The secondary structure of a protein is characterized by regular elements such as alpha (α) helices, beta (β) sheets and irregular elements such as β bulges, tight turns and random coils. Tight turns are one of the three “classical” secondary structures with approximately one-third of all residues in globular proteins comprised in turns that serve to reverse the direction of polypeptide chain. Gamma turns is the second most characterized and commonly found tight turn in proteins after β-turns. γ-turn comprise of 3.4% of amino acids and are defined as 3 residue turns with H-bond between the carbonyl oxygen of residue (i) and the hydrogen of the amide group of residue (i+2). Search and analysis of 54 proteins by Miner-White, E J, et. al. (J. Mol. Bio. 204, 1983, pp. 777-82) indicated nine proteins to have eleven classic γ-turns, and these eleven turns had mean φ and ψ values at residue i+1 of +75.0 and −64, respectively. Seven of these eleven turns are involved in formation of β-hairpins which produce a reversal in the peptide chain.
Gamma turns are present at ligand binding sites or active sites. It has further been postulated by Milner-White, E J, et. al. (J. Mol. Biol. 204, 1990, pp. 385-397) that inverse gamma turns may function as intermediates in folding and thus stabilizing β-strands before they become β-sheets. Recently, gamma-turns have attracted attention through studies that describe incorporation of peptide secondary structure mimetics into small bioactive peptides in development of stable, effective and selective receptor ligands Alkorta, I, et al. (J. Mol. Model, 2, 1996, pp. 16-25). Proteins having a turn like conformation include angiotensin, thermolysin, tobacco necrosis virus protein, flavodoxin, proteinase A, α-lytic protease and other small cyclic peptides.
Angiotensin II (Ang II) is an important peptide regulating cardiovascular hemodynamics and cardiovascular structure. Most of the known effects of Ang II in adult tissues are attributable to the angiotensin II type I (AT1) receptor although it is well known to bind both the AT1 and AT2 receptor. The AT1 and AT2 receptors have differential pharmacological and biochemical properties and appear to exert opposite effects in terms of cardiovascular hemodynamics and cell growth. In addition, the renin-angiotensin and nitric oxide-generating systems appear to interact in the regulation of cardiovascular function. Ang II stimulates angiogenesis and increases micro-vessel density. In nature, angiotensin II is produced by the action of angiotensin converting enzyme on angiotensinogen; the C-terminal-His-Leu is cleaved.
Angiotensin II (Ang II), an octapeptide having sequence, i.e. Asp-Arg-Val-Tyr-Ile-His-Pro-Phe (SEQ ID NO: 4), is an endogenous substrate for AT1 and AT2 receptors. Ang II adopts a turn-like conformation centered at Val3-Tyr4-Ile5 residue while interacting with the angiotensin receptors.
A reverse turn like conformation in angiotensin II has been mimicked by incorporation of thio-linkage, benzodiazepine-derivative, 3-aminobenzoicacid, etc. with enhanced affinity for AT2 receptors. Hallberg et al (J. Med. Chem., 2004, 47 (4), pp 859-70) have disclosed the design and synthesis of three angiotensin II analogues comprising a benzodiazepine-based γ-turn-like scaffold. However, introduction of such chemically synthesized compounds or their derivatives in a peptide sequence of angiotensin II may cause a change in the configuration of the protein and consequently affect the binding ability of the protein. Further issues relating to the cytotoxicity and delivery of the analogues may also arise.
There is a large body of documented work relating to design and synthesis of peptide analogues of peptides such as angiotensin II synthesized by incorporation of chemically synthesized moieties to confer a mimetic gamma turn in the bioactive peptide. However, none of the prior arts suggest or teach incorporation of a peptide based hydrogen bonding directing reverse-turn scaffold.
The present inventors have earlier synthesized a gamma turn mimetic foldamer in tripeptides and tetra-peptides (P. K. Baruah, et al, J. Org. Chem., 2007, 72, 636). The foldamer comprises proline and 3-amino-5-bromo-2-methoxy benzoic acid as alternating subunits that forms repeating γ-turn conformations. However, the γ-turn conformation was demonstrated in only non-polar organic solvent chloroform and their applications were not demonstrated. None of the prior art provides the conformational studies under aqueous solutions essential for physiological functions.
Several therapeutic peptides and peptide analogues designed do not have ideal pharmacokinetics, bioavailability and toxicity profiles. Therefore, chemically synthesized analogues of angiotensin II and other such bioactive peptides have not been used to their complete potential as a therapeutic agent for treatment of neurological or other disorders. Therefore, there is a need in the art to provide a peptide analogue comprising incorporation of peptide-based hydrogen bonding directing reverse-turn scaffold stable under aqueous conditions in a peptide sequence having better bioavailability, low cytotoxicity and better pharmacokinetic profiles.