US2019142963A1PendingUtilityA1
Fgf21 c-terminal peptide optimization
Individually held — no corporate assignee on recordPriority: Apr 15, 2016Filed: Apr 14, 2017Published: May 16, 2019
Est. expiryApr 15, 2036(~9.7 yrs left)· nominal 20-yr term from priority
A61P 3/04A61K 38/03A61K 38/26A61K 38/28A61P 3/10A61K 47/65A61K 47/66A61K 38/1796C07K 14/50
41
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Claims
Abstract
Disclosed herein are modified C-terminal fragments of FGF21 optimized for binding to Klotho β or antagonizing FGF21 activity. FGF21 peptides modified to comprise modifications to the C-terminal amino acid sequence are disclosed that have enhanced activity at the FGF21 receptor. Additionally, conjugates formed between the optimized FGF21 peptide fragments and insulin like peptides or nuclear hormone receptor ligands are provided.
Claims
exact text as granted — not AI-modified1 . A peptide exhibiting antagonist activity against FGF21 binding to Klotho β, said peptide comprising an amino acid sequence of X 1 X 2 X 3 X 4 X 5 SX 7 DPX 10 X 11 X 12 VX 14 GX 16 X 17 X 18 X 19 RSPSX 24 X 25 X 26 (SEQ ID NO: 235),
wherein
X 1 is Pro or absent;
X 2 is Pro or Leu;
X 3 is Asp or Glu;
X 4 is Val or Thr;
X 5 is Gly, Asp, Phe, Leu or Ser;
X 7 is Ser or Met;
X 10 is Leu or Phe;
X 11 is Ser or Gly;
X 12 is Met or Leu;
X 14 is absent or Thr;
X 16 is Pro, Leu, Arg, Glu, or Gly;
X 17 is Ser or Glu;
X 18 is Gln or Ala;
X 19 is Gly or Val;
X 24 is Tyr or Phe;
X 25 is Ala or Glu; and
X 26 is Ala.
2 . The peptide of claim 1 wherein said peptide comprises a sequence selected from the group consisting of
I) LETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 188),
PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191),
PPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 180),
PLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 179),
PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234)
PPDVGSMDPF GLVGR SQGRS PSFEA (SEQ ID NO: 237),
PPDVF SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 238),
PPDVL SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239),
PPDVS SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 240), and
PPDVG SSDPF GLVGP SQGRS PSFEA (SEQ ID NO: 241), or
II) a peptide that differs from SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 180, SEQ ID NO: 179, SEQ ID NO: 234, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240 or SEQ ID NO: 241 by one to two amino acid substitutions selected from positions 1, 2, 5, 7, 11, 14, 15, 16, 17, 18 and 19.
3 . The peptide of claim 2 wherein said peptide is fused to the carboxy terminus of a polypeptide selected from the group consisting of SEQ ID NO: 194, SEQ ID NO:
195 and SEQ ID NO: 196 or a peptide that differs from SEQ ID NO: 194, SEQ ID NO: 195 or SEQ ID NO: 196 by 1 to 3 amino acid substitutions.
4 . (canceled)
5 . A modified FGF21 peptide wherein said modified peptide differs from SEQ ID NO: 173 by a substitution of the C-terminal amino acid with alanine; and
one or more of the following modifications: i) one or more substitutions selected from the group consisting of G161L, G161F, G161S, S163M, L166F, S167G, M168L, P171R, Y179F, and A180E (based on the numbering of the mature FGF21 peptide of SEQ ID NO: 173); or ii) one or more substitutions selected from the group consisting of A31C, G43C, L98D, L100K, N121D, and D127K; or iii) substitution of the native amino acid at position 167 and/or 175 with the corresponding D-isomer of said native amino acid; or iv) substitution of the C-terminal 25 amino acids of SEQ ID NO: 173 with the 25 amino acid sequence of SEQ ID NO: 188; or v) any combination of i) and ii), or any combination of i), ii) and iii), or any combination of ii) and iv).
6 - 7 . (canceled)
8 . The modified FGF21 peptide of claim 5 wherein said modified peptide differs from SEQ ID NO: 173 by each of the following substitutions: S163M, L166F, S167G and M168L, or said modified peptide differs from SEQ ID NO: 173 by each of the following substitutions: S163M, L166F, S167G, M168L, Y179F, and A180E.
9 . (canceled)
10 . The modified FGF21 peptide of claim 8 further comprising the substitution of P171R.
11 . The modified FGF21 peptide of claim 5 wherein the peptide consists of a peptide selected from the group consisting of
i) SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251 and SEQ ID NO:252 or
ii) SEQ ID NO: 192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208 and SEQ ID NO: 209, or
iii) a modified sequence of SEQ ID NO: 192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208 and SEQ ID NO: 209, wherein SEQ ID NO: 192, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208 or SEQ ID NO: 209 are modified to comprise one or more substitutions selected from the group consisting of A31C, G43C, L98D, L100K, N121D, and D127K.
12 - 14 . (canceled)
15 . A pharmaceutical composition comprising a modified FGF21 peptide of claim 5 and a pharmaceutically acceptable carrier, diluent, or excipient.
16 . A method of reducing weight gain or inducing weight loss in a patient in need thereof, comprising administering to said patient in need thereof a pharmaceutical composition of claim 15 in an amount effective to reduce weight gain or induce weight loss.
17 . A method of treating diabetes, comprising administering to a patient in need thereof a pharmaceutical composition of claim 15 in an amount effective to lower blood glucose levels.
18 . A peptide conjugate comprising the general formula:
Q-L-Y wherein Q is a bioactive peptide selected from the group consisting of an insulin peptide, a glucagon peptide, FGF1, FGF2, and a nuclear hormone; Y is a peptide comprising a sequence selected from the group consisting of LETDSMDPFGLVTGLEAVRSPSFEA (SEQ ID NO: 188), PPDVGSSDPLSMVGPSQGRSPSYAA (SEQ ID NO: 191), PPDVGSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 180), PLETDSMDPFGLVGPSQGRSPSFEA (SEQ ID NO: 179), PDVGSMDPFGLVTGLEAVRSPSYAA (SEQ ID NO: 234) PPDVG SMDPF GLVGR SQGRS PSFEA (SEQ ID NO: 237), PPDVF SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 238), PPDVL SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 239), PPDVS SMDPF GLVGP SQGRS PSFEA (SEQ ID NO: 240), and PPDVG SSDPF GLVGP SQGRS PSFEA (SEQ ID NO: 241); and L is a linking group or a bond.
19 . The conjugate of claim 18 wherein
i) Q is a glucagon peptide comprising a sequence from the group consisting of HX 1 QGTFTSDKSKYLDX 2 RAAQDFVQWLMDT (SEQ ID NO: 202),
(SEQ ID NO: 197)
X 3 AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,
(SEQ ID NO: 198)
X 4 AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,
(SEQ ID NO: 199)
X 5 AQGTFTSDKSKYLDERAAQDFVQWLLEGGPSSGAPPPS,
(SEQ ID NO: 200)
X 6 AQGTFTSDKSKYLDERAAQDFVQWLLDAGPSSGAPPPS
and
(SEQ ID NO: 201)
X 7 AQGTFTSDKSKYLDERAAQDFVQWLLEAGPSSGAPPPS,
wherein
X 1 and X 2 are both Aib;
X 3 is Acetyl D-Tyr;
X 4 is Acetyl D-His;
X 5 is Acetyl D-thio Ala, and
X 6 and X 7 are both acetyl-D-Tyr, or
ii) Q is selected from the group consisting of estradiol and derivatives thereof, estrone and derivatives thereof, testosterone and derivatives thereof, and cortisol and derivatives thereof; or
iii) Q is a compound having the general structure
wherein
R 15 is C 1 -C 4 alkyl, —CH 2 (pyridazinone), —CH 2 (OH)(phenyl)F, —CH(OH)CH 3 , halo or H;
R 20 is halo, CH 3 or H;
R 21 is halo, CH 3 or H;
R 22 is H, OH, halo, —CH 2 (OH)(C 6 aryl)F, or C 1 -C 4 alkyl; and
R 23 is —CH 2 CH(NH 2 )COOH, —OCH 2 COOH, —NHC(O)COOH, —CH 2 COOH —NHC(O)CH 2 COOH, —CH 2 CH 2 COOH, or —OCH 2 PO 3 2− , or
iv) Q is a compound having the general structure
wherein
R 15 is C 1 -C 4 alkyl, —CH(OH)CH 3 , I or H
R 20 is I, Br, CH 3 or H;
R 21 is I, Br, CH 3 or H;
R 22 is H, OH, I, or C 1 -C 4 alkyl; and
R 23 is —CH 2 CH(NH 2 )COOH, —OCH 2 COOH, —NHC(O)COOH, —CH 2 COOH, —NHC(O)CH 2 COOH, —CH 2 CH 2 COOH, or —OCH 2 PO 3 2− , or
v) Q is a compound of the general structure of Formula I:
wherein
R 20 , R 21 and R 22 are independently selected from the group consisting of H, OH, halo and C 1 -C 4 alkyl; and
R 15 is halo or H, or
vi) Q is selected from the group consisting of thyroxine T4 (3,5,3′,5′-tetra-iodothyronine), and 3,5,3′-triiodo L-thyronine; or
vii) Q is a ligand that activates the peroxisome proliferator-activated receptors (PPAR) when in an unbound state.
20 - 25 . (canceled)
26 . The conjugate of claim 18 wherein (Q) is an insulin peptide comprising an A chain and a B chain wherein
i) said A chain comprises a sequence
GIVX 4 X 5 CCX 8 X 9 X 10 CX 12 LX 14 X 15 LX 17 X 18 YCX 21 -R 53 (SEQ ID NO: 19), and said B chain comprises a sequence R 62 -X 25 LCGX 29 X 30 LVX 33 X 34 LYLVCGX 41 X 42 GFX 45 (SEQ ID NO: 20), wherein
X 4 is glutamic acid or aspartic acid;
X 5 is glutamine or glutamic acid
X 8 is histidine, threonine or phenylalanine;
X 9 is serine, arginine, lysine, ornithine or alanine;
X 10 is isoleucine or serine;
X 12 is serine or aspartic acid;
X 14 is tyrosine, arginine, lysine, ornithine or alanine;
X 15 is glutamine, glutamic acid, arginine, alanine, lysine, ornithine or leucine;
X 17 is glutamic acid, aspartic acid, asparagine, lysine, ornithine or glutamine;
X 18 is methionine, asparagine, glutamine, aspartic acid, glutamic acid or threonine;
X 21 is selected from the group consisting of alanine, glycine, serine, valine, threonine, isoleucine, leucine, glutamine, glutamic acid, asparagine, aspartic acid, histidine, tryptophan, tyrosine, and methionine;
X 25 is histidine or threonine;
X 29 is selected from the group consisting of alanine, glycine and serine;
X 30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
X 33 is selected from the group consisting of aspartic acid and glutamic acid;
X 34 is selected from the group consisting of alanine and threonine;
X 41 is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
X 42 is selected from the group consisting of alanine, ornithine, lysine and arginine;
X 45 is tyrosine or phenylalanine;
R 62 is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and
R 53 is COOH or CONH 2 , or
ii) said A chain comprises the sequence GIVEQCCX 8 X 9 ICSLYQLENYCX 21 -R 53 (SEQ ID NO: 73) said B chain comprises the sequence R 62 -X 25 LCGX 29 X 30 LVX 33 X 34 LYLVCGX 41 X 42 GFX 45 (SEQ ID NO: 20), wherein
X 8 is histidine or threonine;
X 9 is serine, lysine, or alanine;
X 21 is alanine, glycine or asparagine;
X 25 is histidine or threonine;
X 29 is selected from the group consisting of alanine, glycine and serine;
X 30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
X 33 is selected from the group consisting of aspartic acid and glutamic acid;
X 34 is selected from the group consisting of alanine and threonine;
X 41 is selected from the group consisting of glutamic acid, aspartic acid or asparagine;
X 42 is selected from the group consisting of alanine, ornithine, lysine and arginine;
X 45 is tyrosine or phenylalanine;
R 62 is selected from the group consisting of FVNQ (SEQ ID NO: 12), a tripeptide valine-asparagine-glutamine, a dipeptide asparagine-glutamine, glutamine and an N-terminal amine; and
R 53 is COOH or CONH 2 , or
iii) said A chain comprises a sequence GIVDECCX 8 X 9 SCDLRRLEMX 19 CX 21 -R 53 (SEQ ID NO: 74) and said B chain comprises a sequence R 62 -X 25 LCGAX 30 LVDALYLVCGDX 42 GFY (SEQ ID NO: 75), wherein
X 8 is phenylalanine or histidine;
X 9 is arginine, ornithine or alanine;
X 19 is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine;
X 21 is alanine or asparagine;
X 25 is histidine or threonine;
X 30 is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteic acid and cysteic acid;
X 42 is selected from the group consisting of alanine ornithine and arginine; and R 53 is COOH or CONH 2 ;
R 62 is selected from the group consisting of AYRPSE (SEQ ID NO: 14), FVNQ (SEQ ID NO: 12), PGPE (SEQ ID NO: 11), a tripeptide glycine-proline-glutamic acid, a tripeptide valine-asparagine-glutamine, a dipeptide proline-glutamic acid, a dipeptide asparagine-glutamine, glutamine, glutamic acid and an N-terminal amine; and
R 53 is COOH or CONH 2 , or
iv) said A chain comprises a sequence GIVDECCX 8 X 9 SCDLRRLEMX 19 CX 21 -R 53 (SEQ ID NO: 74) and the B chain sequence comprises the sequence FVKQX 25 LCGSHLVEALYLVCGERGFF-R 63 (SEQ ID NO: 147), or FVNQX 25 LCGSHLVEALYLVCGERGFF-R 63 (SEQ ID NO: 148), wherein
X 8 is phenylalanine or histidine;
X 9 is arginine, ornithine or alanine;
X 19 is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine;
X 25 is selected from the group consisting of histidine and threonine; and
R 63 is selected from the group consisting of YTX 28 KT (SEQ ID NO: 149), YTKPT (SEQ ID NO: 150), YTX 28 K (SEQ ID NO: 152), YTKP (SEQ ID NO: 151), YTPK (SEQ ID NO: 70), YTX 28 , YT, Y and a bond, wherein X 28 is proline, aspartic acid or glutamic acid, or
v) said A chain comprises a sequence GIVDECCX 8 X 9 SCDLRRLEMX 19 CX 21 —R 53 (SEQ ID NO: 74) and the B chain sequence comprises the sequence FVKQX 25 LCGSHLVEALYLVCGERGFFYTEKT (SEQ ID NO: 162), FVNQX 25 LCGSHLVEALYLVCGERGFFYTDKT (SEQ ID NO: 164), FVNQX 25 LCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 165) or FVNQX 25 LCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 161) wherein X 8 is phenylalanine or histidine;
X 9 is arginine, ornithine or alanine;
X 19 is tyrosine, 4-methoxy-phenylalanine or 4-amino-phenylalanine;
X 25 is selected from the group consisting of histidine and threonine; or
vi) said A chain comprises a sequence GIVEQCCTSICSLYQLENYCN-R 53 (SEQ ID NO: 1) and said B chain comprises a sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2), wherein R 53 is COOH or CONH 2 .
27 - 31 . (canceled)
32 . The conjugate of claim 26 , wherein L is stable in vivo, hydrolyzable in vivo, or metastable in vivo.
33 . The conjugate of claim 32 , wherein L comprises an ether moiety, or an amide moiety, an ester moiety, an acid-labile moiety, a reduction-labile moiety, an enzyme-labile moiety, a hydrazone moiety, a disulfide moiety, or a cathepsin-cleavable moiety.
34 . The conjugate of claim 18 , further comprising an acyl group or alkyl group covalently linked to an amino acid side chain of said conjugate.
35 . A pharmaceutical composition comprising a conjugate of claim 18 , or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
36 . The peptide of claim 5 for use in treating diabetes.
37 . The peptide of claim 5 for use in reducing weight gain or inducing weight loss in a patient in need thereof.Join the waitlist — get patent alerts
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