Mini-hepcidin peptides and methods of using thereof

1. An isolated peptide consisting of the following structural formula

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10

wherein

A1 is Asp, Glu, pyroglutamate, Gln, Asn, D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp, Ala, D-Ala, Cys, D-Cys, Phe, D-Phe, Asp or D-Asp, or Dpa or (D)Dpa;

where if Al is Asp or D-Asp, A2 is Cys or D-Cys, if A1 is Phe or D-Phe the n-terminus is optionally attached to a PEG molecule linked to chenodeoxvcholate, ursodeoxvcholate, or palmitoyl, or if A1 is Dpa or (D)Dpa is attached at the n-terminus to palmitoyl,

A2 is Thr, Ser, Val, Ala, D-Thr, D-Ser, D-Val, Tle, Inp, Chg, bhThr, N-MeThr, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or D-Gly;

A3 is His, D-His, Dpa, (D)Dpa, or 2-aminoindane;

A4 is Phe, D-Phe, bhPhe, Dpa, Bip, lNal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;

A5 is Pro, D-Pro, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc;

A6 is Ile, D-Ile, Phg, Chg, Amc, bhIle, Ach, or MeIle;

A7 is Cys, D-Cys, Cys(S-tBut), homoC, Pen, or (D)Pen;

A8 is Ile, D-Ile, Chg, Dpa, bhIle, Ach, or MeIle;

A9 is Phe, Leu, Ile, Tyr, D-Phe, D-Leu, D-He, PheF5, N-MePhe, benzylamide, bhPhe, Dpa, Bip, 1Nal, bhDpa, cyclohexylalanine, Asp, D-Asp, or cysteamide,

wherein Phe or D-Phe are optionally linked at the n-terminus to RA, Asp or D-Asp are optionally linked at the n-terminus to RB, bhPhe is linked to RC,

wherein RA is -CONH-CH2-CH2-S-, or D-Pro linked to Pro-Lys or Pro-Arg, or bhPro linked to Pro linked to Pro-Lys or Pro-Arg, or D-Pro linked to bhPro Lys or bhPro-Arg,

wherein RB is -PEG 11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, or -(PEG 11)-(GPHyp) 10 ,

wherein RC is -D-Pro linked to Pro-Lys or ProArg, or -D-Pro linked to bhPro-Lys or bhPro-Arg; and

A10 is Cys, Ser, Ala, D-Cys, D-Ser, or D-Ala;

wherein the carboxy-terminal amino acid is in amide or carboxy- form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence;

wherein A1, A2, A1 to A2, A10, A9 to A10, or a combination thereof are optionally absent

wherein

bhAsp is 3-aminopentanedioic acid,

Ida is 2,2′-azanediyldiacetic acid,

N-MeAsp is (methylamino)pentanedioic acid,

Tle is L-tert-leucine,

Inp is isonipecotic acid,

Chg is L-α-cyclohexylglycine,

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid,

Dpa is 3,3-diphenyl-L-alanine,

(D)Dpa is 3,3-diphenyl-D-alanine,

bhPhe is (S)-2-amino-4-phenylbutanoic acid,

Bip is L-biphenylalanine,

1Nal is (1-naphthyl)-L-alanine,

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

PheF5 is (S)-2-amino-3-(perfluorophenyl)propanoic acid,

hPhe is (S)-2-amino-4-phenylbutanoic acid,

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid,

Oic is octahydroindole-2-carboxylic acid,

bhPro is L-β-homoproline,

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-5-PhPro is (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid,

Idc is (R)-2-methylindoline,

Phg is L-phenylglycine,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

bhIle is (3R)-3-amino-4-methylhexanoic acid,

Ach is 1-aminocyclohexane-1-carboxylic acid,

MeIle is (3R)-4-methyl-3-(methylamino)hexanoic acid,

Cys(S-tBut) is S-t-Butylthio-L-cysteine,

homoC is L-homocysteine,

Pen is L-Penicillamine, and

(D)Pen is D-Penicillamine GPHyp is Gly-Pro-HydroxyPro.

2. The peptide of claim 1 , wherein A1 is Asp, A2 is Thr, A3 is His, A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys, D-Cys, Cys(S-tBut), homoC, Pen, or (D)Pen, A8 is Ile, A9 is Phe in amide form, and A10 is absent.

3. The peptide of claim 1 , wherein A1 and A2 are absent, A3 is His, A4 is Phe, A5 is Pro, A6 is Ile, A7 is Cys D-Cys, Cys(S-tBut), homoC, Pen, or (D)Pen, A8 is Ile in amide form, and A9and A10 are absent.

4. The peptide of claim 1 , wherein A1 and A2 are absent, A3 is His, A4 is Phe, A 5 is Pro, A6 is Ile, A7 is Cys D-Cys, Cys(S-tBut), homoC, Pen, or (D)Pen, and A8 to A10 are absent, where the C-terminus is in amide form.

5. The peptide of claim 1 , wherein the peptide is a cyclic peptide.

6. The peptide of according to claim 1 , wherein the sequence is retroinverted such that A1 is the C-terminus and A10 is the N-terminus.

7. An isolated peptide consisting of the following structural formula

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10

wherein

A1 is Asp, Glu, pyroglutamate, Gln, Asn, D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp, Ala, D-Ala, Cys, D-Cys, Phe, D-Phe, Asp or D-Asp, or Dpa or (D)Dpa;

where if A1 is Asp or D-Asp, A2 is Cys or D-Cys, if A1 is Phe or D-Phe the n-terminus is optionally attached to a PEG molecule linked to chenodeoxvcholate, ursodeoxvcholate, or palmitoyl, or if A1 is Dpa or (D)Dpa is attached at the n-terminus to palmitoyl,

A2 is Thr, Ser, Val, Ala, D-Thr, D-Ser, D-Val, Tle, Inp, Chg, N-MeThr, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or D-Gly;

A3 is His, D-His, Dpa, (D)Dpa, or 2-aminoindane;

A4 is Phe, D-Phe, bhPhe, Dpa, Bip, lNal, bhDpa, Amc, PheF5, hPhe, Igl, or cyclohexylalanine;

A5 is Pro, D-Pro, Oic, bhPro, trans-4-PhPro, cis-4-PhPro, cis-5-PhPro, or Idc;

A6 is Ile, D-Ile, Phg, Chg, Amc, bhIle, Ach, or MeIle;

A7 is Cys, D-Cys, Cys(S-tBut), homoC, Pen, (D)Pen, or Dap(AcBr);

A8 is Ile, D-Ile, Chg, Dpa, bhIle, Ach, or MeIle;

A9 is Phe, Leu, Ile, Tyr, D-Phe, D-Leu, D-He, PheF5, N-MePhe, benzylamide, bhPhe, Dpa, Bip, 1Nal, bhDpa, cyclohexylalanine, Asp, D-Asp, or cysteamide,

wherein Phe or D-Phe are optionally linked at the n-terminus to RA, Asp or D-Asp are optionally linked at the n-terminus to RB, bhPhe is linked to RC,

wherein RA is -CONH-CH2-CH2-S-, or D-Pro linked to Pro-Lys or Pro-Arg, or bhPro linked to Pro linked to Pro-Lys or Pro-Arg, or D-Pro linked to bhPro Lys or bhPro-Arg,

wherein RB is -PEG 11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, or -(PEG 11)-(GPHyp) 10 ,

wherein RC is -D-Pro linked to Pro-Lys or ProArg, or -D-Pro linked to bhPro-Lys or bhPro-Arg; and

A10 is Cys, Ser, Ala, D-Cys, D-Ser, or D-Ala;

wherein the carboxy-terminal amino acid is in amide or carboxy- form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and

wherein A1, A2, A1 to A2, A10, A9 to A10, or a combination thereof are optionally absent,

wherein the peptide has -CONH 2 -CH 2 -CH 2 -S, Chenodeoxycholate-(PEG11)-, Ursodeoxycholate-(PEG11)-, Palmitoyl-(PEG11)-, 2(Palmitoyl)-diaminopropionic acid-(PEG11)-, -PEG11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, -(PEG11)-(Gly-Pro-hydroxyproline)10, -(D)P-PK, -(D)P-PR, -bhProPK, -bhProPR, (D)P-bhProK, or -(D)P-bhProR at the N-terminus, C-terminus, or both,

wherein

bhAsp is 3-aminopentanedioic acid,

Ida is 2,2′-azanediyldiacetic acid,

N-MeAsp is (methylamino)pentanedioic acid,

Tle is L-tert-leucine,

Inp is isonipecotic acid,

Chg is L-a-cyclohexylglycine,

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid,

Dpa is 3,3-diphenyl-L-alanine,

(D)Dpa is 3,3-diphenyl-D-alanine,

bhPhe is (S)-2-amino-4-phenylbutanoic acid,

Bip is L-biphenylalanine,

1Nal is (1-naphthyl)-L-alanine,

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

PheF5 is (S)-2-amino-3-(perfluorophenyl)propanoic acid,

hPhe is (S)-2-amino-4-phenylbutanoic acid,

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid,

Oic is octahydroindole-2-carboxylic acid,

bhPro is L-β-homoproline,

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-5-PhPro is (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid,

Idc is (R)-2-methylindoline,

Phg is L-phenylglycine,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

bhIle is (3R)-3-amino-4-methylhexanoic acid,

Ach is 1-aminocyclohexane-1-carboxylic acid,

MeIle is (3R)-4-methyl-3-(methylamino)hexanoic acid,

Cys(S-tBut) is S-t-Butylthio-L-cysteine,

homoC is L-homocysteine,

Pen is L-Penicillamine, and

(D)Pen is D-Penicillamine GPHyp is Gly-Pro-HydroxyPro.

8. A peptide selected from the group consisting of

H-F-P-I-C-I,

H-F-P-I-C-I-F,

D-T-H-F-P-I-C-I-D-T-H-F-P-I-C-I-F,

D-T-H-F-P-I-A-I-F-C,

D-T-H-A-P-I-C-I-F,

D-T-H-A-P-I-CS-S-tBut-I-F,

D-T-H-F-P-I-C-I-F,

D-T-H-F-P-I-homoC-I-F,

D-T-H-F-P-I-Pen-I-F,

D-T-H-F-P-I-(D)Pen-I-F,

C-D-T-H-F-P-I-C-I-F,

D-Tle-H-Phg-Oic-Chg-C-Chg-F,

D-Tle-H-P-Oic-Chg-C-Chg-F,

(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D,

(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H,

R2-(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D,

R3-(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D,

(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D-R6,

R4-(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D,

R5-(D)F-(D)I-(D)C-(D)I-(D)P-(D)F-(D)H-(D)T-(D)D,

D-T-H-bhPhe-P-I-C-I-F,

D-T-H-Dpa-P-I-C-I-F,

D-T-H-Bip-P-I-C-I-F,

D-T-H-1Nal-P-I-C-I-F,

D-T-H-bhDpa-P-I-C-I-F,

D-T-H-F-P-I-C-I-bhPhe,

D-T-H-F-P-I-C-I-Dpa,

D-T-H-F-P-I-C-I-Bip,

D-T-H-F-P-I-C-I-1Nal,

D-T-H-F-P-I-C-I-bhDpa,

D-T-H-Dpa-P-I-C-I-Dpa,

D-Dpa-P-I-C-I-F,

D-Dpa-P-I-C-I-Dpa,

D-T-H-Dpa-P-R-C-R-Dpa,

D-T-H-Dpa-P-(D)R-C-(D)R-Dpa,

D-T-H-Dpa-Oic-I-C-I-F,

D-T-H-Dpa-Oic-I-C-I-Dpa,

D-T-H-Dpa-P-C-C-C-Dpa,

D-T-H-F-P-I-C-I-F-R8,

D-T-H-F-P-I-C-I-F-R9,

D-T-H-F-P-I-C-I-F-R10,

D-T-H-F-P-I-C-I-F-R11,

D-T-H-F-P-I-C-I-F-R12,

D-T-H-F-P-I-C-I-F-R13,

D-T-H-F-P-I-C-I-bhPhe-R8,

D-T-H-F-P-I-C-I-bhPhe-R9,

D-T-H-F-P-I-C-I-bhPhe-R12,

D-T-H-F-P-I-C-I-bhPhe-R13,

C-Inp-(D)Dpa-Amc-R-Amc-Inp-Dpa-Cysteamide,

C-P-(D)Dpa-Amc-R-Amc-Inp-Dpa-Cysteamide,

C-(D)P-(D)Dpa-Amc-R -Amc-Inp-Dpa-Cysteamide, and

C-G-(D)Dpa-Amc-R-Amc-Inp-Dpa-Cysteamide,

wherein R1 =-CONH 2 -CH 2 -CH 2 -S, R2 =Chenodeoxycholate-(PEG11)-, R3 =Ursodeoxycholate-(PEG11)-, R4=Palmitoyl-(PEG11)-, R5=2(Palmitoyl)- diaminopropionic acid-(PEG11)- , R6=-PEG11 -GYIPEAPRDGQAYVRKDGEWVLLSTFL, R7=-(PEG11)- (Gly-Pro-hydroxyproline)10,R8=-(D)P-PK, R9=-(D)P-PR, R10=-bhProPK, R11=-bhProPR, R12=-(D)P-bhProK, and R13=-(D)P-bhProR,

wherein

bhAsp is 3-aminopentanedioic acid,

Ida is 2,2′-azanediyldiacetic acid,

N-MeAsp is (methylamino)pentanedioic acid,

Tle is L-tert-leucine,

Inp is isonipecotic acid,

Chg is L-α-cyclohexylglycine,

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid,

Dpa is 3,3-diphenyl-L-alanine,

(D)Dpa is 3,3-diphenyl-D-alanine,

bhPhe is (S)-2-amino-4-phenylbutanoic acid,

Bip is L-biphenylalanine,

1Nal is (1-naphthyl)-L-alanine,

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

PheF5 is (S)-2-amino-3-(perfluorophenyl)propanoic acid,

hPhe is (S)-2-amino-4-phenylbutanoic acid,

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid,

Oic is octahydroindole-2-carboxylic acid,

bhPro is L-β-homoproline,

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-5-PhPro is (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid,

Idc is (R)-2-methylindoline,

Phg is L-phenylglycine,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

bhIle is (3R)-3-amino-4-methylhexanoic acid,

Ach is 1-aminocyclohexane-1-carboxylic acid,

MeIle is (3R)-4-methyl-3-(methylamino)hexanoic acid,

Cys(S-tBut) is S-t-Butylthio-L-cysteine,

homoC is L-homocysteine,

Pen is L-Penicillamine, and

(D)Pen is D-Penicillamine.

9. The peptide according to claim 1 , wherein the peptide exhibits hepcidin activity.

10. The peptide according to claim 1 , wherein the peptide binds ferroportin.

11. A composition which comprises at least one peptide according to claim 1 .

12. A method of binding a ferroportin or inducing ferroportin internalization and degradation which comprises contacting the ferroportin with at least one peptide according to claim 1 .

13. A method of treating a disease of iron metabolism in a subject which comprises administering at least one peptide according to claim 1 to the subject.

14. The method of claim 13 , wherein the disease of iron metabolism is an iron overload disease.

15. A kit comprising at least one peptide according to claim 1 packaged together with a reagent, a device, instructional material, or a combination thereof.

16. A complex comprising at least one peptide according to claim 1 bound to a ferroportin or an antibody.

17. An isolated peptide consisting of the following structural formula

A1-A2-A3-A4-A5-A6-A7-A8-A9-A10

wherein

A1 is Asp, Glu, pyroglutamate, Gln, Asn, D-Asp, D-Glu, D-pyroglutamate, D-Gln, D-Asn, bhAsp, Ida, or N-MeAsp, Ala, D-Ala, Cys, D-Cys, Phe, D-Phe, Asp or D-Asp, or Dpa or (D)Dpa;

where if A1 is Asp or D-Asp, A2 is Cys or D-Cys, if A1 is Phe or D-Phe the n-terminus is optionally attached to a PEG molecule linked to chenodeoxvcholate, ursodeoxvcholate, or palmitoyl, or if A1 is Dpa or (D)Dpa is attached at the n-terminus to palmitoyl,

A2 is Thr, Ser, Val, Ala, D-Thr, D-Ser, D-Val, Tle, Inp, Chg, N-MeThr, D-Ala, Cys, D-Cys, Pro, D-Pro, Gly, or D-Gly;

A3 is His;

A4 is Phe;

A5 is Pro;

A6 is Ile;

A7 is Cys, D-Cys, Cys(S-tBut), or homoC;

A8 is Ile;

A9 is Phe, Leu, Ile, Tyr, D-Phe, D-Leu, D-He, PheF5, N-MePhe, benzylamide, bhPhe, Dpa, Bip, 1Nal, bhDpa, cyclohexylalanine, Asp, D-Asp, or cysteamide,

wherein Phe or D-Phe are optionally linked at the n-terminus to RA, Asp or D-Asp are optionally linked at the n-terminus to RB, bhPhe is linked to RC,

wherein RA is -CONH-CH2-CH2-S-, or D-Pro linked to Pro-Lys or Pro-Arg, or bhPro linked to Pro linked to Pro-Lys or Pro-Arg, or D-Pro linked to bhPro Lys or bhPro-Arg,

wherein RB is -PEG 11-GYIPEAPRDGQAYVRKDGEWVLLSTFL, or -(PEG 11)-(GPHyp) 10 ,

wherein RC is -D-Pro linked to Pro-Lys or ProArg, or -D-Pro linked to bhPro-Lys or bhPro-Arg; and

A10 is Cys, Ser, Ala, D-Cys, D-Ser, or D-Ala;

wherein the carboxy-terminal amino acid is in amide or carboxy- form;

wherein at least one sulfhydryl amino acid is present as one of the amino acids in the sequence; and

wherein A1, A2, A1 to A2, A10, A9 to A10, or a combination thereof are optionally absent

wherein

bhAsp is 3-aminopentanedioic acid,

Ida is 2,2′-azanediyldiacetic acid,

N-MeAsp is (methylamino)pentanedioic acid,

Tle is L-tert-leucine,

Inp is isonipecotic acid,

Chg is L-α-cyclohexylglycine,

N-MeThr is (2S)-3-hydroxy-2-(methylamino)butanoic acid,

Dpa is 3,3-diphenyl-L-alanine,

(D)Dpa is 3,3-diphenyl-D-alanine,

bhPhe is (S)-2-amino-4-phenylbutanoic acid,

Bip is L-biphenylalanine,

1Nal is (1-naphthyl)-L-alanine,

bhDpa is (S)-3-Amino-4,4-diphenylbutanoic acid,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

PheF5 is (S)-2-amino-3-(perfluorophenyl)propanoic acid,

hPhe is (S)-2-amino-4-phenylbutanoic acid,

Igl is (S)-2-amino-2-(2,3-dihydro-1H-inden-2-yl)acetic acid,

Oic is octahydroindole-2-carboxylic acid,

bhPro is L-β-homoproline,

trans-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-4-PhPro is (2S,4S)-4-phenylpyrrolidine-2-carboxylic acid,

cis-5-PhPro is (2S,5R)-5-phenylpyrrolidine-2-carboxylic acid,

Idc is (R)-2-methylindoline,

Phg is L-phenylglycine,

Amc is 4-(aminomethyl)cyclohexane carboxylic acid,

bhIle is (3R)-3-amino-4-methylhexanoic acid,

Ach is 1-aminocyclohexane-l-carboxylic acid,

MeIle is (3R)-4-methyl-3-(methylamino)hexanoic acid,

Cys(S-tBut) is S-t-Butylthio-L-cysteine,

homoC is L-homocysteine,

Pen is L-Penicillamine, and

(D)Pen is D-Penicillamine.

18. The isolated peptide of claim 1 , wherein the peptide binds to ferroportin with an EC 50 of about 1000 nM or less.