Bernard Lammek

Structure―activity relationships of novel peptide agonists of the human bradykinin B2 receptor

The nonapeptide bradykinin (BK) is involved in the genesis of inflammation, edema and in pain mediation. As such, much effort has gone into the development of peptide/non-peptide antagonists to counteract these processes. However, there is an increasing awareness of the potential value of chemically stable BK agonists in the treatment of diabetes and cardiovascular diseases. In this study, a structure-activity relationship study of BK was performed to develop potent and stable peptide mimetics active at the human B2 receptors (hB2R). Twenty-three analogues were produced with substitutions at positions 1, 3, 5, 7, 8 and/or 9 of BK. In vitro binding (on transiently transfected HEK-293T cells) and biological activities (vasomotricity tests on human umbilical veins, MAPK assays on HEK-293T cells) of novel BK peptide derivatives at hB2R were determined alongside with previously reported synthetic agonists (e.g. RMP-7, JMV1609, FR190997). Some peptides were also tested in vivo in rats and rabbits using blood pressure assays. Two compounds, [Hyp(3), Thi(5), Cha(8)]-BK and [Hyp(3), Thi(5), (N)Chg(7), Thi(8)]-BK, exhibited equivalent (or even greater) in vitro affinities and potencies to BK at the naturally expressed and recombinant hB2R. Their potency and duration of action in vivo were highly superior to BK, thus inferring that they can withstand intravascular proteolysis. These novel compounds show promise as candidates for investigating the pharmacology of BK receptors and developing potential therapeutical applications.

Origin of the positive 225–230 nm circular dichroism band in proteins: Its application to conformational analysis

The 225-230 nm circular dichroism band found in many disulfide-containing proteins and peptides is sensitive to environmental changes. This band is assigned to the disulfide bond, the conformation of which influences both the intensity and lambda max of the band. This property can be used to monitor subtle conformation changes observed in many polypeptides. Examples using the alpha-neurotoxins of elapid venoms and neurohypophyseal hormones are discussed.

The Multi-Leu Peptide Inhibitor Discriminates Between PACE4 and Furin And Exhibits Antiproliferative Effects On Prostate Cancer Cells

The proprotein convertases (PCs) play an important role in protein precursor activation through processing at paired basic residues. However, significant substrate cleavage redundancy has been reported between PCs. The question remains whether specific PC inhibitors can be designed. This study describes the identification of the sequence LLLLRVKR, named Multi-Leu (ML)-peptide, that displayed a 20-fold selectivity on PACE4 over furin, two enzymes with similar structural characteristics. We have previously demonstrated that PACE4 plays an important role in prostate cancer and could be a druggable target. The present study demonstrates that the ML-peptide significantly reduced the proliferation of DU145 and LNCaP prostate cancer-derived cell lines and induced G0/G1 cell cycle arrest. However, the ML-peptide must enter the cell to inhibit proliferation. It is concluded that peptide-based inhibitors can yield specific PC inhibitors and that the ML-peptide is an important lead compound that could potentially have applications in prostate cancer.

Potentiometric and spectroscopic studies of the Cu(II) complexes of Ala-Arg8-vasopressin and oxytocin : two vasopressin-like peptides

The results are reported of a potentiometric and spectroscopic study of the H+ and Cu2+ complexes of Ala-Arg8-vasopressin (Ala-AVP) and oxytocin at 25 degrees C and an ionic strength of 0.10 mol dm-3 (KNO3). The coordination chemistry of oxytocin and Cu(II) has been shown to be virtually identical to that of Arg8-vasotocin, forming unusually stable complexes with four nitrogen coordination (4N complexes) below pH 7. Spectroscopic evidence suggests weak interaction between Cu(II) and the sulphur atom of the -Cys6- residue in the 2N species (pH congruent to 6) but this is absent in the 4N complex. Evidence is also presented for perturbation of electronic transitions within the aromatic ring of the Tyr residue by Cu(II). While the physiological potency of Ala-AVP is very high, its coordination chemistry differs significantly from that of Arg8-vasopressin. With Cu(II) it forms complexes of similar stability to those with tetraglycine, demonstrating that the addition of an alanyl residue to the amino-terminal of the peptide destroys the conformation which is particularly favorable for rapid 4N coordination.

Effects of bradykinin and prostaglandin inhibition on systemic and regional hemodynamics in conscious normotensive rats.

These experiments were designed to study the effects of inhibition of endogenous bradykinin and/or prostaglandins on systemic and regional hemodynamics in conscious normotensive rats, using the radioactive microsphere technique. Administration of either a bradykinin antagonist (50 micrograms/min for 5 min) or indomethacin (10 mg/kg) alone, decreased the cardiac output by an average of 17.58 and 25.94%, respectively (P less than 0.05), without significant change in total peripheral resistance. Regional blood flow decreased and local vascular resistance increased in most organs. Coronary and renal blood flow decreased by an average of 16.45 and 17.26% with bradykinin antagonist and 23.85 and 19.80% with indomethacin, respectively (P less than 0.05). Indomethacin but not bradykinin antagonist also decreased cerebral blood flow by an average of 47.57% (P less than 0.01). Infusion of the bradykinin antagonist following prior prostaglandin inhibition with indomethacin did not induce further changes in either systemic or regional hemodynamics, except in the liver where a significant additional increment in vascular resistance was observed. Our data suggest that most organs have similar patterns of regional vascular sensitivity to bradykinin and prostaglandins (including the heart, kidney, testis, stomach, skin and adrenal). The major differences were in the cerebral vasculature, which was much more sensitive to prostaglandins, and in the liver, spleen and small intestine, which were more sensitive to bradykinin. We conclude that both hormonal systems participate to a varying extent in the maintenance of resting vascular tone in most organs; however, in some cases, their effects may be additive and in others they may work in opposite directions.

New bradykinin analogs in contraction of rat uterus

In this study, we evaluated 20 of our previously synthesized peptides on isolated rat uterus by Holtons procedure with minor modifications, and compared their activity with that assessed previously by their ability to inhibit vasodepressor response to exogenous bradykinin (BK) in conscious rats. We used [D-Arg(0), Hyp(3), Thi(5, 8), (D-Phe)(7)]BK, the B(2) antagonist of Vavrek and Stewart as a model when designing our analogs. We observed that, in the case of the rat uterus test, the activity of peptides modified by acylation of the N-terminus with various bulky groups depends substantially on the chemical character of the substituent. We also learned that, contrary to previous examples, acylation of the N-terminus of antagonists, which contain a sterically restricted fragment in the C-terminal part, may not improve their antagonistic potencies. Besides an improved characterization of a series BK analogs, our studies have provided new information on the structure-activity relationship, which in turn may be of value in the design of more potent and selective bradykinin antagonists. The results of our studies appear to support the hypothesis of others about the presence of different subtypes of B(2) receptors in rat uterus and blood vessels.

Design, Synthesis, and Structure−Activity Relationship Studies of a Potent PACE4 Inhibitor

PACE4 plays an important role in the progression of prostate cancer and is an attractive target for the development of novel inhibitor-based tumor therapies. We previously reported the design and synthesis of a novel, potent, and relatively selective PACE4 inhibitor known as a Multi-Leu (ML) peptide. In the present work, we examined the ML peptide through detailed structure-activity relationship studies. A variety of ML-peptide analogues modified at the P8-P5 positions with leucine isomers (Nle, DLeu, and DNle) or substituted at the P1 position with arginine mimetics were tested for their inhibitory activity, specificity, stability, and antiproliferative effect. By incorporating d isomers at the P8 position or a decarboxylated arginine mimetic, we obtained analogues with an improved stability profile and excellent antiproliferative properties. The DLeu or DNle residue also has improved specificity toward PACE4, whereas specificity was reduced for a peptide modified with the arginine mimetic, such as 4-amidinobenzylamide.

Analogues of arginine vasopressin modified in position 2 or 3 with naphthylalanine : Selective antagonists of oxytocin in-vitro

In this study we describe the synthesis and some pharmacological properties of six new analogues of arginine vasopressin (AVP).

Unusual gain in the coordination ability of vasopressin-like peptides towards Cu2+ ions by insertion of the highly hydrophobic side chain

Potentiometric and spectroscopic data show an unusual gain in the stability constants of Cu2+ complexes with vasopressin analogues having the highly hydrophobic naphthalene-alanine residue inserted in position three (Nal3). The naphthalene derivative is a much more powerful ligand for binding Cu2+ ions that the parent peptide. Theoretical calculations indicate the effective hydrophobic protection of the metal site by Tyr2 and Nal3 aromatic side-chains. The interaction of the guanidine moiety of Arg4 with naphthalene can also increase distinctly the stability of the respective 4N complex.

Synthesis of three NH2-terminally extended arginine-vasopressins with prolonged biological activities

The synthesis of three novel AVP-analogues, extended by 1–3 amino acids at their NH2-termini in accordance with the sequence of the bovine arginine-vasopressin neurophysin II precursor, is reported. The compounds were assayed for their antidiuretic and vasopressor activities with particular attention to the duration of the effects. All compounds showed high potency, based on the intensity, and prolonged effects in both test systems compared with AVP.