Eunice J. York

Bradykinin antagonists : present progress and future prospects

Bradykinin (BK) antagonist peptides have been powerful tools for delineating roles of kinins in both normal and pathological physiology and offer promise of drug development for a variety of inflammatory conditions and cancers. At the present time, potent peptide antagonists are available that are either specific for BK B1 or B2 receptors, or are effective on both receptor classes. Non-peptide BK B2 antagonists are now being announced and are under investigation in several companies. The best peptide B1-B2 peptide antagonist is stable against all kininases, is orally available, and has a very long lifetime in vivo. Certain dimers of this antagonist, as well as several smaller molecules, are active against several cancers, both in vitro and in vivo.

The Glu 2- ... Arg 10+ side-chain interaction in the C-peptide helix of ribonuclease A.

Previous studies have identified Lys 1, Glu 2, and His 12 as the charged residues responsible for the pH-dependent stability of the helix formed by the isolated C-peptide (residues 1-13 of ribonuclease A). Here we examine whether the helix-stabilizing behavior of Glu 2- results from a Glu 2- ... Arg 10+ interaction, which is known to be present in the crystal structure of ribonuclease A. The general approach is to measure the helix content of C-peptide analogs as a function of three variables: pH (titration of ionizing groups), amino acid identity (substitution test), and NaCl concentration (ion screening test). In order to interpret the results of residue replacement, several factors in addition to the putative Glu 2- ... Arg 10+ interaction have been studied: intrinsic helix-forming tendencies of amino acids; interactions of charged residues with the alpha-helix macrodipole; and helix-lengthening effects. The results provide strong evidence that the Glu 2- ... Arg 10+ interaction is linked to helix formation and contributes to the stability of the isolated C-peptide helix. NMR evidence supports these conclusions and suggests that this interaction also acts as the N-terminal helix stop signal. The implications of this work for protein folding and stability are discussed.

Minimum structure opioids—Dipeptide and tripeptide analogs of the enkephalins

Through a systematic reduction of peptide structure, a series of 25 tripeptide and 5 dipeptide amide and alcohol analogs of enkephalin were synthesized and assayed in vitro on the stimulated guinea pig ileum. Tyr-Pro-Phe-NH2, Tyr-D-Ala-Phe-NH2, Tyr-D-Ala-Phe-ol and Tyr-D-Phe-Phe-NH2 had 20-25% the potency of Met-enkephalin. Four aromatic alkylamides of the dipeptide Tyr-D-Ala were made with benzylamine, phenethylamine, phenylpropylamine and phenylbutylamine. All had full naloxone reversible enkephalin-like activity in the ileum assay. Tyr-D-Ala-phenylpropylamide has about 80% the potency of Met-enkephalin in vitro, and is equipotent with Tyr-D-Ala-Gly-Phe-Met-NH2 in producing analgesia in mice after intraventricular administration. Tyr-D-Phe-NH2 is the smallest peptide to show full intrinsic enkephalin-like activity in vitro, although its potency is very low.

Bradykinin antagonists as new drugs for prostate cancer

Bradykinin (BK) is an autocrine growth factor for lung and prostate cancers. BK also facilitates tumor extension by increasing tissue permeability and stimulating angiogenesis. Peptide BK antagonists are in development as potential new drugs for lung cancer. Newer nonpeptide BK antagonists have even higher potency against lung cancer, in vitro and in vivo. These compounds have now been applied to the study of prostate cancers, and have been found to be effective. Prostate cancer cell line PC3 is derived from a late-stage, hormone-independent, metastatic tumor; its growth is difficult to inhibit. Our established BK antagonists, while less effective against this line of prostate cancer in xenografts in nude mice than against lung cancer, are active and have led the way to development of new peptide and nonpeptide agents for prostate cancer. In addition to inhibiting cancer cell growth directly, they inhibit angiogenesis mediated by vascular endothelial growth factor, and inhibit increased tissue permeability mediated by membrane metalloproteases in these tumors. This class of compounds offers hope for development of new drugs for refractory prostate cancer.

Position effect on apparent helical propensities in the C-peptide helix

A search has been made for position effects on apparent helix propensities when another amino acid is substituted for alanine in the C-peptide helix of ribonuclease A. Three internal alanine residues (Ala4, Ala5, Ala6) are used as sites for substitution. Five amino acids, Glu, His, Arg, Lys and Phe, are substituted singly in individual peptides at each of these three positions, and the pH profiles of helix content for the substituted peptides have been determined. The effect of using an acetyl or a succinyl amino-terminal-blocking group has also been determined for each substitution. A strong position effect is found at Ala5: the helix content of the substituted peptide is significantly higher for substitution at position 5 than at positions 4 or 6 in almost all cases. The reason for the position 5 effect is unknown. The results also show that electrostatic interactions often influence substitution experiments, and they provide data on the variability of substitution experiments made with a natural sequence peptide.

Metabolism-resistant bradykinin antagonists: development and applications.

Abstract Bradykinin plays many roles in normal and pathological physiology, but rapid enzymatic degradation made elucidation of its functions extremely difficult. Development of effective degradation-resistant antagonists made it possible to delineate these roles and to open the way for development of new drugs to control pathology due to excess production of bradykinin. Presently available peptide bradykinin antagonists are extremely potent, are completely resistant to enzymatic degradation, and are orally available. Non-peptide bradykinin antagonists have also been discovered. Development of bradykinin antagonists as drugs for cancer, inflammation and trauma is anticipated.

Potent tetrapeptide enkephalins

Abstract Small peptides with opiate-like activity have generally had structures closely resembling that of the opioid pentapeptide enkephalin: Tyr-Gly-Gly-Phe-Met-COOH. Single deletions of any one of the amino acids has been demonstrated to reduce opiate activity drastically. In this work we show that the potency losses resulting from the removal of glycine 3 can be fully attenuated by substitution of D-alanine in position two and derivatization of the acid to the amide. This tetrapeptide (Tyr-DAla-Phe-Met-NH 2 ) has narcotic activity similar to the parent pentapeptide in the guinea pig ileum and mouse tail-flick tests. This enhanced potency, relative to the unaltered tetrapeptide, is theorized to arise from increased resistance to enzymatic destruction. the data presented show that a five amino acid sequence is not mandatory for the expression of opiate activity in enkephalin analogs.

Lysine substitution increases mu-selectivity of potent di- and tri-peptide enkephalin analogs

A general trend toward increased mu-selectivity has been reported in two classes of minimal structure enkephalin analogs which contain the essential structural and functional information for potent enkephalin-like biological activity. A single Lys residue added to the amino terminal of several tripeptide amides, and to the potent dipeptide amide Tyr-DAla-phenylpropylamide, causes a further increase in mu-selectivity as determined in the stimulated guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, and in receptor binding studies in homogenates of guinea pig brain. The effect of Lys substitution was a significant decrease in the delta-related MVD potencies.

An NMR Conformational Analysis of Cyclic Bradykinin Mimics. Evidence for a β-Turn

Abstract A detailed NMR study is carried out in acetonitrile/water solutions on three novel cyclic bradykinin antagonist analogues, BKM-824, BKM-870, and BKM-872, to examine their solution structures, and to correlate the structures with bradykinin antagonist and anti-cancer activities. The solution structures of the cyclic peptides are correlated with the structural data for known linear bradykinin antagonists. The sequences are: BKM-824 c[Ava-Igl-Ser-DF5F-Oic- Arg] where Ava is 5-aminovaleric acid, Igl is α-(2-indanyl)glycine, F5F is pentafluorophenylalanine, and Oic is (2S,3aS,7aS)-octahydroindole-2-carboxylic acid; BKM-870; c[DArg-Arg-Add-DF5F-Oic-Arg] where Add is 12-aminododecanoic acid; and BKM-872; c[DArg-Arg-Eac-Ser-DF5F-Oic-Arg] where Eac is 6-aminocaproic acid. BKM-824 was the only peptide within this series that possessed a discernable solution structure. The NMR data indicate the presence of a type I β-turn between residues F5F4 and Ava1, a C-terminal-like end. Molecular dynamics calculations show that a type I β-turn from DF5F4 to Ava1 does exist although the turn was somewhat distorted. This result differs from the structures seen in linear bradykinin antagonists, which usually possess a type II II′β-turn at the C-terminal end and the presence of a defined turn is correlated with bradykinin antagonist activity. There is no solution structure for BKM-870 and BKM-872 but a correlation between the primary sequence Argterminal-DArg1-Arg2-long chain aliphatic amino acid and anti-cancer activity is evident.

An nmr conformational analysis of a synthetic peptide Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from the New World Centruroides noxius 2 (Cn2) scorpion toxin: comparison of the structure with those of the Centruroides scorpion toxins.

The solution structure of a synthetic peptide, Cn2(1-15)NH2-S-S-acetyl-Cn2(52-66)NH2 from toxin 2 (Cn2) of the New World scorpion Centruroides noxius was determined using nmr and molecular dynamics calculations. The peptide has no significant secondary structure such as an alpha-helix or a beta-sheet, yet it has a fixed conformation for the first chain. The backbone secondary structure involving residues 6-12 in this peptide shows an excellent overlap with the structures of natural neurotoxins from Centruroides sculpturatus Ewing. Residues 6-9 form a distorted type I beta-turn and residues 10-12 form a gamma-turn. As residues 7-10 in the Centruroides toxins correspond to one of the regions of highest sequence variability, it may account for the species specificity and/or selectivity of toxic action. The conformation of this region evidently plays an important role in receptor recognition and in binding to the neutralizing monoclonal antibody BCF2 raised against the intact toxin.