Constrained helical peptides and methods of making same

Details Constrained helical peptides and methods of making same Constrained helical peptides and methods of making same

1997-11-05

2001-08-07

Jones, Dwayne C. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C514S013800, C514S014800, C514S015800, C514S016700, C514S017400, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000

Reexamination Certificate

active

06271198

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the conformational constraint of peptides. In particular, the invention relates to constraining peptides to an &agr;-helical conformation. This invention also relates to the rational design and preparation of HIV vaccines based on HIV gp41 polypeptide sequences. This invention further relates to improved methods for HIV infection diagnosis and immunogens which induce antibodies useful in the diagnostic methods.
A variety of methods for stabilizing &agr;-helical peptides have been described previously. Addition of trifluoroethanolor hexafluoroisopropanol has frequently been used to stabilize &agr;-helices in aqueous solution. Dimerization of &agr;-helices at hydrophobic interfaces has also provided exogenous stabilization. Short &agr;-helical peptides have been stabilized by incorporating groups at the termini to stabilize the intrinsic helix dipole. Naturally occurring capping motifs as well as organic templates have been used to stabilize &agr;-helices by end-nucleation. Several non-covalent side chain constraints have been investigated for &agr;-helix stabilization, including hydrophobic interactions, salt bridges, and metal ion chelation by both natural and unnatural amino acids.
Finally, &agr;-helices have been stabilized by covalent side chain tethers. Chorev et al.,
Biochemistry,
30: 5968-5974 (1991), Osapay et al.,
J. Am. Chem. Soc.,
112: 6046-6051 (1990), Osapay et al.,
J. Am. Chem. Soc.,
114: 6966-6973 (1990),Bracken et al.,
J. Am. Chem. Soc.,
116: 6431-6432 (1994), and Houston et al.,
J. Peptide Science,
1: 274-282 (1995) described the stabilization of &agr;-helices by side chain to side chain lactamization. Ravi et al.,
J. Am. Chem. Soc.,
105: 105-109 (1983) and Jackson et al.,
J. Am. Chem. Soc.,
113: 9391-9392 (1991) described the constraint of peptides by disulfide bonds between residues. The naturally occurring peptide apamin has been used as a scaffold for the presentation of &agr;-helical peptide sequences constrained in helical conformation by disulfide bonds to scaffold cysteine residues.
Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus identified as the human immunodeficiency virus (HIV). There have been intense efforts to develop a vaccine that induces a protective immune response based on induction of antibodies or cellular responses. Recent efforts have used subunit vaccines where an HIV protein, rather than attenuated or killed virus, is used as the immunogen in the vaccine for safety reasons. Subunit vaccines generally include gp120, the portion of the HIV envelope protein which is on the surface of the virus.
The HIV envelope protein has been extensively described, and the amino acid and nucleic acid sequences encoding HIV envelope from a number of HIV strains are known (Myers, G. et al., 1992. Human Retroviruses and AIDS. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, Los Alamos, N.M.). The HIV envelope protein is a glycoprotein of about 160 kd (gp160) which is anchored in the membrane bilayer at its carboxyl terminal region. The N-terminal segment, gp120, protrudes into the aqueous environment surrounding the virion and the C-terminal segment, gp41, spans the membrane. Via a host-cell mediated process, gp160 is cleaved to form gp120 and the integral membrane protein gp41. As there is no covalent attachment between gp120 and gp41, free gp120 is sometimes released from the surface of virions and infected cells.
gp120 has been the object of intensive investigation as a vaccine candidate for subunit vaccines, as the viral protein which is most likely to be accessible to immune attack. At present, clinical trials using gp120 MN strain are underway.
However, effective vaccines based on gp120 or another HIV protein for protection against additional strains of HIV are still being sought to prevent the spread of this disease.
SUMMARY OF THE INVENTION
The invention provides a method for constructing a constrained helical peptide comprising the steps of: (1) synthesizing a peptide, wherein the peptide comprises a sequence of eight amino acid residues, wherein the sequence of eight amino acid residues has a first terminal residue and a second terminal residue, wherein the first terminal residue and the second terminal residue flank an internal sequence of six amino acid residues, and wherein the first terminal residue has a side chain containing an amide bond-forming substituent and the second terminal residue has a side chain containing an amide bond-forming substituent; (2) providing a difunctional linker having a first functional group capable of forming an amide linkage with the side chain amide bond-forming substituent of the first terminal residue and having a second functional group capable of forming an amide linkage with the side chain amide bond-forming substituent of the second terminal residue; and (3) cyclizing the peptide by reacting the side chain amide bond-forming substituent of the first terminal residue with the first functional group of the difunctional linker to form an amide linkage and reacting the side chain amide bond-forming substituent of the second terminal residue with the second functional group of the difunctional linker to form an amide linkage, yielding a constrained helical peptide.
The invention also provides a method for constructing a constrained helical peptide comprising the steps of: (1) synthesizing a peptide, wherein the peptide comprises a sequence of eight amino acid residues, wherein the sequence of eight amino acid residues has a first terminal residue and a second terminal residue, wherein the first terminal residue and the second terminal residue flank an internal sequence of six amino acid residues, wherein the first terminal residue has a side chain containing an amide bond-forming substituent and the second terminal residue has a side chain containing an amide bond-forming substituent, and wherein the side chain amide bond-forming substituent of the first terminal residue is protected with a first protecting group and the side chain amide bond-forming substituent of the second terminal residue is protected with a second protecting group such that the first protecting group and the second protecting group are differentially removable; (2) removing the first protecting group such that the side chain amide bond-forming substituent of the first terminal residue is deprotected and the side chain amide bond-forming substituent of the second terminal residue is not deprotected; (3) providing a difunctional linker having a first functional group capable of forming an amide linkage with the side chain amide bond-forming substituent of the first terminal residue and having a second functional group capable of forming an amide linkage with the side chain amide bond-forming substituent of the second terminal residue; (4) reacting the peptide with the difunctional linker to form an amide linkage between the first functional group of the difunctional linker and the side chain amide bond-forming substituent of the first terminal residue; (5) removing the second protecting group to deprotect the side chain amide bond-forming substituent of the second terminal residue; and (6) cyclizing the peptide by intramolecularly reacting the side chain amide bond-forming substituent of the second terminal residue with the second functional group of the difunctional linker to form an amide linkage and yield a constrained helical peptide.
The invention further provides a method for constructing a constrained helical peptide, comprising the steps of: (a) synthesizing a peptide, wherein the peptide comprises a sequence of eight amino acid residues, wherein the sequence of eight amino acid residues has a first terminal residue and a second terminal residue, wherein the first terminal residue and the second terminal residue flank an internal sequence of six amino acid residues, wherein the first terminal residue has a side chain containing an amide bond-forming substituent and the second terminal residue has sid

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