Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

In vitro and in vivo activities of reduced-size antagonists of luteinizing hormone-releasing hormone.

A novel series of octapeptide LHRH antagonists was designed on the basis of the structure of the (2-9) fragment of a LHRH agonist. By adopting a systematic SAR study, we were able to improve first the in vitro activity and then the in vivo LH suppression, raising them up to the range of the decapeptide antagonists NalGlu (51) and A-75998 (50), resulting in A-76154 (49). The octapeptide antagonist A-76154 is the most potent reduced-size LHRH antagonist reported. It suppresses LH in the castrated rat by over 80% for a period of 4 h following sc bolus administration of 30 micrograms/kg.

Differential orientation of a GnRH agonist and antagonist in the pituitary GnRH receptor.

In the present study we have used a high affinity photoaffinity label (PAL) agonist (pGlu-His-Trp-Ser-125I-iodoTyr-D-Lys(para-N3-Benzoyl)-Leu-Arg-P roNHEt) and a PAL antagonist (NAcD2Nal-D4ClPhe-D3Pal-Ser-125I-iodo-NMeTyr-D-L ys(para-N3-Benzoyl)Leu-Lys(Isp) - Pro-D-Ala-NH2) to covalently label the GnRH receptor. Rat pituitary membranes were incubated 3 h with either the radioiodinated agonist or antagonist in the dark, exposed to UV light, then electrophoresed in SDS. The PAL agonist and antagonist labeled broad bands (estimated molecular weight [M(r)] 46K-60K). Labeling by either PAL agonist or antagonist was displaced by unlabeled agonist or antagonist, indicating that the agonist and antagonist bind to the same molecule. The broad band, believed to reflect differential glycosylation, was divided into six sections corresponding M(r) to 60K, 56K, 52K, 48K, 46K and 44K for the agonist and 62K, 58K, 54K, 52K, 48K and 45K for the antagonist; these were electroeluted with recovery > 80% based on radioactivity. Each could be re-electrophoresed to the same location from which it was eluted. Other eluted samples were treated with trypsin. The M(r) of the samples labeled with the agonist PAL were shifted to M(r) 48K, 42K, 40K, 37K, 35K and 33K by proteolysis. The observation that each section shifted approximately the same M(r) after trypsin treatment suggests that the backbone of the labeled proteins in each gel section is identical. Samples labeled with the antagonist PAL were shifted to M(r) < 10,000 in all cases. These data indicate that the agonist and antagonist PALs bind to different regions of the GnRH receptor and, therefore, are likely oriented differently with respect to the receptor and support the view that different strategies should be used for the design of agonists and antagonists.

Effect of N-methyl substitution of the peptide bonds in luteinizing hormone-releasing hormone agonists.

Each peptide bond in leuprolide (1), deslorelin (13), and nafarelin (24) was separately substituted with N-methyl. The synthesized compounds were tested for in vitro receptor binding, LH release, and stability against chymotrypsin and intestinal degradation. The NMe-Ser4 (30), NMe-Leu7 (33), and Sar10 (35) analogues of nafarelin had pD2 values 2-, 20-, 9-fold higher than their respective parent. All the other N-methyl agonists were less active. For the first time, conversion of LHRH agonists to antagonists was observed as a result of N-methyl substitution in the peptide backbone. [NMe-Phe2,DLeu6,Pro9NHEt]LHRH (4), [NMe-1Nal3,DLeu6,Pro9NHEt]LHRH (6), [NMe-His2,DTrp6,Pro9NHEt]LHRH (14), [NMe-Phe2,DNal6]LHRH (27), and [D2Nal6,NMe-Arg8]LHRH (34) exhibited antagonist responses. Substitutions of NMe-1Nal3, NMe-Ser4, or NMe-Tyr5 in leuprolide rendered the 3-4 peptide bond in these compounds completely stable to chymotrypsin. Examination of the three-dimensional structure of leuprolide when bound to the active site of chymotrypsin, reveals the NH's of residues 3 and 5 are involved in hydrogen bond interactions with the enzyme. N-Methylation at these positions is not only disrupting the hydrogen bond interactions, but is also sterically preventing the substrate from fitting in the enzyme's active site. All the compounds in the leuprolide series were also tested against intestinal degradation using an in vitro rat jejunum sac assay. In this model the pattern of stabilization was similar, but not identical, to that against chymotrypsin. The pharmacokinetics of all the analogues in the leuprolide series and of several others in the deslorelin and nafarelin series were determined. The clearance values of all the three NMe-Tyr5 analogues, 8, 20, and 31 were lower than their respective parents. These slower clearances suggest lower rates of metabolism.

Stabilization of the N-terminal residues of luteinizing hormone-releasing hormone agonists and the effect on pharmacokinetics.

To stabilize leuprolide (1) against chymotrypsin and intestinal degradation several agonists of LHRH (2-12), modified at position 1, 2, or 3 and/or containing N-alpha-methyl at positions 1, 2, or 4, were synthesized by SPPS. These agonists were tested in vitro for (a) rat pituitary LHRH receptor binding, (b) LH release from rat pituitary cells, (c) stability against chymotrypsin, and (d) stability against rat intestinal degradation. The clearances of the compounds in the rat were determined using a RIA. Complete stabilization against chymotrypsin (t1/2) and lumenal degradation (T1/2) was achieved with substitution of NMe-Ser4 in leuprolide; however, with an increase in clearance. Substitution with 1-Nal3 increased both t1/2 and T1/2, while substitution with NAc-Sar1 increased only T1/2. [NAcSar1,NMeSer4,D-Trp6,Pro9NHEt]LHRH (12), the doubly stabilized analogue, was tested in the rat by both iv and id administrations, and its bioavailabilities were measured. No significant improvement in id absorption over leuprolide was observed.