acids. In one
embodiment, the
allosteric inhibitor peptide is less than 1,300 Da. In one embodiment, the
allosteric inhibitor
peptide ranges between approximately 466-1067 Da. In one embodiment, the
allosteric
inhibitor peptide ranges between approximately 175-1,000 Da.
In one embodiment, the present invention contemplates a method, comprising: a)
administering a PCSK9 allosteric enhancer peptide to a subject, wherein said
subject has at
least one symptom of a cardiovascular disease; and b) reducing said at least
one symptom of
cardiovascular disease by said PCSK9 allosteric inhibitor peptide
administration. In one
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embodiment, the cardiovascular disease comprises hypercholesterolemia. In one
embodiment,
said at least one symptom comprises reduced circulating cholesterol. In one
embodiment, said
at least one symptom comprises elevated high density lipoprotein. In one
embodiment, the at
least one symptom comprises reduced low density lipoprotein. In one
embodiment, the at least
one symptom comprises low blood pressure. In one embodiment, said at least one
symptom is
reduced between 10% - 85%. In one embodiment, said at least one symptom is
reduced
between 20% - 65%. In one embodiment, said at least one symptom is reduced
between 30% -
55%. In one embodiment, the administering comprises an effective dose of said
PCSK9
allosteric inhibitor peptide. In one embodiment, said administering further
comprises a
delivery system selected from the group including, but not limited to,
liposomes, microparticles
and nanoparticles. In one embodiment, the effective dose comprises a
pharmaceutical
composition. In one embodiment, the pharmaceutical composition comprises
salts. In one
embodiment, the pharmaceutical composition is formulated for oral
administration. In one
embodiment, the allosteric enhancer peptide comprises between approximately 3
¨ 8 amino
acids. In one embodiment, the allosteric enhancer peptide is six amino acids.
In one
embodiment, the allosteric enhancer peptide is less than 1,300 Da. In one
embodiment, the
allosteric enhancer peptide ranges between approximately 466-1067 Da. In one
embodiment,
the allosteric enhancer peptide ranges between approximately 175-1,000 Da.
In one embodiment, the present invention contemplates a method, comprising: a)
administering a PCSK9 allosteric synthetic peptide to a subject, wherein said
subject has at
least one symptom of a liver disease; and b) reducing said at least one
symptom of liver disease
by said PCSK9 allosteric peptide administration. In one embodiment, the at
least one symptom
comprises elevated low density lipoprotein receptor density. In one embodiment
the at least
one symptom comprises reduced low density lipoprotein receptor density. In one
embodiment,
said at least one symptom is reduced between 10% - 85%. In one embodiment,
said at least
one symptom is reduced between 20% - 65%. In one embodiment, said at least one
symptom
is reduced between 30% - 55%. In one embodiment, the PCSK9 allosteric
synthetic peptide
comprises a PCSK9 allosteric enhancer peptide. In one embodiment, the PCSK9
allosteric
synthetic peptide comprises a PCSK9 allosteric inhibitor peptide. In one
embodiment, the
administering comprises an effective dose of said PCSK9 allosteric peptide. In
one
embodiment, said administering further comprises a delivery system selected
from the group
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including, but not limited to, liposomes, microparticles and nanoparticles. In
one embodiment,
the effective dose comprises a pharmaceutical composition. In one embodiment,
the
pharmaceutical composition comprises salts. In one embodiment, the
pharmaceutical
composition is formulated for oral administration. In one embodiment, the
allosteric synthetic
peptide comprises between approximately 3 ¨ 8 amino acids. In one embodiment,
the
allosteric synthetic peptide is six amino acids. In one embodiment, the
allosteric synthetic
peptide is less than 1,300 Da. In one embodiment, the allosteric synthetic
peptide ranges
between approximately 466-1067 Da. In one embodiment, the allosteric synthetic
peptide
ranges between approximately 175-1,000 Da.
In one embodiment, the present invention contemplates a method comprising: a)
providing; i) a PCSK9 protein, wherein said protein comprises an allosteric
modulation site
and an orthosteric low density lipoprotein receptor (LDLR) binding site; and
ii) an allosteric
synthetic peptide capable of binding said allosteric modulation site; b)
binding said allosteric
synthetic peptide to said allosteric modulation site, wherein said allosteric
synthetic peptide
induces a conformational shift of said orthosteric LDLR binding site. In one
embodiment, said
binding of said allosteric synthetic peptide to said allosteric modulation
site, inhibits an
induced fit conformational shift of said orthosteric LDLR binding site. In one
embodiment, the
binding induces a conformational shift of said PCSK9 protein. In one
embodiment, the
resulting PCSK9 conformational shift reduces the binding affinity of said
orthosteric LDLR
binding site interaction to a LDLR, wherein low density lipoprotein clearance
is increased. In
one embodiment, the conformational shift enhances dissociation of said
orthosteric low density
lipoprotein receptor binding site from a low density lipoprotein receptor. In
one embodiment,
the confotmational shift reduces the orthosteric Cis-His Rich Domain (CHRD)
binding site to a
binding ligand (e.g., for example, to facilitate vesicle trafficking at low
pH; DeVay et al.,
"Characterization of proprotein convertase subtilisin/kexin type 9 (PCSK9)
trafficking reveals
a novel lysosomal targeting mechanism via amyloid precursor-like protein 2
(APLP2)" J Biol
Chem. 288(15):10805-10818 (2013). In one embodiment, the orthosteric low
density
lipoprotein receptor binding site conformational shift comprises an induced
fit inhibition. In
one embodiment, the binding of said allosteric synthetic peptide reduces the
conformational
shift required for the induced fit of the orthosteric LDLR binding site of
PCSK9, inhibiting the
binding affinity of said orthosteric LDLR interaction, wherein low density
lipoprotein
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clearance is increased. In one embodiment, the inducing of said orthosteric
low density
lipoprotein receptor binding site conformational shift is biomechanical. In
one embodiment,
the conformational shift results in biomechanical stiffening of the connecting
loop between a
PCSK9 catalytic domain and a PCSK9 C-terminal domain. In one embodiment, the
biomechanical conformational shift comprises a translocational and/or
rotational movement of
amino acid alanine443 side chain and/or backbone. In one embodiment, the
biomechanical
conformational shift comprises a translocational and/or rotational movement of
amino acid
valine"' side chain and/or backbone. In one embodiment, the biomechanical
conformational
shift comprises a translocational and/or rotational movement of amino acid
aspartic acid' side
chain and/or backbone. In one embodiment, the biomechanical conformational
shift
comprises a translocational and/or rotational movement of amino acid
threonine' side chain
and/or backbone. In one embodiment, the biomechanical conformational shift
comprises a
translocational and/or rotational movement of amino acid proline445 side chain
and/or backbone.
In one embodiment, the biomechanical conformational shift comprises a
translocational and/or
rotational movement of amino acid proline 446 side chain and/or backbone. In
one embodiment,
the biomechanical conformational shift comprises a reorientation and
translocation of
histidine449. In one embodiment, the biomechanical mechanism comprises the
inhibition of the
translocational and/or rotational movement of amino acid alanine443 side chain
and/or
backbone. In one embodiment, the biomechanical mechanism comprises the
inhibition of the
translocational and/or rotational movement of amino acid valine441 side chain
and/or backbone.
In one embodiment, the biomechanical mechanism comprises the inhibition of the
translocational and/or rotational movement of amino acid aspartic acid422 side
chain and/or
backbone. In one embodiment, the biomechanical mechanism comprises the
inhibition of the
translocational and/or rotational movement of amino acid threonine162 side
chain and/or
backbone. In one embodiment, the biomechanical mechanism comprises the
inhibition of the
translocational and/or rotational movement of amino acid proline445 side chain
and/or
backbone. In one embodiment, the biomechanical mechanism comprises the
inhibition of the
translocational and/or rotational movement of amino acid proline 446 side
chain and/or
backbone. In one embodiment, the biomechanical shift comprises the inhibition
of the
translocational and/or rotational movement of histidine449 side chain and/or
backbone. In one
embodiment, the allosteric synthetic peptide is VYVRFW. In one embodiment, the
allosteric
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synthetic peptide is VLELYW. In one embodiment, the allosteric synthetic
peptide is
ISDLSY. In one embodiment, the allosteric synthetic peptide comprises between
approximately 3 ¨ 8 amino acids. In one embodiment, the allosteric synthetic
peptide is six
amino acids. In one embodiment, the allosteric synthetic peptide is less than
1,300 Da. In one
embodiment, the allosteric synthetic peptide ranges between approximately 466-
1067 Da. In
one embodiment, the allosteric synthetic peptide ranges between approximately
175-1,000 Da.
In one embodiment, the present invention contemplates a compound of the
formula:
0 S1
HN
R3 R1
I II= MI =
R2 0 S2 0
n-2
wherein: i) n, the number of amino acid residues, is an integer in the range 3-
8; ii) the
constituent amino acids are single enantiomers of independently selected
natural or unnatural
amino acids; iii) R2 and R3, are independently selected from the group
consisting of hydrogen,
a lower alkyl, a branched alkyl, a hydroxyalkyl, a cycloalkyl, a heterocycle,
aryl, heteroaryl,
acyl, substituted or unsubstituted benzoyl, alkyl or aryl sulfonyl (e.g. mesyl
or tosyl) and
carbamoyl (e.g. BOG); iv) R1 is selected from the group consisting of ¨OH and -
NR4-R5; v)
R4 and R5, independently, are selected from the group consisting of hydrogen;
a lower alkyl,
an aryl, a cycloalkyl, an aromatic heterocycle, pyridine, tetrazole;
alternatively, R4 and R5 are
joined as a heterocyle, such as piperidine; pyrrolidine; morpholine;
piperazine; a substituted
heterocycle, such as 4-methylpiperazine; or a fused heterocycle, such as
dihydroquinoline or
indoline. In one embodiment, the compound further comprises a negatively
charged polar
group. In one embodiment, the negatively charged polar group includes, but is
not limited to,
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0-phosphate, 0-sulfate, or 5-0- or 5-N- tetrazole incorporated in the side-
chain Si, S2, or S3.
In one embodiment, the side chain Si, S2 or S3 comprises a phosphoserine. In
one
embodiment, the side chain Si comprises -CH2-NH-tetrazole. In one embodiment,
the C-
terminus comprises a glycine. In one embodiment, the compound comprises
between
approximately 3 ¨ 8 amino acids. In one embodiment, the compound is six amino
acids. In one
embodiment, the compound is less than 1,300 Da. In one embodiment, the
compound ranges
between approximately 466-1067 Da. In one embodiment, the compound ranges
between
approximately 175-1,000 Da. In one embodiment, the compound comprises a
synthetic
peptide.
In one embodiment, the present invention contemplates a compound of the
formula:
Val-Tyr-Val-Arg-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
(3-Ala-Phe(3-CH2NH2)-Val-D-Ser(p)-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Asp(NHCH2Ph)-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Hph-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Val-Leu-Glu-Leu-Tyr-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Leu-Asp-Leu-Phe-Phe-Ser.
In one embodiment, the present invention contemplates a compound of the
formula: Ile-
Leu-Asp-Leu-Ser-Tyr.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Trp-Ser-Ser(p).
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In one embodiment, the present invention contemplates a compound of the
foiniula:
Ac-Trp-Ala-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Trp(5-F)-Ala-Ser(p)-morpholine.
In one embodiment, the present invention contemplates a compound of the
formula:
Thr-Leu-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Tyr-Trp-Gly.
In one embodiment, the present invention contemplates a compound of the
formula:
Phe(4-Ph)-Ala-Ser(p)-morpholine.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Val-Tyr-Val-Arg-Phe-Trp-NH2, Ala-Phe(3-CH2NH2)-Val-D-Ser(p)-Phe-
Trp-NH2,
Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p)-NH2, Thr-Leu-Asp(NHCH2Ph)-Thr-Trp-
Ser-
Ser-Ser(p)-NH2, Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p)-NH2, Thr-
Leu-
Hph-Thr-Trp-Ser-Ser-Ser(p)-NH2, Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ser-
Ser(p)-
NH2, Val-Leu-Glu-Leu-Tyr-Trp-NH2, Leu-Asp-Leu-Phe-Phe-Ser-NH2, Ile-Leu-Asp-Leu-
Ser-
Tyr-NH2, Ac-Trp-Ser-Ser(p)-NH2, Ac-Trp-Ala-Ser(p)-NH2, Ac-Trp(5-F)-Ala-Ser(p)-
NH2
and Thr-Leu-Thr-Trp-Ser-Ser-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Trp-Ser-Ser(p)-NHCH3, Ac-Trp-Ala-Ser(p)-NHCH3, Ac-Trp-Ala-
Ser(p)-
morpholine, Ac-Trp-Ala-Ser(p)-4-methylpiperizine, Ac-Trp-Ala-Ser(p)-
piperidine, Ac-Trp-
Ala-Ser(p)-pyrrolidine.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-
Asp(NHCH2Ph)-
Thr-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-
Ser(p), Ac-
Thr-Leu-Hph-Thr-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-
Ser-
Ser(p), Ac-Val-Leu-Glu-Leu-Tyr-Trp, Ac-Leu-Asp-Leu-Phe-Phe-Ser, Ac-Ile-Leu-Asp-
Leu-
Ser-Tyr and Ac-Thr-Leu-Thr-Trp-Ser-Ser-Ser(p).
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ala-Ser(p), Thr-Leu-Asp(NHCH2Ph)-
Thr-Trp-
Ser-Ala-Ser(p), Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ala-Ser(p), Thr-Leu-
Hph-
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Thr-Trp-Ser-Ala-Ser(p), Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ala-Ser(p) and
Thr-Leu-
Thr-Trp-Ser-Ala-Ser(p).
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ala-Ser(p)-NH2, Thr-Leu-
Asp(NHCH2Ph)-
Thr-Trp-Ser-Ala-Ser(p)-NH2, Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ala-
Ser(p)-
NH2, Thr-Leu-Hph-Thr-Trp-Ser-Ala-Ser(p)-NH2, Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-
Ser-
Ala-Ser(p)-NH2 and Thr-Leu-Thr-Trp-Ser-Ala-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Tip-Ser-Ala-Ser(p), Ac-Thr-Leu-
Asp(NHCH2Ph)-
Thr-Trp-Ser-Ala-Ser(p), Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ala-
Ser(p), Ac-
Thr-Leu-Hph-Thr-Trp-Ser-Ala-Ser(p), Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-
Ala-
Ser(p) and Ac-Thr-Leu-Thr-Trp-Ser-Ala-Ser(p).
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Thr-Leu-Cys(C112-Ph)-Thr-Trp-Ser-Ala-Ser(p)-NH2, Ac-Thr-Leu-
Asp(NHCH2Ph)-Thr-Trp-Ser-Ala-Ser(p)-NH2, Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-
Trp-Ser-Ala-Ser(p)-NH2, Ac-Thr-Leu-Hph-Thr-Trp-Ser-Ala-Ser(p)-NH2, Ac-Thr-Leu-
Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ala-Ser(p)-NH2, Ac-Thr-Leu-Thr-Trp-Ser-Ala-
Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser(p), Thr-Leu-Asp(NHCH2Ph)-Thr-
Trp-Ser-
Ser(p), Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser(p), Thr-Leu-Hph-Thr-Trp-
Ser-
Ser(p), Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ser(p) and Thr-Leu-Thr-Trp-Ser-
Ser(p).
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Thr-Leu-Cys(CH2-Ph)-'Thr-Trp-Ser-Ser(p)-NH2, Ac-Thr-Leu-
Asp(NHCH2Ph)-
Thr-Trp-Ser-Ser(p)-NH2, Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser(p)-
NH2,
Ac-Thr-Leu-Hph-Thr-Trp-Ser-Ser(p)-NH2, Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-
Ser-
Ser(p)-NH2 and Ac-Thr-Leu-Thr-Trp-Ser-Ser(p)-N112.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ala-Ser(p), Thr-Leu-Asp(NHCH2Ph)-Thr-
Trp-Ala-
Ser(p), Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ala-Ser(p), Thr-Leu-Hph-Thr-Trp-
Ala-
Ser(p), Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ala-Ser(p) and Thr-Leu-Thr-Trp-Ala-
Ser(p).
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In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ala-Ser(p)-NH2, Ac-Thr-Leu-
Asp(NHCH2Ph)-
Thr-Trp-Ala-Ser(p)-NH2, Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ala-Ser(p)-
NH2,
Ac-Thr-Leu-Hph-Thr-Trp-Ala- Ser(p)-NH2, Ac-Thr-Leu-C ys (CH2-Ph)-Thr-Trp (3 -
Me)-Ala-
Ser(p)-NH2 and Ac-Thr-Leu-Thr-Trp-Ala-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Ala-Trp-Ser-Ser-Ser(p), Thr-Leu-Asp(NHCH2Ph)-
Ala-Trp-
Ser-Ser-Ser(p), Thr-Leu-Gly(CH2CH2cyclohexyl)-Ala-Trp-Ser-Ser-Ser(p), Thr-Leu-
Hph-Ala-
Trp-Ser-Ser-Ser(p), Ac-T1u--Leu-Cys(CH2-Ph)-Ala-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-
Asp(NHCH2Ph)-Ala-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Ala-Trp-
Ser-
Ser-Ser(p), Ac-Thr-Leu-Hph-Ala-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Cys(CH2-Ph)-Ala-
Trp-Ser-
Ser-Ser(p)-NH2, Ac-Thr-Leu-Asp(NHCH2Ph)-Ala-Ttp-Ser-Ser-Ser(p)-NH2, Ac-Thr-Leu-
Gly(CH2CH2cyclohexyl)-Ala-Trp-Ser-Ser-Ser(p)-NH2 and Ac-Thr-Leu-Hph-Ala-Trp-
Ser-
Ser-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH2-Ph)-Ser-Trp-Ser-Ser-Ser(p), Thr-Leu-Asp(NHCH2Ph)-
Ser-Trp-
Ser-Ser-Ser(p), Thr-Leu-Gly(CH2CH2cyclohexyl)-Ser-Trp-Ser-Ser-Ser(p), Thr-Leu-
Hph-Ser-
Trp-Ser-Ser-Ser(p), Ac-Tbr-Leu-Cys(CH2-Ph)-Ser-Trp-Ser-Ser-Ser(p), Ae-Thr-Leu-
Asp(NHCH2Ph)-Ser-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Gly(CH2CH2cyclohexyl)-Ser-Trp-
Ser-
Ser-Ser(p), Ac-Thr-Leu-Hph-Ser-Trp-Ser-Ser-Ser(p), Ac-Thr-Leu-Cys(CH2-Ph)-Ser-
Trp-Ser-
Ser-Ser(p)-NH2, Ac-Thr-Leu-Asp(NHCH2Ph)-Ser-Trp-Ser-Ser-Ser(p)-NH2, Ac-Thr-Leu-
Gly(CH2CH2cyclohexyl)-Ser-Trp-Ser-Ser-Ser(p)-NH2 and Ac-Thr-Leu-Hph-Ser-Trp-
Ser-Ser-
Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Ac-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p), Ac-Asp(NHCH2Ph)-Thr-Trp-Ser-
Ser-
Ser(p), Ac-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p), Ac-Hph-Thr-Trp-Ser-
Ser-Ser(p),
Ac-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p)-NH2, Ac-Asp(NHCH2Ph)-Thr-Trp-Ser-Ser-
Ser(p)-
NH2, Ac-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p)-NH2 and Ac-Hph-Thr-Trp-
Ser-
Ser-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, BOC-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p), BOC-Asp(NHCH2Ph)-Thr-Trp-
Ser-
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Ser-Ser(p), BOC-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p), BOC-Hph-Thr-Trp-
Ser-
Ser-Ser(p), BOC-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p)-NH2, BOC-Asp(NHCH2Ph)-Thr-
Trp-
Ser-Ser-Ser(p)-N112, BOC-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p)-NH2 and
BOC-
Hph-Thr-Trp-Ser-Ser-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound including,
but not
limited to, Thr-Leu-Cys(CH3)-Thr-Trp-Ser-Ser-Ser(p), Thr-Leu-Cys(CH(CH3)2)-Thr-
Trp-Ser-
Ser-Ser(p), Thr-Leu-Cys(CH2-3,4-difluoropheny1)-Thr-Trp-Ser-Ser-Ser(p), Thr-
Leu-
Cys(CH2-3-hydroxyphenye-Thr-Trp-Ser-Ser-Ser(p), Thr-Leu-Cys(CH2-3-methypheny1)-
Thr-
Trp-Ser-Ser-Ser(p) and Ac-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p)-NH2.
In one embodiment, the present invention contemplates a compound having the
formula
of Phe(4-Ph)-Gly(Et)-Ser(p)-morpholine.
In one embodiment, the present invention contemplates a compound selected from
the
group consisting of Ac-Tyr-Trp(6-0Me)-Gly, Ac-Tyr(3-F)-Trp-Gly, pivaloyl-Tyr-
Trp-Gly,
mesyl-Tyr-Trp-Gly, BOC-Tyr-Trp-Gly,
00 OH
N
0
N H
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NH
4111
0 0
N HN N
H
0 0
NH
140
NH
= = OH
0 0 0 0
H
H2N . N NH NfreN
0 0 0 "'",`,,
/0H OH NH
NH
, and
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NH
OH
0 0 0 0
H2NNOH
H H
0 0
0OH 0
O s OH
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Trp-D-Phe(3CF3)-D-Arg-NH2.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Trp-D-Phe(3C1)-D-Arg-NH2.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Trp-D-Phe-D-Arg-NH2.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Trp-D-Phe-D-Arg.
In one embodiment, the present invention contemplates a compound of the
formula:
NAc-NMe-D-Arg-D-Phe(30H)-D-Trp-NH2.
In one embodiment, the present invention contemplates a compound of the
foimula:
Ac-Arg-Phe(3CF3)-Gly.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Ala-Val-Arg-N(Me)(Ph3CF3).
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Arg-D-Phe(30H)-D-Trp.
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In one embodiment, the present invention contemplates a compound of the
formula:
Ac-D-Arg-D-Phe(30H)-D-Trp-NH2.
In one embodiment, the present invention contemplates a compound of the
formula:
Propionyl-D-Arg-D-Phe(30H)-D-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Val-Arg-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Ac-Tyr-Val-Arg-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Val-Tyr-Asp-Arg-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Val-Tyr-Glu-Arg-Phe-Trp.
In one embodiment, the present invention contemplates a compound of the
formula:
Val-Tyr-Val-Cit-Phe-W (Cit = Citrulline).
In one embodiment, the present invention contemplates a pharmaceutical
composition
comprising a compound of the formula:
JIN111
0 Si
N
R3 R2
HijNir R1
0 0 S2
n-2
and a carrier, wherein: i) n, the number of amino acid residues, is an integer
in the range 3-8;
ii) the constituent amino acids are single enantiomers of independently
selected natural or
unnatural amino acids; iii) R2 and R3, are independently selected from the
group consisting of
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hydrogen, a lower alkyl, a branched alkyl, a hydroxyalkyl, a cycloalkyl, a
heterocycle, aryl,
heteroaryl, acyl, substituted or unsubstituted benzoyl, alkyl or aryl sulfonyl
(e.g. mesyl or
tosyl) and carbamoyl (e.g. BOC); iv) R1 is selected from the group consisting
of ¨OH and -
NR4-R5; v) R4 and R5, independently, are selected from the group consisting of
hydrogen; a
lower alkyl, an aryl, a cycloalkyl, an aromatic heterocycle, pyridine,
tetrazole; alternatively, R4
and R5 are joined as a heterocyle, such as piperidine; pyrrolidine;
morpholine; piperazine; a
substituted heterocycle, such as 4-methylpiperazine; or a fused heterocycle,
such as
dihydroquinoline or indoline. In one embodiment, the pharmaceutical
composition further
comprises a negatively charged polar group. In one embodiment, said negatively
charged polar
group is selected from at least one of the group consisting of 0-phosphate, 0-
sulfate, or 5-0-
or 5-N- tetrazole incorporated in the side-chain Si, S2, or S3. In one
embodiment, the side
chain Si, S2 or S3 comprises a phosphoserine. In one embodiment, the side
chain Si
comprises -CH2-NH-tetrazole. In one embodiment, the C-terminus comprises a
glycine. In
one embodiment, the pharmaceutical composition further comprises a statin. In
one
embodiment, the statin includes, but is not limited to, atorvastatin,
rosuvastatin and/or
simvastatin. In one embodiment, the pharmaceutical composition comprises an
anti-diabetic
drug. In one embodiment, the pharmaceutical composition comprises a
cardiovascular drug.
In one embodiment, the pharmaceutical composition comprises ezetimibe (Zetie).
In one
embodiment, the pharmaceutical composition comprises an anti-hypertensive
including, but
not limited to, amlodipine besylate (Norvase). In one embodiment the anti-
diabetic drug
includes, but not limited to, sitagliptin (Januvia ) and/or metformin. In one
embodiment, the
compound comprises between approximately 3 ¨ 8 amino acids. In one embodiment,
the
compound is six amino acids. In one embodiment, the compound is less than
1,300 Da. In one
embodiment, the compound ranges between approximately 466-1067 Da. In one
embodiment,
the compound ranges between approximately 175-1,000 Da. In one embodiment, the
compound comprises a synthetic peptide.
Definitions
The term "compound" or "ligand" as used herein, refers to any exogenous
molecule
comprising natural amino acids capable of interacting with (i.e., for example,
attaching,
binding etc.) to a binding partner thereby altering the biological function of
the binding partner.
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Compounds/ligands may include, but are not limited to, an amino acid chain
comprising at
least two peptide bonds, antibodies, proteins, peptides, and/or tripeptides.
Such
compounds/ligands may be derivatized by substitutents including, but not
limited to,
hydroxyls, sulfurs, amines, amides, ethers, esters, aliphatic chains, aromatic
rings, aliphatic
rings, subtituted aromatic rings and/or substituted aliphatic rings. Such
compounds/ligands
may be an inhibitor compound/ligand, or an enhancer compound/ligand. A
compound/ligand
may also include a "drug", thereby referring to any pharmacologically active
substance capable
of being administered, which achieves a desired effect. Drugs or
compounds/ligands can be
synthetic or naturally occurring
The term "synthetic ligand" as used herein, refers to a molecule comprising
amino acids
which is a ligand, and was designed ex vivo and is subsequently synthesized
using in vitro, in
vivo, or a combination of in vitro and in vivo means to produce a molecule of
pre-specified
characteristics (e.g., charge, shape, molecular weight) and is bound by
another naturally
occurring biomolecule to form a complex. Preferably these synthetic ligands
are smaller than a
target natural biomolecule, more preferably these synthetic ligands are less
than 1,300 Da, and
more preferably are between 350 and 1,250 Da.
The term "synthetic peptide" as used herein, refers to non-natural amino acid
sequence
of approximately 3-8 amino acids and ranging between approximately 350-1,500
Da.
Preferably a non-natural amino acid sequence of approximately 4-5 amino acids
and ranging
between approximately 550¨ 1,000 Da. For example, a synthetic peptide is six
amino acids
and less than 1,300 Da, for example, ranging between approximately 466-1067
Da.
Preferably, a synthetic peptide is made in accordance with Example V.
The term "allosteric site" as used herein, refers to a ligand binding site,
other than the
native chemically active/receptor binding site that, when bound to an
exogenous ligand,
changes the shape and activity of a protein (as an enzyme). For example, an
"allosteric
enhancer peptide" refers to a ligand binding to an allosteric site that may
increase the native
activity and/or respective affinity(ies) of the protein (e.g., for example, a
PCSK9 allosteric
enhancer peptide). Alternatively, an "allosteric inhibitor peptide" refers to
a ligand binding to
an allosteric site that may decrease the native activity and/or respective
affinity(ies) of the
protein (e.g., for example, a PCSK9 allosteric inhibitor peptide). For
example, the binding site
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, , ,
Lys421 pro446 Trp453 un454, , , , Gin628 Giy629 Asn652
comprises His417, and Thr653 of the PCSK9
protein.
The term "orthosteric site" as used herein, refers to a primary, unmodulated
binding site
of a ligand (e.g., for example, a peptide) to a receptor, binding and/or a
catalytic site.
The term "conformation" as used herein, refers to a three-dimensional
stereochemical
configuration of an amino acid sequence. For example, any specific
conformation results from
a theimodynamic balance between steric interactions, hydrophobic interactions,
hydrogen
bonding, electrochemical bonding and/or salt bridge interactions between
individual amino
acids in an amino acid sequence.
The term "conformational shift" as used herein, refers to the introduction of
an
exogenous force or molecule (e.g., an inhibitor peptide) that may alter a
first thermodynamic
balance (conformation 1) into a second thermodynamic balance (confonnation 2),
or enhances
the dynamic range and/or the type and/or the number of metastable folding
states within a lone
protein, and/or a protein-ligand complex, and/or a protein-protein complex.
Furthermore, a
conformation shift may be predominantly exhibited under certain specific
external conditions
(pH, temperature, etc.) and/or during specific periods within the lifetime of
a lone protein or
multi-part complex, including but not limited to conditions preferential for
molecular
recognition, initial binding interaction, induced fit interaction, functional
activity, and/or
dissociation.
The term "EGFA" as used herein, refers to the most amino EGF-like domain of
the low
density lipoprotein receptor. For example, the EGF-like domain may comprise an
extracellular
portion of the LDLR receptor.
The term "LDL-R" and "LDLR" as used herein, refers to an abbreviation for the
low
density lipoprotein receptor. The abbreviation may be in reference to the
entire LDL-R
receptor protein or any portion thereof. LDL-Rs reside on a cell surface and
can bind to low
density lipoproteins such that the LDL-R/LDL complex become internalized
within a cell (i.e.,
for example, a hepatocyte), wherein the LDL is released and the LDL-R is
recycled back to the
cell surface.
The ten-n, "binding interface" as used herein, refers to any collection of
attractive
interactions (i.e., for example, hydrogen bonding, electrostatic interactions,
hydrophobic
interactions, etc) between the functional groups (i.e., for example, hydroxyl,
amide, amine,
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carboxyl, amidine, guanidine, hydrocarbon, sulfonyl etc.) of at least two
different molecules.
The collection of attractive forces forms a stable molecular plane thereby
forming a 'binding
interface' between the at least two molecules.
The term "induced fit" as used herein, refers to any acceptance of a peptide
requiring a
change in receptor conformation. Such a conformation may be facilitated by a
translational /
rotational movement of amino acid side chains and flexible loops, thereby
rearranging the
electrostatic and/or hydrophobic fields.
The term "complex" or "composition" as used herein, refers to any chemical
association of two or more ions or molecules joined usually by weak
electrostatic bonds rather
than by covalent bonds. For example, a complex or composition may be formed
between a
peptide as described herein and a PCSK9 amino acid sequence, thereby creating
a
peptide/PCSK9 amino acid sequence complex or composition. Optionally, such
complexes or
compositions may also include, but are not limited to, an LDLR amino acid
sequence or any
portion thereof, including but not limited to the EGFA region.
The term "hydrogen bond" as used herein, an electrostatic attraction between a
hydrogen atom in one polar molecule (as of water) and a small electronegative
atom (as of
oxygen, nitrogen, or fluorine) in usually another molecule of the same or a
different polar
substance.
The term "salt bridge" as used herein, refers to any interaction or a
combinations of
interactions, such as hydrogen bonding and/or electrostatic interactions,
which align cationic
and anionic chemical structures in such a way that the charged moieties
overlap.
The term "interaction" as used herein, refers to any effect that one molecule
and/or
functional group may have on another molecule and/or functional group. Such
effects may
include, but are not limited to, steric (i.e., for example, physical),
electrostatic (i.e., for
example, electrical attraction or repulsion), electromagnetic, hydrophilic, or
hydrophobic
effects.
The term "overlap" as used herein, refers to any positioning of molecules in
such a way
that the electronic structure of one molecule is on top of, and extending past
the border of
another molecule, or be positioned in this way.
The term "hypercholesterolemia" as used herein, refers to any medical
condition
wherein blood cholesterol levels are elevated above the clinically recommended
levels. For
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example, if cholesterol is measured using low density lipoproteins (LDLs),
hypercholesterolemia may exist if the measured LDL levels are above, for
example,
approximately 70 mg/d1. Alternatively, if cholesterol is measured using free
plasma
cholesterol, hypercholesterolemia may exist if the measured free cholesterol
levels are above,
for example, approximately 200-220 mg/d1.
The term "at risk for" as used herein, refers to a medical condition or set of
medical conditions exhibited by a patient which may predispose the patient to
a
particular disease or affliction. For example, these conditions may result
from
influences that include, but are not limited to, behavioral, emotional,
chemical,
biochemical, or environmental influences.
The term "effective amount" as used herein, refers to a particular amount of a
pharmaceutical composition comprising a therapeutic agent that achieves a
clinically
beneficial result (i.e., for example, a reduction of symptoms). Toxicity and
therapeutic
efficacy of such compositions can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for determining the
LD50 (the
dose lethal to 50% of the population) and the ED50 (the dose therapeutically
effective
in 50% of the population). The dose ratio between toxic and therapeutic
effects is the
therapeutic index, and it can be expressed as the ratio LD50/ED50. Compounds
that
exhibit large therapeutic indices are preferred. The data obtained from these
cell
culture assays and additional animal studies can be used in formulating a
range of
dosage for human use. The dosage of such compounds lies preferably within a
range of
circulating concentrations that include the ED50 with little or no toxicity.
The dosage
varies within this range depending upon the dosage form employed, sensitivity
of the
patient, and the route of administration.
The term "symptom", as used herein, refers to any subjective or objective
evidence of disease or physical disturbance observed by the patient. For
example,
subjective evidence is usually based upon patient self-reporting and may
include, but is
not limited to, pain, headache, visual disturbances, nausea and/or vomiting.
Alternatively, objective evidence is usually a result of medical testing
including, but
not limited to, body temperature, complete blood count, lipid panels, thyroid
panels,
blood pressure, heart rate, electrocardiogram, tissue and/or body imaging
scans.
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The term "disease" and/or "disorder", as used herein, refers to any impairment
of the normal state of the living animal or plant body or one of its parts
that interrupts
or modifies the performance of the vital functions. Typically manifested by
distinguishing signs and symptoms, it is usually a response to: i)
environmental factors
(as malnutrition, industrial hazards, or climate); ii) specific infective
agents (as worms,
bacteria, or viruses); iii) inherent defects of the organism (as genetic
anomalies); and/or
iv) combinations of these factors
The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent"
and grammatical equivalents (including "lower," "smaller," etc.) when in
reference to
the expression of any symptom in an untreated subject relative to a treated
subject,
mean that the quantity and/or magnitude of the symptoms in the treated subject
is
lower than in the untreated subject by any amount that is recognized as
clinically
relevant by any medically trained personnel. In one embodiment, the quantity
and/or
magnitude of the symptoms in the treated subject is at least 10% lower than,
at least
25% lower than, at least 50% lower than, at least 75% lower than, and/or at
least 90%
lower than the quantity and/or magnitude of the symptoms in the untreated
subject.
The terms "increase," "enhance," "elevate," and grammatical equivalents
(including "higher," "larger," etc.) when in reference to the expression of
any symptom
in an untreated subject relative to a treated subject, mean that the quantity
and/or
magnitude of the symptoms in the treated subject is greater than in the
untreated
subject by any amount that is recognized as clinically relevant by any
medically trained
personnel. In one embodiment, the quantity and/or magnitude of the symptoms in
the
treated subject is at least 10% greater than, at least 25% greater than, at
least 50%
greater than, at least 75% greater than, and/or at least 90% greater than the
quantity
and/or magnitude of the symptoms in the untreated subject.
The term "attached" as used herein, refers to any interaction between a medium
(or carrier) and a drug. Attachment may be reversible or irreversible. Such
attachment
includes, but is not limited to, covalent bonding, ionic bonding, Van der
Waals forces
or friction, and the like. A drug is attached to a medium (or carrier) if it
is
impregnated, incorporated, coated, in suspension with, in emulsion with, in
solution
with, mixed with, etc.
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The term "administered" or "administering", as used herein, refers to any
method of
providing a composition to a patient such that the composition has its
intended effect on the
patient. An exemplary method of administering is by a direct mechanism such
as, local tissue
administration (i.e., for example, extravascular placement), oral ingestion,
transdermal patch,
topical, inhalation, suppository etc.
The term "patient" or "subject", as used herein, is a human or animal and need
not be hospitalized. For example, out-patients, persons in nursing homes are
"patients." A patient may comprise any age of a human or non-human animal and
therefore includes both adult and juveniles (i.e., children). It is not
intended that the
term "patient" connote a need for medical treatment, therefore, a patient may
voluntarily or involuntarily be part of experimentation whether clinical or in
support of
basic science studies.
The term "affinity" as used herein, refers to the measure of the thermodynamic
tendency of two or more molecules to assemble to form a multi-part complex and
to remain
assembled in said complex. For example, the SRX55 ligand has a high affinity
for PCSK9 and
is thermodynamically favored to form a complex. It is understood that a change
in conditions
(e.g., pH during the receptor internalization process) For example, a decrease
in the LDL
affinity for LDLR and the two molecules may dissociate, or separate, from one
another.
The term "derived from" as used herein, refers to the source of a compound or
amino
acid sequence. In one respect, a compound or amino acid sequence may be
derived from an
organism or particular species. In another respect, a compound or amino acid
sequence may be
derived from a larger complex or sequence. In another respect, a compound or
sequence may
be derived by chemical modification of part or all of an amino acid sequence
found in nature.
The term "protein" as used herein, refers to any of numerous naturally
occurring
extremely complex substances (as an enzyme or antibody) that consist of amino
acid residues
joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen,
oxygen, usually
sulfur. In general, a protein comprises amino acids having an order of
magnitude within the
hundreds.
The term "peptide" as used herein, refers to any of various amides that are
derived from
three or more amino acids by combination of the amino group of one acid with
the carboxyl
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group of another and are usually obtained by partial hydrolysis of proteins.
In general, a
peptide comprises amino acids having an order of magnitude within the tens or
smaller.
The term "pharmaceutically" or "phatniacologically acceptable", as used
herein, refer to
molecular entities and compositions that do not produce adverse, allergic, or
other untoward
reactions when administered to an animal or a human.
The term, "pharmaceutically acceptable carrier", as used herein, includes any
and all
solvents, or a dispersion medium including, but not limited to, water,
ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable
mixtures thereof, and dimethylsulfoxide, vegetable oils, coatings, isotonic
and absorption
delaying agents, liposome, commercially available cleansers, and the like.
Supplementary
bioactive ingredients also can be incorporated into such carriers.
The term, "purified" or "isolated", as used herein, may refer to a peptide
composition
that has been subjected to treatment (i.e., for example, fractionation) to
remove various other
components, and which composition substantially retains its expressed
biological activity.
Where the term "substantially purified" is used, this designation will refer
to a
composition in which the protein or peptide foul's the major component of the
composition,
such as constituting about 50%, about 60%, about 70%, about 80%, about 90%,
about 95% or
more of the composition (i.e., for example, weight/weight and/or
weight/volume). The term
"purified to homogeneity" is used to include compositions that have been
purified to 'apparent
homogeneity" such that there is single protein species (i.e., for example,
based upon SDS-
PAGE or HPLC analysis). A purified composition is not intended to mean that
some trace
impurities may remain.
As used herein, the term "substantially purified" refers to molecules, such as
amino acid
sequences, that are removed from their natural environment, isolated or
separated, and are at
least 60% free, preferably 75% free, and more preferably 90% free from other
components
with which they are naturally associated. An "isolated polypeptide" is
therefore a substantially
purified polypeptide.
The term "biocompatible", as used herein, refers to any material does not
elicit a
substantial detrimental response in the host. There is always concern, when a
foreign object is
introduced into a living body, that the object will induce an immune reaction,
such as an
inflammatory response that will have negative effects on the host. In the
context of this
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invention, biocompatibility is evaluated according to the application for
which it was designed:
for example; a bandage is regarded a biocompatible with the skin, whereas an
implanted
medical device is regarded as biocompatible with the internal tissues of the
body. Preferably,
biocompatible materials include, but are not limited to, biodegradable and
biostable materials.
The teaus "amino acid sequence" and "polypeptide sequence" as used herein, are
interchangeable and to refer to a sequence of amino acids.
A "variant" of a protein is defined as an amino acid sequence which differs by
one or
more amino acids from a polypeptide sequence or any homolog of the polypeptide
sequence.
The variant may have "conservative" changes, wherein a substituted amino acid
has similar
structural or chemical properties, e.g., replacement of leucine with
isoleucine. More rarely, a
variant may have ''nonconservative" changes, e.g., replacement of a glycine
with a tryptophan.
Similar minor variations may also include amino acid deletions or insertions
(i.e., additions), or
both.
A "deletion" is defined as a change in amino acid sequence in which one or
more amino
acid residues, respectively, are absent.
An "insertion" or "addition" is that change in an amino acid sequence which
has
resulted in the addition of one or more amino acid residues.
The term "derivative' as used herein, refers to any chemical modification of
an amino
acid. Illustrative of such modifications would include, but are not limited
to, replacement of
hydrogen by an alkyl, aryl, hydroxyl, sulfhydryl, sulfoxyl, sulfonyl, acyl,
phosphoryl, alkoxyl,
amino or amino heterocyclic group. For example, tyrosine is a 4-hydroxyl amino
acid
derivative of phenylalanine, and phosphoserine is an 0-phosphoric derivative
of serine. Other
possible chemical modifications might include, but are not limited to, C-
terminal amides, and
acyl or sulfonyl N-terminal modifications.
The term "bind" as used herein, includes any physical attachment or close
association,
which may be permanent or temporary. Generally, an interaction of hydrogen
bonding,
hydrophobic forces, van der Waals forces, covalent and ionic bonding etc.,
facilitates physical
attachment between the molecule of interest and the analyte/target being
measuring/affected.
The "binding" interaction may be brief as in the situation where binding
causes a chemical
reaction to occur. That is typical when the binding component is an enzyme and
the
analyte/target is a substrate for the enzyme. Reactions resulting from contact
between the
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binding agent and the analyte/target are also within the definition of binding
for the purposes of
the present invention.
Brief Description Of The Figures
Figure 1 shows exemplary data of WT PCSK9 inhibition as measured by FACS in
HuH7 cells. HuH7 cells were incubated for 18h in the absence (Cnt) or presence
of 0.75 ug/ml
PCSK9-WT protein alone (WT) or mixed with 1001.tM of various SRX peptides. The
level of
LDLR at the cell surface was measured by FACS using anti human LDLR Ab and a
suitable
secondary Ab labeled with Alexa 647. Cell surface LDLR is reported relative to
Cnt. %
inhibition of activity was calculated as [SRX ¨ WT] / [Cnt ¨ WT] x 100.
Figure 2 shows exemplary data of WT PCSK9 activity by numerous PCSK9
allosteric
modulation peptides. HuH7 cells were incubated in a 96-well plate for a total
of 20h in the
absence (Cnt) or presence of 1.01.1g/m1PCSK9-WT protein alone (WT) or mixed
with 100 uM
of various SRX peptides. After 16h, dil-LDL (5 ug/ml) was added to the
incubation mixtures.
After 4h, fluorescence was measured (Ex: 520 nm/ Em: 575nm; cutoff: 550nm).
Dil-LDL
uptake is calculated as RFU corrected for the number of cells.
Figure 3 shows exemplary data of a mutated PCSK9 protein ("gain of function"
(G0F)-
D374Y) modulation by numerous PCSK9 allosteric modulation peptides. HuH7 cells
were
incubated in a 96-well plate for a total of 20h in the absence (Cnt) or
presence of 0.5 jig/m1
PCSK9-D374Y protein alone (DY) or mixed with 100 uM of various SRX peptides.
After 16h,
dil-LDL (5 ug/ml) was added to the incubation mixtures. After 4h, fluorescence
was measured
(Ex: 520 nm/ Em: 575nm; cutoff: 550nm). Dil-LDL uptake is calculated as RFU
corrected for
the number of cells.
Figure 4 shows exemplary data of a mutated PCSK9 protein ('gain of function"
GOF-
D374Y) modulation showing dose dependent inhibition by SRX55, as measured by
dil-LDL
uptake in HuH7 cells. HuH7 cells were incubated in a 96-well plate for a total
of 20h in the
absence (Cnt) or presence of 0.5 ug/m1PCSK9 GOF-D374Y protein alone (DY) or
mixed with
increasing concentrations of various SRX peptides. After 16h, dil-LDL (5
ug/ml) was added to
the incubation mixtures. After 4h, fluorescence was measured (Ex: 520 nm/ Em:
575nm;
cutoff: 550nm). Dil-LDL uptake is calculated as RFU corrected for the number
of cells.
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Figure 5 shows exemplary data of a mutated PCSK9 protein ("gain of function"
GOF-
D374Y) modulation showing dose-dependent inhibition by SRX55, as measured by
dil-LDL
uptake in HepG2 cells. HepG2 cells were incubated in a 96-well plate for a
total of 20h in the
absence (Cnt) or presence of 21Ag/m1PCSK9 GOF-D374Y protein alone (DY) or
mixed with
increasing concentrations of SRX55 peptide. After 16h, dil-LDL (5 ug/ml) was
added to the
incubation mixtures. After 4h, fluorescence was measured (Ex: 520 nm/ Em:
575nm; cutoff:
550nm). Dil-LDL uptake is calculated as RFU corrected for the number of cells.
Figure 6 shows exemplary data of HepG2 cells were incubated in a 96-well plate
for a
total of 20h in the absence (Cnt) or presence PCSK9 protein alone (D374Y: 0.6
ug/ml; WT: 1.2
ug/ml) or mixed with increasing concentrations of SRX55 peptide. After 16h,
dil-LDL (5
ug/ml) was added to the incubation mixtures. After 4h, fluorescence was
measured (Ex: 520
nm/ Em: 575nm; cutoff: 550nm). Dil-LDL uptake is calculated as RFU corrected
for the
number of cells. The PCSK9 and -7+ SRX55 mixtures were pre-incubated for 3 hrs
at 37C
prior to addition to the cells.
Figure 7 shows exemplary data of FL-83B cells were incubated in a 96-well
plate for a
total of 20h in the absence (Cnt) or presence PCSK9 protein alone (D374Y: 0.6
ug/ml; WT: 1.2
ug/ml) or mixed with increasing concentrations of SRX55 peptide. After 16h,
dil-LDL (5
ug/ml) was added to the incubation mixtures. After 4h, fluorescence was
measured (Ex: 520
nm/ Em: 575nm; cutoff: 550nm). Dil-LDL uptake is calculated as RFU corrected
for the
number of cells. The PCSK9 and -7+ SRX55 mixtures were pre-incubated for 3
firs at 37C
prior to addition to the cells.
Figure 8 presents an illustrative embodiment showing the binding of an
allosteric
modulatory synthetic peptide (e.g., SRX55) to a PCSK9 protein. The prodomain
is shown in
light blue. The two halves of the PCSK9 "catalytic" domain are shown as yellow
and dark
blue, respectively. The EGF-A binding site is shown as blue and yellow
spacefill. SRX55
(green) is shown binding to the allosteric ligand binding site. The N-terminal
helix is shown in
white.
Detailed Description Of The Invention
This invention is related to the field of hypercholesterolemia. In particular,
the
invention provides compositions and methods to modulate circulating levels of
low density
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lipoproteins by altering the conformation of the protein PCSK9 using a
synthetic peptide
and/or a synthetic peptide derivative sequences of 3-8 amino acids ranging
between 350 ¨
2,000 Da. Altering the conformation of PCSK9 affects the interaction between
PCSK9 and an
endogenous low density lipoprotein receptor, and can lead to reduced or
increased levels of
circulating LDL-cholesterol. High LDL-cholesterol levels are associated with
increased risk for
heart disease. Low LDL-cholesterol levels may be problematic in other
conditions, such as
liver dysfunction; thus, there is also utility for peptides which can raise
LDL levels.
I. Physiological Role Of Native PCSK9
Proprotein convertase subtilisin/kexin type 9, also known as PCSK9, is an
enzyme that
in humans is encoded by the PCSK9 gene. Seidah et al., "The secretory
proprotein convertase
neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and
neuronal
differentiation" Proc. Natl. Acad. Sci. U.S.A. 100 (3): 928-933 (2003).
Similar genes
(orthologs) are found across many species. Many enzymes, including PSCK9, are
inactive
when they are first synthesized, because they have a section of peptide chains
that blocks their
activity; proprotein convertases remove that section to activate the enzyme.
An illustrative embodiment shows the binding of an allosteric modulatory
synthetic
peptide (e.g., SRX55) to a PCSK9 protein. See, Figure 8. The prodomain is
shown in light
blue. The two halves of the PC SK9 "catalytic" domain are shown as yellow and
dark blue,
respectively. The EGF-A binding site is shown as blue and yellow spacefill.
SRX55 (green) is
shown binding to the allosteric ligand binding site. The N-terminal helix is
shown in white.
The PSCK9 gene encodes a proprotein convertase belonging to the proteinase K
subfamily of the secretory subtilase family. The encoded protein is
synthesized as a soluble
zymogen that undergoes autocatalytic intramolecular processing in the
endoplasmic reticulum.
The protein may function as a proprotein convertase. For example, a human
PCSK9 amino
acid sequence is:
001 mgtvssrrsw wp1p111111111gpagara qededgdyee lvlalrseed glaeapehgt
061 tatfhrcakd pwrlpgtyvv vlkeethlsq sertarrlqa qaarrgyltk ilhvfhgllp
121 gflykmsgd1 lelalldphv dyieedssvf aqsipwnler itppryrade yqppdggslv
181 evylldtsiq stihreiegry mvtdfenvpe edgtrfhrqa skcdshgthl agvvsgrdag
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241 vakgasmrsl rvincqgkgt vsgtliglef irksqlvqpv gplvvllpla ggysrvinaa
301 cqrlaragvv lvtaagnfrd daclyspasa pevitvgatn aqdqpvtlgt lgtnfgrcvd
361 lfapgediig assdcstcfv sqsgtsqaaa hvagiaamml saepeltlae lrqrlihfsa
421 kdvineawfp edqrvltpnl vaalppsthg agwqlfcrtv wsahsgptrm atavarcapd
481 eellscssfs rsgkrrgerm eaqggklvcr ahnafggegv yaiarccllp qancsvhtap
541 paeasmgtrv hchqqghvlt gcsshweved lgthkppvlr prgqpnqcvg hreasihasc
601 chapgleckv kehgipapqe qvtvaceegw tltgcsalpg tshvlgayav dntcvvrsrd
661 vsttgstseg avtavaiccr srhlaqasqe lq (Accession No. NP 777596).
PSCK9 is believed to play a regulatory role in cholesterol homeostasis. For
example,
PCSK9 can bind to the epidermal growth factor-like repeat A (EGF-A) domain of
the low-
density lipoprotein receptor (LDL-R) resulting in LDL-R internalization and
degradation.
Clearly, it would be expected that reduced LDL-R levels result in decreased
metabolism of
LDL-C, which could lead to hypercholesterolemia.
As it is estimated that approximately 9 million Americans have a high or very
high risk
for heart-related problems that could benefit from PCSK9 inhibitors
(especially when in
combination with statins). PCSK9 inhibitors could result in such widespread
usage having the
potential to replace statins in certain conditions. PCSK9 has medical
significance because it
acts in cholesterol homeostasis. Drugs that block PCSK9 biological actions are
believed to
lower circulating low-density lipoprotein cholesterol (LDL-C) levels (i.e.,
for example, by
increasing the availability of LDL-Rs and, consequently, LDL-C clearance).
Such drugs are
beginning Phase III clinical trials to assess their safety and efficacy in
humans, and to
determine if they can improve outcomes in heart disease.
Drugs that inhibit LDL-R/PCSK9 complex formation have been suggested to lower
cholesterol much more than conventionally available cholesterol-lowering drugs
(i.e., for
example, statins). It is biologically plausible that this would also lower
heart attacks and other
diseases caused by raised cholesterol. Studies with humans, including phase
III clinical trials
now underway, are focused as to whether PCSK9 inhibition actually does lower
cardiovascular
disease, with acceptable side effects. Lopez D., "Inhibition of PCSK9 as a
novel strategy for
the treatment of hypercholesterolemia" Drug News Perspect. 21(6): 323¨e30
(2008); Steinberg
et al., "Inhibition of PCSK9: a powerful weapon for achieving ideal LDL
cholesterol levels"
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Proc. Natl. Acad. Sci. U.S.A. 106(24): 9546-9547 (2009); Mayer, "Annexin A2 is
a C-
teiminal PCSK9-binding protein that regulates endogenous low density
lipoprotein receptor
levels" J. Biol. Chem, 283(46): 31791-31801 ((2008); and Anonomyous, "Bristol-
Myers
Squibb selects Isis drug targeting PCSK9 as development candidate for
prevention and
treatment of cardiovascular disease" Press Release. FierceBiotech. 2008-04-08.
Currently, it has been reported that PCSK9 antibody drugs are in clinical
trials (e.g., for
example, Sanofi/Regeneron, Amgen, Pfizer, Novartis, Roche). However, one
disadvantage of
antibody therapy is that the administration is performed by subcutaneous or
intravenous
injection. A number of monoclonal antibodies that bind to PCSK9 near the
catalytic domain
that interact with the LDL-R and hence inhibit LDL-R/PCSK9 complex formation
are
currently in clinical trials. These antibodies include AMG145 (Amgen), 1D05-
IgG2 (Merck &
Co.), and SAR236553/REGN727 (Aventis/Regeneron). Lambert et al., "The PCSK9
decade"
J. Lipid Res. 53(12): 2515-2524 (2012).
Peptides that mimic the EGF-A domain of the LDL-R have been developed to
inhibit
LDL-R/PCSK9 complex formation. Shan et al., "PCSK9 binds to multiple receptors
and can be
functionally inhibited by an EGF-A peptide". Biochem. Biophys. Res. Commun.
375(1): 69-73
(2008). Peptidic PCSK9 inhibitors of the EGF-A binding site were identified by
screening
both linear and disulfide-constrained phage-displayed peptide libraries. This
approach
identified a 13-amino acid peptide (Pep2-8) that includes structural mimicry
of the natural
binding domain of LDL receptor. The peptide inhibitor binding site was
determined to largely
overlap with that of the EGF(A) domain; therefore, Pep2-8 acts a competitive
inhibitor of LDL
receptor binding. This is akin to the inhibition mechanism of anti-PC SK9
monoclonal
antibodies, which also disrupt the interaction of the LDL receptor-EGF(A)
domain with
PCSK9. Zhang et al., "Identification of a Small Peptide That Inhibits PCSK9
Protein Binding
to the Low Density Lipoprotein Receptor' J Biol Chem 289:942-955 (2014).
PCSK9 antisense oligonucleotides (Isis Pharmaceuticals) have been shown to
increase
expression of the LDL-R and decrease circulating total cholesterol levels in
mice. Graham et
al., "Antisense inhibition of proprotein convertase subtilisin/kexin type 9
reduces serum LDL
in hyperlipidemic mice" J. Lipid Res. 48(4): 763-767 (2007). It has also been
reported that a
locked nucleic acid (Santaris Pharma) reduced PCSK9 mRNA levels in mice. Gupta
et al., "A
locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and
enhances LDLR
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expression in vitro and in vivo" PLoS ONE 5(5): el 0682 (2010); and Lindholm
et al., "PCSK9
LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol
in nonhuman
primates". Mol. Ther. 20(2):376-381 (2012). Initial clinical trials of an RNAi
(ALN-PCS,
Alnylam Pharmaceuticals) has shown positive results as an effective means of
inhibiting LDL-
R/PCSK9 complex formation. Frank-Kamenetsky et al., "Therapeutic RNAi
targeting PCSK9
acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman
primates"
Proc. Natl. Acad. Sci. U.S.A. 105(33): 11915-11920 (2008).
PCSK9 Allosteric Site Modulation Peptides
Variants of PCSK9 can reduce or increase circulating cholesterol. Abifadel et
al.,
"Mutations in PCSK9 cause autosomal dominant hypercholesterolemia" Nat. Genet.
34 (2):
154-156 (2003). LDL-C is normally removed from the blood when it binds to an
LDL-R on
the surface of liver cells, and is internalized within the hepatocyte as a
receptor-ligand
complex. However, when PCSK9 binds to an LDL-R, the LDL-R is concomitantly
degraded
along with the complexed LDL particle. However, if a PCSK9 is not bound to an
LDL-R, the
LDL-R is recycled after internalization thereby returning to the surface of
the cell for removal
of more cholesterol.
In some embodiments, the invention relates to synthetic peptide sequences of 3-
8
amino acids in length, and less than approximately 1,300 Da, having a
modulation effect on
PCSK9's ability to form an LDL-R/PCSK9 complex. In some embodiments, the
synthetic
peptides comprise a lipophilie N-terminal amino acid (e.g., phenylalanine). In
some
embodiments, the present invention contemplate the use of peptides that bind
to a PCSK9
allosteric site. In some embodiments, the peptides decrease LDL-R/PCSK9
complex formation
and are thereby useful to treat various diseases comprising lipid
dysregulation. In some
embodiments, the peptides increase LDL-R/PCSK9 complex formation and are
thereby useful
in research and development of therapies relevant to LDL dysregulation.
Although it is not necessary to understand the mechanism of an invention, it
is believed
that "gain-of-function" (GOF) PCSK9 mutants may result in conditions
including, but not
limited to, hypercholesterolemia. For example, peptides (e.g., synthetic
peptides and/or
synthetic peptide derivatives) that bind to a PCSK9 allosteric site and
increase the affinity of
PCSK9's low density lipoprotein receptor for a low density lipoprotein
receptor on the surface
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of a cell (e.g., a hepatocyte) would be expected to increase the symptoms of
hypercholesterolemia by increasing low density lipoprotein receptor
internalization and
degradation.
Although it is not necessary to understand the mechanism of an invention, it
is believed
that "loss-of-function" (LOF) PCSK9 mutants may result in conditions
comprising reduced
low density lipoproteins and would be expected to result in
hypocholesterolemia thereby
reducing the risk of cardiovascular diseases, including but not limited to,
coronary heart
disease. For example, peptides that bind to a PCSK9 allosteric site that
decrease the affinity of
PCSK9's low density lipoprotein receptor binding site for a low density
lipoprotein receptor on
the surface of a cell (e.g., a hepatocyte) would be expected to reduce the
symptoms of
hypercholesterolemia by promoting low density lipoprotein internalization and
clearance due
to concomitant recycling of the low density lipoprotein receptor.
The presently disclosed embodiments of PCSK9 allosteric peptides have several
advantages over current therapeutic strategies to control LDL discussed above.
For example,
small PCSK9 allosteric peptides, as contemplated herein, have the advantage
that these
peptides can be administered orally without immunological reactions seen with
antibody
administration, or systemic degradation problems as seen with nucleic acid
administration (i.e.,
antisense or locked nucleic acids). Nonetheless, as these small peptides have
long half-lives,
encapsulation drug delivery systems, such as liposomes or other biodegradable
protective
compositions, will lengthen these half-lives to a greater extent than either
antibodies or nucleic
acids.
The data presented herein exemplifies sixteen (16) synthetic peptides having
various
effects on PCSK9's ability to bind to LDL-R mediated by binding to a PCSK9
allosteric site.
For example, three synthetic peptides were able to increase cell surface
expression of LDL-R
by 60-95%, by preventing WT PCSK9 /LDL-R complex formation, as measured by
FACS in
HuH7 cells. In particular, one synthetic peptide (SRX55) was able to increase
cell surface
expression of LDL-R by 100%, by changing WT PCSK9/LDL-R complex affinity. See,
Figure 1. These same three synthetic peptides were detelmined to increase LDL
internalization
by 30-50 %, as measured by dil-LDL uptake in Hull? cells. In another study,
one peptide was
able to inhibit the activity of GOF PCSK9-D374Y by 100%, as measured by diI-
LDL uptake in
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HepG2 cells, and four peptides showed a 20-30 %, as measured by dil-LDL uptake
in HuH7
cells. Some peptides also show inhibitory activity in mouse hepatocyte diI-LDL
uptake.
In particular, the present data shows an ability of PSCK9 allosteric synthetic
peptides to
modulate LDLR cell surface levels by binding a peptide to PCSK9. See, Figure
1. In that
experiment, the LDLR levels of a hepatocyte culture model (HuH7 cells) were
measured by
fluorescence activated cell sorting (FACS) in accordance with Example III.
Cell surface
LDLR is reported as a percentage of Basal levels of LDLR, indicated by the
Cnt_Amm.Bic,
Cnt_Ac.Acid, and Cnt bars in the top graph. See, Figure 1 (top panel). LDLR
levels in the
presence of exogenous PCSK9 is indicated as WT_Amm.Bic, WT-Ac.Acid, and WT,
and
Exogenous PCSK9 in combination with a tested peptide is indicated as SRX##.
The measured
LDLR levels are reported as % versus basal controls (Cnt) of the respective
group. Examples
of peptides (e.g., an allosteric synthetic inhibitor peptide) which positively
modulate (increase)
LDLR cell surface level include SRX55, SRX56, SRX60, and SRX62, and exemplary
peptides
(e.g., an allosteric synthetic enhancer peptide) which negatively modulate
(decrease) LDLR
cell surface levels include SRX69, SRX72, and SRX73. This was further shown a
percent
inhibition (% inhibition was calculated as [SRX - WT] / [Cnt-WT] x 100%) where
positive
modulation (increase) of LDLR level is reported as positive % inhibition, and
negative
modulation (decrease) of LDLR level is reported as negative % inhibition. See,
Figure 1
(bottom panel).
The ability to modulate hepatocyte LDL internalization by the binding of a
ligand to the
PCSK9:LDLR complex is demonstrated in Figures 2 through 7. LDL internalization
was
measured by uptake of a fluorescently tagged LDL molecule (diI-LDL) in the
absence of
exogenous PCSK9 (Cnt), in the presence of exogenous PCSK9 (normal PCSK9 = WT,
D374Y
mutant PCSK9 = DY), or in the presence of PCSK9 and a tested peptide
(indicated as SRX##,
or SRX if a single peptide results is shown in a graph).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be modulated
in a
model hepatocyte cell line (HuH7) in the presence of the tested SRX peptides.
See, Figure 2
(top panel). LDL internalization was shown to be positively modulated
(increased) by
allosteric synthetic inhibitor peptides such as SRX55, SRX 56, SRX60, and
SRX67. LDL
internalization can be negatively modulated (decreased) by allosteric
synthetic enhancer
peptides such SRX36, SRX61, SRX64, SRX65, SRX66, and SRX73. The percent
inhibition is
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shown, where positive modulation (increase) in LDL internalization is reported
as >0%
inhibition, and negative modulation (decrease) in LDL internalization is
reported as <0%
inhibition. See, Figure 2 (bottom panel).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be modulated
in a
model hepatocyte cell line (HuH7) by the presence of the tested SRX peptides
in combination
with a clinically relevant pathologic gain-of-function D374Y exogenous PCSK9
(DY). See,
Figure 3 (top panel). LDL internalization was shown to be positively modulated
(increased) by
allosteric synthetic inhibitor peptides such as SRX55, SRX 56, SRX60, SRX63,
SRX64, and
SRX66. LDL internalization can be negatively modulated (decreased) by
allosteric synthetic
enhancer peptides such SRX36, SRX71, SRX72, and SRX73. The percent inhibition
is shown,
where positive modulation (increase) in LDL internalization is reported as >0%
inhibition, and
negative modulation (decrease) in LDL internalization is reported as <0%
inhibition. See,
Figure 3 (bottom panel).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be positively
modulated (increased) by the presence of allosteric synthetic inhibitor
peptides (SRX55, SRX
60, SRX66 and SRX56) in combination with a clinically relevant pathologic gain-
of-function
D374Y PCSK9 (DY). SRX55 was shown to have a positive modulation in a dose
dependent
manner. See, Figure 4 (top panel). The percent inhibition is shown, where
positive modulation
(increase) in LDL internalization is reported as >0% inhibition, note that
SRX55 at 11.1 uM is
within sampling noise of 0%. See, Figure 4 (bottom panel).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be positively
modulated (increased) in a second model hepatocyte cell line (HepG2) in
combination with a
clinically relevant pathologic gain-of-function D374Y PCSK9 (DY) in a dose
dependent
manner with SRX55. See, Figure 5 (top panel). This positive modulation is
further shown as a
percent inhibition, where positive modulation (increase) in LDL
internalization is reported as
>0% inhibition. See, Figure 5 (bottom panel).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be positively
modulated (increased) in a second hepatocyte cell line (HepG2) when pre
incubated in
combination with a clinically relevant pathologic gain-of-function D374Y PCSK9
(DY) or
normal PCSK9 (WT) in a dose dependent manner with SRX55. See, Figure 6 (top
panel).
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This positive modulation is further shown as percent inhibition, where
positive modulation
(increase) in LDL internalization is reported as >0% inhibition. See, Figure 6
(bottom panel).
LDL internalization, as reported by diI-LDL % uptake vs Cnt, can be positively
modulated (increased) in a third hepatocyte cell line (FL83B) when pre
incubated in
combination with a clinically relevant pathologic gain-of-function D374Y PCSK9
(DY) or
normal PCSK9 (WT) in a dose dependent manner with SRX55. See, Figure 7 (top
panel).
This positive modulation is further shown as percent inhibition, where
positive modulation
(increase) in LDL internalization is reported as >0% inhibition. See, Figure 7
(bottom panel).
The most efficacious peptides (e.g., for example, SRX55; Compound 1) performed
in
consistent order across all assays and PCSK9 phenotypes. Improved peptides
were then
designed that were expected to have better drug-like properties, as they were
designed based
upon an analysis of the preliminary results. Typically, the design of these
improved peptides
have at least one of the first three amino acids from the C-terminus
incorporated with a
negatively charged polar group, such as a phosphate, a sulfate, a tetrazole or
a carboxylic acid.
For example, in Compound 3, the polar group comprises a phosphate group:
01111 411
H
10,
OH
0 0 0 0
NH
H2N N N . OH
0
""OH 0
OH 0
0
FOH
P.
0 "NON
Compound 3: Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p).
Alternatively, in Compound 14, the C-terminal glycine comprises a polar group:
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Compound 14: Ac-Tyr-Trp-Gly.
OH
0 0
OH
N N
0 0
NH
The constituent amino acids may be of defined stereochemistry, usually the
natural "L"
enantiomer and may have naturally occurring or synthetic side chains. The
peptide "N"
terminus may be free, alkylated, sulfonated, or acylated. The "C" terminus may
be the
carboxylic acid or an amide.
Various natural and unnatural amino acids may be contemplated. Tryptophan
indole
side chains may be substituted with alkyl, alkoxy, halo, carboxy, etc. to form
other analogs.
Phenyalanine, tyrosine, and homophenylalanine phenyl moieties may have
additional phenyl
substitution, such as alkyl, alkoxy, halo, carboxy, etc. Serine may be
substituted in some
examples by alanine. Threonine may be substituted by serine or alanine.
Valine, leucine, and
isoleucine may be interchanged in some analogs. Amino acids with carboxylic
acid side chains,
such as aspartic acid, may have the side chain derivatized as an amide.
III. Clinical Therapeutics
In some embodiments, the present invention contemplates the administration of
a
PCSK9 allosteric inhibitor peptide to a subject having a symptom of a
cardiovascular disease.
In one embodiment, the cardiovascular disease comprises hypercholesterolemia.
In one
embodiment, the cardiovascular disease comprises hypertension. In one
embodiment, the
hypercholesterolemia comprises elevated low density lipoprotein levels.
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In some embodiments, the present invention contemplates the administration of
a
PCSK9 allosteric inhibitor peptide to a subject having a symptom of a
metabolic disease. In
one embodiment, the metabolic disease comprises diabetes.
Although it is not necessary to understand the mechanism of an invention, it
is believed
that the administration of a PCSK9 allosteric inhibitor synthetic peptide
(i.e., for example,
SRX55) induces a conformational shift of the PCSK9 protein such that the
affinity of the low
density lipoprotein binding site for a low density lipoprotein receptor is
decreased, wherein
PCSK9/LDL-R complex formation is decreased. The decrease in PCSK9/LDL-R
complex
formation results in an increase in the bioavailability of LDL-R receptors for
binding to
circulating LDL, thereby increasing the internalization and clearance of LDL
by LDL-R. It is
further believed that PCSK9 allosteric inhibitor peptides result in increased
bioavailability of
hepatocyte cell LDL-Rs.
A. Hypercholesterolemia
Hypercholesterolemia (also spelled hypercholesterolaemia) is the presence of
high
levels of cholesterol in the blood. It is a form of "hyperlipidemia" (elevated
levels of lipids in
the blood) and "hyperlipoproteinemia" (elevated levels of lipoproteins in the
blood).
Durrington, P "Dyslipidaemia" The Lancet 362(9385):717-731.
Hypercholesterolemia is
typically due to a combination of environmental and genetic factors.
Environmental factors
include obesity and dietary choices. Genetic contributions are usually due to
the additive
effects of multiple genes, though occasionally may be due to a single gene
defect such as in the
case of familial hypercholesterolaemia. A number of secondary causes exist
including: diabetes
mellitus type 2, obesity, alcohol, monoclonal gammopathy, dialysis, nephrotic
syndrome,
obstructive jaundice, hypothyroidism, Cushing's syndrome, anorexia nervosa,
medications
(thiazide diuretics, ciclosporin, glucocorticoids, beta blockers, retinoic
acid). Bhatnagar et al.,
(2008) "Hypercholesterolaemia and its management" BMJ337: a993. Genetic
abnormalities
are in some cases completely responsible for hypercholesterolemia, such as in
familial
hypercholesterolemia where there is one or more genetic mutations in the
autosomal dominant
APOB gene, the autosomal recessive LDLRAP1 gene, autosomal dominant familial
hypercholesterolemia (HCHOLA3) variant of the PCSK9 gene, or the LDL receptor
gene.
"Hypercholesterolemia" Genetics Home Reference U.S. National Institutes of
Health,
ghr.nlm.nih.gov/condition=hypercholesterolemia. Even when there is no single
mutation
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responsible for hypercholesterolemia, genetic predisposition still plays a
major role in
combination with sedentary lifestyle, obesity, or an atherogenic diet.
Citkowitz et al., (2010)
"Polygenic Hypercholesterolemia". eMedicine Medscape, emedicine.medscape.com/
article/121424-overview.
Cholesterol is a sterol. It is one of three major classes of lipids which all
animal cells
utilize to construct their membranes and is thus manufactured by all animal
cells. Plant cells do
not manufacture cholesterol. It is also the precursor of the steroid hormones,
bile acids and
vitamin D. Since cholesterol is insoluble in water, it is transported in the
blood plasma within
protein particles (lipoproteins). Lipoproteins are classified by their
density: very low density
lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density
lipoprotein (LDL) and
high density lipoprotein (HDL). Biggerstaff et al., (2004). "Understanding
lipoproteins as
transporters of cholesterol and other lipids" Adv Physiol Educ 28 (1-4): 105-
6. All the
lipoproteins carry cholesterol, but elevated levels of the lipoproteins other
than HDL (termed
non-HDL cholesterol), particularly LDL-cholesterol are associated with an
increased risk of
atherosclerosis and coronary heart disease. Carmena et al., (2004)
"Atherogenic lipoprotein
particles in atherosclerosis" Circulation 109(23 Suppl 1): 1112-7. In
contrast, higher levels of
HDL cholesterol are protective. Kontush et al., (2006) "Antiatherogenic small,
dense HDL--
guardian angel of the arterial wall?" Nat Clin Pract Cardiovasc Med 3(3):144-
153. Elevated
levels of non-HDL cholesterol and LDL in the blood may be a consequence of
diet, obesity,
inherited (genetic) diseases (such as LDL receptor mutations in familial
hypercholesterolemia),
or the presence of other diseases such as diabetes and an underactive thyroid.
Total cholesterol
is the amount of all of the fats in your blood. These fats are called lipids.
There are different
types of lipid that make up your total cholesterol. The two most important
types are: low
density lipoprotein (LDL) -"bad" cholesterol and high density lipoprotein
(HDL) -"good"
cholesterol. High cholesterol, especially "bad" cholesterol (LDL), can clog
your arteries. This
may reduce blood flow to your heart. It can lead to heart disease, stroke, or
heart attack.
Cholesterol is measured in milligrams per deciliter (mg/dL). In conditions
such as heart
disease or diabetes, LDL cholesterol should stay below 100 mg/dL. If there is
a risk for heart
disease, LDL cholesterol should be lower than 130 mg/dL. In general, LDL
cholesterol should
be lower than 160 - 190 mg/dL. Alternative, HDL "good" cholesterol should be
high. For
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example, HDL levels in men should be above 40 mg/dL, while HDL levels should
be above 50
mg/dL for women.
One symptom of hypercholesterolemia comprises a longstanding elevation of
serum
cholesterol that can lead to atherosclerosis. Bhatnagar et al., (2008)
''Hypercholesterolaemia
and its management" BMJ 337: a993. Over a period of decades, chronically
elevated serum
cholesterol contributes to formation of atheromatous plaques in the arteries.
This can lead to
progressive stenosis (narrowing) or even complete occlusion (blockage) of the
involved
arteries. Alternatively smaller plaques may rupture and cause a clot to form
and obstruct blood
flow. Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R (July 2010).
"Concept of
vulnerable/unstable plaque'' Arterioscler. Thromb. Vase. Biol. 30(7): 1282-
1292. A sudden
occlusion of a coronary artery results in a myocardial infarction or heart
attack. An occlusion
of an artery supplying the brain can cause a stroke. If the development of the
stenosis or
occlusion is gradual blood supply to the tissues and organs slowly diminishes
until organ
function becomes impaired. At this point that tissue ischemia (restriction in
blood supply) may
manifest as specific symptoms including, but not limited to, temporary
ischemia of the brain
(commonly referred to as a transient ischemic attack) may manifest as
temporary loss of vision,
dizziness and impairment of balance, aphasia (difficulty speaking), paresis
(weakness) and
paresthesia (numbness or tingling), usually on one side of the body.
Insufficient blood supply
to the heart may manifest as chest pain, and ischemia of the eye may manifest
as transient
visual loss in one eye. Insufficient blood supply to the legs may manifest as
calf pain when
walking, while in the intestines it may present as abdominal pain after eating
a meal. Grundy et
al., (1998) "Primary prevention of coronary heart disease: guidance from
Framingham: a
statement for healthcare professionals from the AHA Task Force on Risk
Reduction. American
Heart Association" Circulation 97(18):1876-1887.
B. Hypocholesterolemia
Hypocholesterolemia is the presence of abnormally low (hypo-) levels of
cholesterol in
the blood (-emia). Although the presence of high total cholesterol (hyper-
cholesterolemia)
correlates with cardiovascular disease, a defect in the body's production of
cholesterol can lead
to adverse consequences as well. Cholesterol is an essential component of
mammalian cell
membranes and is required to establish proper membrane permeability and
fluidity. It is not
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clear if a lower than average cholesterol level is directly harmful; it is
often encountered in
particular illnesses.
Possible causes of low cholesterol include, but are not limited to, statins,
hyperthyroidism, or an overactive thyroid gland, adrenal insufficiency, liver
disease,
malabsorption (inadequate absorption of nutrients from the intestines), such
as in celiac
disease, malnutrition, abetalipoproteinemia (a genetic disease that causes
cholesterol readings
below 50 mg/di), hypobetalipoproteinemia (a genetic disease that causes
cholesterol readings
below 50 mg/di, manganese deficiency, Smith-Lemli-Opitz syndrome, Marfan
syndrome,
leukemias and other hematological diseases.
Demographic studies suggest that low cholesterol is associated with increased
mortality, mainly due to depression, cancer, hemorrhagic stroke, aortic
dissection and
respiratory diseases. Jacobs et al., (1992). "Report of the Conference on Low
Blood
Cholesterol: Mortality Associations" Circulation 86 (3): 1046-1060; and Suarez
E.C., (1999)
"Relations of trait depression and anxiety to low lipid and lipoprotein
concentrations in healthy
young adult women". Psychosom Med 61(3): 273-279. It is also possible that
whatever causes
the low cholesterol level also causes mortality, and that the low cholesterol
is simply a marker
of poor health.
C. Diabetes
Diabetes affects more than 20 million Americans. Over 40 million Americans
have pre-
diabetes (which often develops before type 2 diabetes).Diabetes is usually a
lifelong (chronic)
disease in which there is a high level of sugar in the blood. Insulin is a
hormone produced by
the pancreas to control blood sugar. Diabetes can be caused by too little
insulin, resistance to
insulin, or both. To understand diabetes, it is important to first understand
the normal process
by which food is broken down and used by the body for energy.
Several things happen when food is digested. A sugar called glucose enters the
bloodstream. Glucose is a source of fuel for the body. An organ called the
pancreas makes
insulin. The role of insulin is to move glucose from the bloodstream into
muscle, fat, and liver
cells, where it can be used as fuel.
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People with diabetes have high blood sugar because their body cannot move
sugar into
fat, liver, and muscle cells to be stored for energy. This is because either
their pancreas does
not make enough insulin or their cells do not respond to insulin normally.
There are two major types of diabetes. The causes and risk factors are
different for each
type. Type 1 diabetes can occur at any age, but it is most often diagnosed in
children, teens, or
young adults. In this disease, the body makes little or no insulin. Daily
injections of insulin are
needed. The exact cause is unknown. Type 2 diabetes makes up most diabetes
cases. It most
often occurs in adulthood. But because of high obesity rates, teens and young
adults are now
being diagnosed with it. Many people with type 2 diabetes do not know they
have it.
Gestational diabetes is high blood sugar that develops at any time during
pregnancy in a
woman who does not have diabetes.
Diabetes symptoms may result from high blood sugar level and include, but are
not
limited to, blurry vision, excess thirst, fatigue, hunger, urinating often and
weight loss.
IV. Pharmaceutical Compositions
The present invention further provides pharmaceutical compositions (e.g.,
comprising
the peptides described above). The pharmaceutical compositions of the present
invention may
be administered in a number of ways depending upon whether local or systemic
treatment is
desired and upon the area to be treated. Administration may be topical
(including ophthalmic
and to mucous membranes including vaginal and rectal delivery), pulmonary
(e.g., by
inhalation or insufflation of powders or aerosols, including by nebulizer;
intratracheal,
intranasal, epidetnial and transdermal), oral or parenteral. Parenteral
administration includes
intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular
injection or infusion;
or intracranial, (e.g., intrathecal or intraventricular), administration.
Pharmaceutical compositions and formulations for topical administration may
include
transdermal patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases,
thickeners and
the like may be necessary or desirable.
Compositions and formulations for oral, sublingual or buccal administration
include
powders or granules, suspensions or solutions in water or non-aqueous media,
capsules, gels,
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drops, strips, gums, sachets or tablets. Thickeners, flavoring agents,
diluents, emulsifiers,
dispersing aids or binders may be desirable.
Compositions and formulations for parenteral, intrathecal or intraventricular
administration may include sterile aqueous solutions that may also contain
buffers, diluents and
other suitable additives such as, but not limited to, penetration enhancers,
carrier compounds
and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not
limited to,
solutions, emulsions, and liposome-containing formulations. These compositions
may be
generated from a variety of components that include, but are not limited to,
preformed liquids,
self-emulsifying solids and self-emulsifying semisolids.
In some embodiment, the pharmaceutical compositions may further comprise other
drugs, hormones, and/or peptides. For example, the pharmaceutical composition
may further
comprise a statin drug. Statins (or HMG-CoA reductase inhibitors) are a class
of drugs used to
lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which
plays a role in
the production of cholesterol in the liver. Increased cholesterol levels have
been associated
with cardiovascular diseases, and statins are therefore used in the prevention
of these diseases.
Lewington et al., "Blood cholesterol and vascular mortality by age, sex, and
blood pressure: a
meta-analysis of individual data from 61 prospective studies with 55,000
vascular deaths"
Lancet 370(9602): 1829-1839 (2007). Research has found that statins are most
effective for
treating cardiovascular disease (CVD) as a secondary prevention strategy, with
questionable
benefit in those with elevated cholesterol levels but without previous CVD.
Taylor et al.
"Statins for the primary prevention of cardiovascular disease". In: Taylor,
Fiona. Cochrane
Database Syst Rev (1) (2011). Statins have rare but severe adverse effects,
particularly muscle
damage.
Specific examples of statins include, but are not limited to, atorvastatin
(Lipitor and
Torvasn, fluvastatin (Lescol ), lovastatin (Mevacor , Altocor , Altoprev ),
pitavastatin
(Livalo , Pitava ), pravastatin (Pravachol , Selektine , Lipostae),
rosuvastatin (Crestor ) and
simvastatin (Zocor , Lipex ). Several combination preparations of a statin and
another agent,
such as ezetimibe/simvastatin, are also available.
Specific examples of cardiovascular drugs include, but are not limited to,
propranolol,
digitalis, amlodipine besylate, and nifedipine.
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Specific examples of other phanuaceutical compositions may further include,
but are
not limited to, exetimibe (Zetiaa'), amlodipine besylate (Norvase), niacin,
sitagliptin
(Januvie), metfonnin or orlistat (Alli /Xenical ).
The pharmaceutical formulations of the present invention, which may
conveniently be
presented in unit dosage form, may be prepared according to conventional
techniques well
known in the pharmaceutical industry. Such techniques include the step of
bringing into
association the active ingredients with the pharmaceutical carrier(s) or
excipient(s). In general
the formulations are prepared by uniformly and intimately bringing into
association the active
ingredients with liquid carriers or finely divided solid carriers or both, and
then, if necessary,
shaping the product.
The compositions of the present invention may be formulated into any of many
possible
dosage forms such as, but not limited to, tablets, capsules, liquid syrups,
soft gels,
suppositories, and enemas. The compositions of the present invention may also
be formulated
as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may
further
contain substances that increase the viscosity of the suspension including,
for example, sodium
carboxyrnethylcellulose, sorbitol and/or dextran. The suspension may also
contain stabilizers.
In one embodiment of the present invention the pharmaceutical compositions may
be
formulated and used as foams. Pharmaceutical foams include formulations such
as, but not
limited to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar
in nature these formulations vary in the components and the consistency of the
final product.
The compositions of the present invention may additionally contain other
adjunct
components conventionally found in pharmaceutical compositions. Thus, for
example, the
compositions may contain additional, compatible, pharmaceutically-active
materials such as,
for example, antipruritics, astringents, local anesthetics or anti-
inflammatory agents, or may
contain additional materials useful in physically formulating various dosage
forms of the
compositions of the present invention, such as dyes, flavoring agents,
preservatives,
antioxidants, opacifiers, thickening agents and stabilizers. However, such
materials, when
added, should not unduly interfere with the biological activities of the
components of the
compositions of the present invention. The formulations can be sterilized and,
if desired, mixed
with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting
agents, emulsifiers,
salts for influencing osmotic pressure, buffers, colorings, flavorings and/or
aromatic substances
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and the like which do not deleteriously interact with the active
pharmaceutical ingredient(s) of
the formulation.
Dosing is dependent on severity and responsiveness of the disease state to be
treated,
with the course of treatment lasting from several days to several months, or
until a cure is
effected or a diminution of the disease state is achieved. Optimal dosing
schedules can be
calculated from measurements of drug accumulation in the body of the patient.
The
administering physician can easily determine optimum dosages, dosing
methodologies and
repetition rates. Optimum dosages may vary depending on the relative potency
of individual
oligonucleotides, and can generally be estimated based on EC50s found to be
effective in in
vitro and in vivo animal models or based on the peptides described herein. In
general, dosage is
from 0.01 p,g to 100 g per kg of body weight, and may be given once or more
daily, weekly,
monthly or yearly. The treating physician can estimate repetition rates for
dosing based on
measured residence times and concentrations of the drug in bodily fluids or
tissues. Following
successful treatment, it may be desirable to have the subject undergo
maintenance therapy to
prevent the recurrence of the disease state, wherein the peptide is
administered in maintenance
doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more
daily, to once every
years.
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Experimental
Example I
Cell Culture And Transfections
HepG2/shPCSK9 or HuH7/shPCSK9 cells (1) lacking endogenous PCSK9 were seeded
at 1 x 105cells/well in a 12 well microplate (Greiner Bio-One). These cells
were then incubated
for 4h or overnight with 0.7 Rg/m1 of either V5-tagged PCSK9 or its gain-of-
function PCSK9-
D374Y pre-incubated, or not, for 4h with each peptide at 50 jtM (or less if
needed for the most
active peptides). The cells were then lysed in lx RIPA buffer (150 mM NaC1, 50
mM Tris-
HC1, pH 8.0), containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS
supplemented with lx complete protease inhibitor mixture (Roche Applied
Science), and
analyzed by Western blot.
Example II
Western Blot Analyses
Proteins in the cell lysates were resolved by 10% Tris-Glycine SDS-PAGE. The
gels
were blotted onto polyvinylidene difluoride (PVDF, Perkin Elmer Life Sciences)
membranes
(GE Healthcare), blocked for lh in TBS-T (50mM Tris-HC1, pH 7.5, 150mM NaC1,
0.1%
Tween 20) containing 5% nonfat milk and immunoblotted with a homemade
polyclonal human
PCSK9 antibody (1:1000) (13), human LDLR antibody (1:1000, R&D Systems), beta-
Actin
(1:5000; Sigma) and monoclonal antibody (mAb) V5-HRP (1:5000; Sigma).
Appropriate
horseradish peroxidase-conjugated secondary antibodies (1:10000, Sigma) were
used for
detection with enhanced chemiluminescence using the ECL Plus kit (GE
Healthcare).
Quantitation of protein bands was obtained using Image J software.
Example III
FACS Analysis
HuH7/shPCSK9 cells were incubated at 37 C for 4h as above with PCSK9 pre-
incubated, or not, with each of the exemplified peptides used at 5004 (or less
if needed for
the most active peptides). Benjannet et al., "Effects of the prosegment and pH
on the activity of
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PCSK9: evidence for additional processing events" J Biol Chem. 285(52): 40965-
40978
(2010).
The cells were then washed 3x with solution A (calcium/magnesium-free
Dulbecco's
PBS (Invitrogen) containing 0.5% bovine serum albumin (Sigma) and 1g/liter
glucose)). The
cells were then incubated for 10 min at room temperature with lx Versene
solution
(Invitrogen) followed by the addition of 5 ml of solution A. The cells were
then incubated for
40 min in solution A containing a human LDLR mAb-C7 (1:100; Santa Cruz
Biotechnology).
Following washes, the cells were then incubated for 20 mm in solution A
containing a
secondary antibody (Alexa Fluor 647 donkey anti-mouse antibody; 1:250;
Molecular Probes).
Following suspension in PBS containing 0.2% of propidium iodide, the cells
were
analyzed by FACS for both propidium iodide (dead cells) and LDLR in live cells
with Alexa
Fluor 647 using the FACS BD LSR (BD Biosciences).
Cell Activity of Exemplified Peptides
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10 -
Compound 11 -
Compound 12 (+)
Compound 13 -
Compound 14 (+)
Compound 15 -
Compound 16 (+)
+ implies > 30% inhibition above control at 100 uM
- implies inhibition within error range
(+)implies inhibition >30% below control at 100 uM
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Example IV
Cellular diI-LDL Uptake Assay
Cells, such as HepG2, HuH7, FL83B or a cell line transfected with a short-
hairpin
PCSK9 knockdown sequence such as HepG2/shPCSK9, HuH7/shPCSK9, FL83B/shPCSK9,
were seeded at 2 x 104 cells/well in a 96-well plate and cultured at 37 degC
in RPMI + 10%
FB S. After approximately 24 hours, the cell media was aspirated off and
replaced with RPMI
+ 3-5 mg/mL LPDS (Lipoprotein Deficient Serum, Millipore) media for further
experimentation. Benjannet et al., "Effects of the prosegment and pH on the
activity of PC SK9:
evidence for additional processing events" J Biol Chem. 285(52): 40965-40978
(2010).
Peptide activity was assessed by culturing cells with: i) no SRX peptide/PC
SK.9 protein
complex (control, Cnt); ii) PCSK9 protein; and iii) SRX peptide/PCSK9 complex.
Various
permutations of these experimental conditions were also used, including: i)
the addition of wild
type PCSK9 (WT); ii) a mutant PCSK9 (e.g., D374Y mutant PCSK9, DY); iii)
various SRX
peptides and/or PCSK9 at the same concentration and/or combinations; iv)
various SRX
peptides and/or PCSK9 at different concentrations and/or combinations; v) the
use of different
cells, as mentioned above, with or without a transfected short-hairpin
sequence; vi) a pre-
incubation of the PCSK9 and SRX peptide (e.g., 1, hour, 2 hours, 3 hours, 4
hours etc.); vii)
various temperatures including, but not limited to, body temperature (e.g., 37
C),
supraphysiologic temperature (e.g., 39 C); and viii) with/without agitation
(e.g., shaker or
gentle periodic vortexing).
Cells were cultured using one of the combinations of conditions described in
the
preceding paragraph for 16 hours. After 16 hours, a quantity of diI-LDL (Low
density
lipoprotein coupled with 1,1'-dioctadecy1-3,3,3',3'-
tetramethylindocarbocyanine perchlorate)
needed to bring the media concentration to 5 ug/mL of diI-LDL was added to the
culture well
and cells continued to be cultured under these new conditions for 4 additional
hours. At the
end of the 4-hour incubation period (20 total hours of cell culture), the
cellular uptake was
halted with the addition of 4% formaldehyde in 10 uM Hoechst 33342 in a
solvent such as
deionized autoclaved water or PBS, and specimens were incubated at 20 C for
20 minutes.
Cell specimens were rinsed twice with PBS and then fluorescence measured with
excitation at 360 nm and emission detected at 460 nm to measure DNA content.
Cell
specimens were then be incubated with a 0.1% SDS in a 0.1 N NaOH solution
while being
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shaken for 10 minutes. Fluorescence of the diI-LDL in the specimens were
quantified using
excitation at 530 nm and resulting emission at 580 nm.
Fluorescence measurements of diI-LDLR were normalized to estimated cell
numbers,
determined from the Hoechst fluorescence. Data was analyzed for the different
experimental
conditions and reported as percentage relative fluorescence units (RFU) of the
Cnt specimen.
Percent inhibition was calculated as the difference in RFU of a peptide
exposed specimen to
the RFU of PCSK9-no peptide, divided by the RFU difference in PCSK9-no peptide
to RFU of
Cnt specimen, also expressed as [(SRX:RFU) ¨ (PCSK9-no peptide:RFU)] / [(PCSK9-
no
peptide:RFU) ¨ (Cnt:RFU)] x 100.
Example V
Methods Of Making PCSK9 Allosteric Inhibitor Peptides
This example presents several methods of identifying and synthesizing peptides
of the
present invention. R. B. Merrifield (1963). "Solid Phase Peptide Synthesis. I.
The Synthesis of
a Tetrapeptide". J. Am. Chem. Soc. 85 (14): 2149-2154; Albericio, F. (2000).
Solid-Phase
Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. p. 848. ISBN 0-
8247-0359-6;
and Albericio F, Carpino LA., "Coupling reagents and activation" Methods
Enzyrnol.
1997;289:104-126.
All peptides were manufactured using Fmoc (9-fluorenylmethyloxycarbonyl)
chemistry
(21st Century Biochemicals, 260 Cedar Hill St., Marlboro, MA 01752). In brief,
the peptides
are made using a polystyrene resin, functionalized with an appropriate linker,
and the peptides
are then manufactured using an Intavis RS Peptide Synthesizer (Germany) or
manufactured by
hand using glass peptide synthesis vessels fitted with coarse glass frits for
removing reactants
by vacuum (Chemglass).
In either case, the amino acids are added sequentially as follows: the amino
acids are
dissolved in either NMP (N-Methyl-2- pyrrolidone) or DMF (Dimethylformamide);
these
solvents are also used for washing the resin following each step. The Fmoc-
protected amino
acid to be added is activated using either HATU (0-(7-azabenzotriazol-1-y1)-
N,N,N,N1-
tetramethyluronium hexafluorophosphate) or HCTU (2-(6-Chloro-1H-benzotriazole-
1-y1)-
1,1,3,3-tetramethylaminium hexafluorophosphate); for a 4-fold stochiometric to
be added
(relative to the resin), a 3.95-fold excess of HATU or HCTU is used to create
the active ester.
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Along with an 8-fold excess of DIPEA (N,N-Diisopropylethylamine) as the base,
these
reagents catalyze the addition of the next amino acid. Once the amino acid is
coupled (each
cycle includes a double coupling cycle to insure efficient coupling) the resin
is exposed to 20%
acetic anhydride to terminate ("cap-off') any peptide chains that have no
added the next amino
acid. The resin is then washed using DMF (3X), Methanol (Me0H, 2X) and DMF
again, 2X.
Piperidine is used to remove the Fmoc group at the end of each coupling cycle
which exposes
the N-terminal amine and allows the next amino acid to be added.
Once synthesis of each step is completed, the peptides (on resin) were dried
using
Me0H (3X) and DCM (3X), cleaved and deprotected using 92% TFA, 2% water, 2%
triisopropylsilane, 2% thioanisole and 2% ethanedithiol for 3-4h at room
temperature. Peptides
were precipitated in cold diethyl ether, centrifuged (2,000 RPM) and the
pellets washed 2X
with cold ether. After drying the peptides were solubilized in water
containing 0.1% TFA
(buffer A) and subjected to RP-HPLC using C18 columns (buffer B = 95%
acetonitrile/0.1%
TFA).
Some PCSK9 allosteric synthetic peptides, and their physical characteristics,
are listed
below:
Compound 1 (SRX-55): Val-Tyr-Val-Arg-Phe-Trp, Calc'd m/z: 868.46; Obs.: 869.00
Compound 2: (SRX-56) 3-A1a-Phe(3-CH2NH2)-Val-D-Ser(p)-Phe-Trp, Calc'd m/z:
864.36; Obs.: 864.80
Compound 3 (SRX-60): Thr-Leu-Cys(CH2-Ph)-Thr-Trp-Ser-Ser-Ser(p), Calc'd m/z:
1053.39; Obs.: 1053.80
Compound 4 (SRX-61): Thr-Leu-Asp(NHCH2Ph)-Thr-Trp-Ser-Ser-Ser(p), Calc'd
m/z: 1064.42; Obs.: 1064.90
Compound 5: (SRX-62) Thr-Leu-Gly(CH2CH2cyclohexyl)-Thr-Trp-Ser-Ser-Ser(p),
Calc'd m/z: 1027.46; Obs.:
Compound 6: (SRX-63) Thr-Leu-Hph-Thr-Trp-Ser-Ser-Ser(p), Calc'd m/z: 1021.42;
Obs.: 1022.30
Compound 7: (SRX-64) Thr-Leu-Cys(CH2-Ph)-Thr-Trp(3-Me)-Ser-Ser-Ser(p), Calc'd
m/z: 1067.40; Obs.: 1067.80
Compound 8: (SRX-65) Val-Leu-Glu-Leu-Tyr-Trp, Calc'd m/z: 821.43; Obs.: 821.90
Compound 9: (SRX-66) Leu-Asp-Leu-Phe-Phe-Ser, Calc'd m/z: 740.37; Obs.: 740.80
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Compound 10: (SRX-67) Ile-Leu-Asp-Leu-Ser-Tyr, Calc'd m/z: 722.39; Obs.:
722.80
Compound 11: (SRX-68) Ac-Trp-Ser-Ser(p), Calc'd m/z: 500.13; Obs.: 500.15
Compound 12: (SRX-69) Ac-Trp-Ala-Ser(p), Calc'd m/z: 484.14; Obs.: 484.40
Compound 13: (SRX-70) Ac-Trp(5-F)-Ala-Ser(p)-morpholine, Calc'd m/z: 571.18;
Obs.: 571.00
Compound 14: (SRX-72) Ac-Tyr-Trp-Gly, Calc'd m/z: 466.19; Obs.: 466.47
Compound 15: (SRX-36) Thr-Leu-Thr-Trp-Ser-Ser-Ser(p), Calc'd m/z: 860.33;
Obs.:
860.00
Compound 16: (SRX-73) Phe(4-Ph)-Ala-Ser(p)-morpholine, Calc'd m/z: 548.20;
Obs.:
548.00
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