Covalent attachment of ligands to nucleophilic proteins guided by non-covalent
View Patent ↗A covalently reactive ligand analogue (CAL) of formula (1): wherein, L 1 . . . L x . . . L m are components defining a ligand determinant, L x is a component unit of the ligand determinant selected from the group consisting of an amino acid residue, sugar residue, a fatty acid residue and a nucleotide, L′ is a functional group of L x , Y″ is atom, covalent bond or linker, Y′ is an optional charged or neutral group Y is a covalently reactive electrophilic group that reacts specifically with a receptor that binds to said ligand determinant, and n is an integer from 1 to 1000 m is an integer from 1 to 30.
1. A ligand analog that binds covalently to a nucleophilic receptor (NuR), wherein said ligand analog has the formula
L−E
wherein L is a ligand that binds noncovalently to a NuR, wherein L comprises a continuous or discontinuous epitope that binds noncovalently to said NuR, and E is an electrophilic group conjugated to a functional group of an amino acid side chain of L having the formula
Y−Y′−Y″−
wherein
Y is an electrophilic group that reacts covalently with NuR,
Y′ is a charged or neutral group, or is absent, and
Y″ is a linker, covalent bond or atom.
2. The ligand analog of claim 1 , wherein Y″ is a linker.
3. The ligand analog of claim 1 , wherein Y′ is a charged or neutral group.
4. The ligand analog of claim 1 , wherein the side chain functional group is selected from the group consisting of —NH 2 , —COOH, —SH, and —OH.
5. The ligand analog of claim 1 , wherein the amino acid side chain is a side chain of alanine, leucine, isoleucine, valine, methionine, cysteine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, serine, threonine or proline.
6. The ligand analog of claim 1 , wherein Y″ is a suberoyl, pimeroyl, aminohexanoyl, aminoacetyl, poly(ethylene oxide), α, ω-dicarboxyl or acetylenedicarboxyl group.
7. The ligand analog of claim 1 , wherein Y′ is a charged group selected from amino(4-amidinophenyl)methyl, 2,6-diaminopentyl, 1-amino-4-guanidinobutyl, 1-amino-3-carboxylpropyl and amino(4-carboxylphenyl)methyl.
8. The ligand analog of claim 1 , wherein Y′ is a neutral group selected from amino(phenyl)methyl, 1-amino-2-phenylethyl, 1-amino-2-methylbutyl, aminomethyl, 2-aminoethyl and 1-aminocyclohexyl.
9. The ligand analog of claim 1 , wherein Y comprises an electrophilic atom Z attached to one or more substituents R.
10. The ligand analog of claim 9 , wherein substituent R is an electron withdrawing group.
11. The ligand analog of claim 10 , wherein R is selected from phenoxyl, 4-nitrophenoxyl, 4-cyanophenoxyl, pentachlorophenoxyl, 4-nitrophenyl, 4-cyanophenyl, cyanomethoxyl, trifluoromethoxyl and 4-nitrophenylmercaptyl.
12. The ligand analog of claim 9 , wherein R is an electron donating group.
13. The ligand analog of claim 12 , wherein R is selected from 4-methoxyphenoxyl, 4-methylphenoxyl, methoxymethoxyl, 4-methoxyphenyl, 4-methylphenyl, methoxymethyl and 4-methoxyphenylmercaptyl.
14. The ligand analog of claim 9 , wherein Z is a phosphorus, carbon, boron or vanadium atom.
15. The ligand analog of claim 14 , wherein Y has the formula:
wherein R 1 is an oxygen or sulfur atom, and R 2 and R 3 are independently hydrogen, oxygen, fluorine, chlorine, bromine, iodine, sulfur, hydroxyl, sulfhydryl, amino, alkoxy or phenoxy.
16. The ligand analog of claim 9 , wherein R is glyoxylpeptide or aminoacylpeptide.
17. The ligand analog of claim 1 , wherein L comprises an epitope from a protein selected from the group consisting of vasoactive intestinal peptide (VIP), Factor VIII, β-amyloid peptide, CD4, extracellular domain of epidermal growth factor erceptor (EGFR), human immunodeficiency virus (HIV) gp120; HIV gp160, Lexl repressor, gag, pol, hepatitis B surface antigen, diptheria toxin, C. tetani toxin, C. botulinum toxin, and pertussis toxin.
18. The ligand analog of claim 1 , wherein NuR is an antibody.
19. The ligand analog of claim 18 , wherein the antibody is a member of the group consisting of alloantibodies to Factor VIII, red blood cell antigens, platelet antigens, kidney antigens, heart antigens and lung antigens.
20. The ligand analog of claim 1 , wherein NuR is gp120.
21. The ligand analog of claim 20 , wherein L-E is electrophilic gp120.
22. The ligand analog of claim 1 , wherein:
the amino acid side chain is a side chain of alanine, leucine, isoleucine, valine, methionine, cysteine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, serine, threonine or proline;
Y″ is a suberoyl, pimeroyl, aminohexanoyl, aminoacetyl, poly(ethylene oxide), α, ω-dicarboxyl or acetylenedicarboxyl group; and
Y′ is a amino(4-amidinophenyl)methyl, 2,6-diaminopentyl, 1-amino-4-guanidinobutyl, 1-amino-3-carboxylpropyl, amino(4-carboxylphenyl)methyl, amino(phenyl)methyl, 1-amino-2-phenylethyl, 1-amino-2-methylbutyl, aminomethyl, 2-aminoethyl or 1-aminocyclohexyl group.
23. The ligand analog of claim 22 , wherein L comprises vasoactive intestinal peptide (VIP), Factor VIII, β-amyloid peptide, CD4, extracellular domain of epidermal growth factor receptor (EGFR), human immunodeficiency virus (HIV) gp120; HIV gp160, Lexl repressor, gag, pol, hepatitis B surface antigen, diptheria toxin, C. tetani toxin, C. botulinum toxin, or pertussis toxin.
24. An isolated covalent complex comprising:
a ligand analog according to claim 1 that binds noncovalently to a nucleophilic receptor; and
the nucleophilic receptor,
wherein the ligand analog and the nucleophilic receptor are covalently bound to each other.
25. A pharmaceutical composition, comprising:
a ligand analog according to claim 1 ; and
a pharmaceutically acceptable carrier.
26. The ligand analog of claim 5 , wherein the side chain functional group of L is a side chain functional group of lysine, aspartic acid, glutamic acid, cysteine, serine, threonine, or tyrosine.
27. The ligand analog of claim 1 , wherein L comprises an epitope from a protein selected from the group consisting of vasoactive intestinal peptide (VIP), Factor VIII, β-amyloid peptide, CD4, extracellular domain of epidermal growth factor receptor (EGFR), and human immunodeficiency virus (HIV) gp120.
28. The ligand analog of claim 22 , wherein L comprises an epitope from a protein selected from the group consisting of vasoactive intestinal peptide (VIP), Factor VIII, β-amyloid peptide, CD4, extracellular domain of epidermal growth factor receptor (EGFR), and human immunodeficiency virus (HIV) gp120.