Methods and compositions for inhibiting ADAM 9 biological activities
Provided are ADAM9 modulating peptides and methods for using the same to modulate ADAM9 biological activities in vitro and/or in vivo, to inhibit ADAM9 biological activities associated with diseases or disorders in subjects, to decrease inflammation, and to inhibit undesirable cellular proliferation, fibrosis, and angiogenesis. In some embodiments, the ADAM9 modulating peptides include modifications of one or more amino acids of the human ADAM9 prodomain amino acid sequence, and in some embodiments the ADAM9 modulating peptides include other modifications such as but not limited to the addition of PEG groups.
1. A peptide comprising SEQ ID NO: 3, wherein relative to the amino acid sequence set forth in SEQ ID NO: 2, the peptide includes one or more amino acid substitutions and/or chemical modifications at amino acid position(s) 85, 104, and 146, wherein the substitution(s) are selected from substitution of the cysteine with serine, alanine, or glycine.
2. The peptide of claim 1 , wherein relative to the amino acid sequence set forth in SEQ ID NO:2, the peptide further includes one or more amino acid substitutions and/or modifications at amino acid position(s) 6, 7, 24, 26, 27, 61, 62, 137, 138, 160, 161, 162, 163, and/or 164 such that the peptide is less inhibitory towards meprin, is less sensitive to furin cleavage, or a combination thereof, as compared to a peptide without the one or more amino acid substitutions and/or modifications.
3. The peptide of claim 1 , wherein, as compared to SEQ ID NO: 2, the amino acid sequence comprises:
a chemical modification of one, two, or all three of cysteines 85, 104, and 146,
a substitution of cysteine 85 to serine, alanine, or glycine and a chemical modification of one or two of cysteines 104 and 146;
a substitution of cysteine 104 to serine, alanine, or glycine and a chemical modification of one or two of cysteines 85 and 146;
a substitution of cysteine 146 to serine, alanine, or glycine and a chemical modification of one or two of cysteines 85 and 104; or
a substitution of any two of cysteines 85, 104, and 146 to serine, alanine, or glycine with a chemical modification of the cysteine which is not substituted at positions 85, 104, or 146.
4. The peptide of claim 3 , wherein relative to the amino acid sequence set forth in SEQ ID NO:2, the peptide further comprises (vi) a substitution of arginine 24 to another amino acid; (vii) a substitution of arginine 26 to another amino acid; (viii) a substitution of arginine 27 to another amino acid; or (ix) any combination of (vi)-(viii).
5. The peptide of claim 4 , wherein
(vi) the arginine at position 24 is substituted with alanine, serine, glycine, or lysine;
(vii) the arginine at position 26 is substituted with alanine, serine, glycine, or lysine;
(viii) the arginine at position 27 is substituted with alanine, serine, glycine, or lysine;
or any combination thereof.
6. The peptide of claim 1 wherein, as compared to SEQ ID NO: 2, the peptide comprises
(i) a substitution at each of amino acids 85, 104, and 146, wherein the substitution(s) are selected from substitution of the cysteine with serine, alanine, or glycine; or
(ii) a substitution at amino acid 85, wherein the substitution is selected from substitution of the cysteine with serine, alanine, or glycine, and a chemical modification at amino acid 104 or amino acid 146 or both amino acids 104 and 146; or
(iii) a substitution at amino acid 104, wherein the substitution is selected from substitution of the cysteine with serine, alanine, or glycine, and a chemical modification at amino acid 85 or amino acid 146 or both amino acids 85 and 146; or
(iv) a substitution at amino acid 146, wherein the substitution is selected from substitution of the cysteine with serine, alanine, or glycine, and a chemical modification at amino acid 85 or amino acid 104 or both amino acids 85 and 104.
7. The peptide of claim 6 , wherein relative to the amino acid sequence set forth in SEQ ID NO:2, the amino acid sequence comprises (xvii) a substitution at amino acid 24 and a substitution at amino acid 26; or (xviii) a substitution at amino acid 24 and a substitution at amino acid 27; or (ix) a substitution at amino acid 26 and a substitution at amino acid 27; or (xx) a substitution at amino acid 24; a substitution at amino acid 26 and a substitution at amino acid 27; or (xxi) a substitution at one, two, or all three of amino acids 161, 163, and 164; or (xxii) a substitution at one, two, or all three of amino acids 162-164; or (xxiii) a substitution at one or both of amino acids 61 and 62; or (xxiv) a substitution at one or both of amino acids 137 and 138; or (xxv) any combination of (xvii)-(xxiv).
8. The peptide of claim 7 , wherein the amino acid sequence comprises (xvii) the amino acid at position 24 and the amino acid at position 26 are independently selected from alanine, serine, glycine, or lysine; or (xviii) the amino acid at position 24 and the amino acid at position 27 are independently selected from alanine, serine, glycine, or lysine; or (ix) the amino acid at position 26 and the amino acid at position 27 are independently selected from alanine, serine, glycine, or lysine; or (xx) the amino acid at position 24, the amino acid at position 26, and the amino acid at position 27 are independently selected from alanine, serine, glycine, or lysine; or (xxi) the amino acid at one, two, or all three of positions 161, 163, and 164 asparagine, glycine, alanine, or serine; or (xxii) the amino acid at position at one, two, or all three of positions 162-164 is asparagine, glycine, alanine, or serine; or (xxiii) the amino acid at positions 61 and 62 is asparagine, alanine, serine, or glycine; or (xxiv) the amino acid at position at one or both of positions 137 and 138 is asparagine, alanine, serine, or glycine; or (xxv) any combination of (xvii)-(xxiv).
9. The peptide of claim 1 , wherein, as compared to SEQ ID NO:2, the peptide comprises: (i) an alanine at amino acid 26 and serines at amino acids 85, 104, and 146; or (ii) an alanine at amino acid 24 and serines at amino acids 85, 104, and 146; or (iii) an alanine at amino acid 27 and serines at amino acids 85, 104, and 146; or (iv) serines at amino acids 85, 104, and 146; or (v) an alanine at amino acid 26, serines at amino acids 85, 104, and 146, and an alanine at amino acid 62; or (vi) a glycine at amino acid 27 and serines at amino acids 85, 104, and 146; or (vii) a serine at amino acid 27 and serines at amino acids 85, 104, and 146; or (viii) an alanine at amino acid 27; serines at amino acids 85, 104, and 146; and a serine at amino acid 138; or (ix) an alanine at amino acid 27, serines at amino acids 85, 104, and 146, and asparagines at amino acids 161 and 163; or (x) an alanine at amino acid 26, serines at amino acids 85, 104, and 146, and an addition of a GSGSC (SEQ ID NO: 27) pentapeptide C-terminal to amino acid 174 of SEQ ID NO: 3; or (xi) an alanine, glycine, or serine at amino acid 27, serines at amino acids 85, 104, and 146, and an addition of a GSCGS (SEQ ID NO: 26) pentapeptide N-terminal to amino acid 1 of SEQ ID NO: 3; or (xii) an alanine, glycine, or serine at amino acid 27, serines at amino acids 85, 104, and 146, and an addition of a GSGSC (SEQ ID NO: 27) pentapeptide C-terminal to amino acid 174 of SEQ ID NO: 3.
10. The peptide of claim 1 , wherein the sequence comprises a cysteine at one, two, or all three of amino acid positions 85, 104, and 146, and one, two, or all three cysteine(s) is/are chemically modified at a sulfhydryl group.
11. The peptide of claim 10 , wherein the sulfhydryl group(s) is/are chemically modified by addition of a maleimide ester, an α-halocarbonyl, a thiosulfonate, or any combination thereof.
12. The peptide of claim 1 , wherein (a) the amino acid at position 146 is pegylated cysteine and/or (b) the peptide further comprises a pegylated cysteine added to the N-terminus, to the C-terminus, or both, and/or (c) the peptide further comprises SEQ ID NO: 26 and/or SEQ ID NO: 27 having a pegylated cysteine.
13. The peptide of claim 1 , wherein one or more of cysteines at positions 85, 104, and/or 146 comprises a chemical modification with a maleimide ester.
14. The peptide of claim 1 , wherein one or more of cysteines at positions 85, 104, and/or 146 comprises a chemical modification resulting from reacting the one or more cysteines with a disulfide.
15. A pharmaceutical composition comprising the peptide of claim 1 .
16. The peptide of claim 1 , wherein the peptide is conjugated to a therapeutic moiety, a diagnostic moiety, a detectable moiety, or any combination thereof.
17. The peptide of claim 16 , wherein the moiety is a chemotherapeutic agent.
18. A peptide comprising an amino acid sequence having at least 87% sequence identity to the full-length sequence of SEQ ID NO: 13, wherein relative to the amino acid sequence set forth in SEQ ID NO: 2, the peptide includes an amino acid substitution and/or modification at amino acid position(s) 85, 104, and 146.
19. A peptide comprising an amino acid sequence having at least 95% sequence identity to the full-length sequence of SEQ ID NO: 13, wherein relative to the amino acid sequence set forth in SEQ ID NO: 2, the peptide includes an amino acid substitution and/or modification at amino acid position(s) 85, 104, and 146.
20. A peptide comprising 100% sequence identity to SEQ ID NO: 13 over its full length.
21. A peptide fragment comprising amino acids 7-174 of SEQ ID NO: 3, wherein as compared to SEQ ID NO: 2, the amino acid sequence of the fragment comprises an alanine at amino acid 27; serines at amino acids 85, 104, and 146; and the fragment does not comprise amino acids 1-6 of SEQ ID NO: 3.