A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor.

Analysis of synthetic tri- and tetrapeptides has previously indicated that N-formylation is required for high biological activity when they react with the phagocyte N-formylpeptide receptor, suggesting that the natural ligand for the receptor is from bacterial and/or mitochondrial sources. To explore this requirement further, we synthesized the pentapeptide methionyl-norleucyl-leucyl-phenylalanyl-phenylalanine (MNleLFF) and studied the effects of different NH2-terminal modifications on its activity. N-formyl-MNleLFF induced transient alterations of [Ca2+]i and superoxide production in human neutrophils with 10- and 100-fold greater potency, respectively, than the proto-type N-formylpeptide, N-formylmethionyl-leucyl-phenylalanine (fMLF). Surprisingly, N-acetyl-MNleLFF was a potent as N-formyl-MNleLFF. Moreover, the unacylated counterpart H-MNleLFF was also highly active, having an EC50 for calcium mobilization of 10 nM, and for respiratory burst activation of 100 nM. All three pentapeptides could completely desensitize calcium transients elicited by stimulation of neutrophils with fMLF, whereas the neutrophil chemoattractants C5a and interleukin 8 only weakly affected fMLF-induced transients, suggesting that they activate neutrophils via the same receptor as fMLF. Finally, all three pentapeptides activated the recombinant human N-formylpeptide receptor expressed in frog oocytes, but did not effectively activate related phagocyte receptors. These data broaden the potential sources of natural ligands for the N-formyl-peptide receptor from N-formylated bacterial and mitochondrial products to other nonformylated endogenous peptides.

The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils.

24 di-, tri-, and tetrapeptides have been synthesized as a start of a systematic study of the structural requirements for chemotactic activity and lysosomal enzyme-releasing ability in rabbit neutrophils. All but two of them are N-formyl methionyl peptides. Using the method of Zigmond and Hirsch (10), two representative peptides, F-Met-Leu-Phe and F-Met-Met-Met, were shown to stimulate directed, as well as, random locomotion; thus, they were truly chemotactic. The various peptides showed a wide spread in activity. F-Met-Leu-Phe, the most active peptide studied, had an ED50 for induced migration of 7 X 10(-11) M and for lysozyme and beta-glucuronidase release of 2.4 X 10(-10) M and 2.6 X 10(-10) M, respectively; the least active, Met-Leu-Glu was 26 million times less active in these respects. The relation of activity to structure is exceedingly specific, very small changes in structure making large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on its constituent amino acids but on the position of the amino acid in the peptide chain. Most striking in this last regards is the high activity conferred by phenylalanine when it is in the carboxyl terminal position of a tripeptide, whereas, as the second amino acid from the NH2 terminal end whether in a tripeptide or a dipeptide, it contributes no more to the activity than other amino acids with hydrophobic side chains such as leucine or methionine. The high activity and the specificity and nature of the structural requirements strongly suggest that the primary interaction of peptide and neutrophil leading to either chemotaxis or lysosomal enzyme release is a binding of the peptide with a stereospecific receptor on the neutrophil surface. Whether all chemotactic factors act through the same receptor is not known. An essentially exact correlation exists between the concentrations of the various synthetic peptides required to induce migration and their ability to induce release of lysozyme or beta-glucuronidase. This implies that these two neutrophil functions are triggered by teh same primary interaction; possibly, the binding of the peptides to the same putative receptor. A higher concentration of a given peptide is required to stimulate lysosomal enzyme release than a corresponding migratory response. A slightly but significantly higher concentration of peptide is required to induce beta-glucuronidase secretion than lysozyme release.

Retro-all-D and retro isomers of a formyl-methionyl peptide chemoattractant: an insight into the mode of binding at the receptor on rabbit neutrophils.

The retro-all-D analog and retro isomer of the formyl-methionyl-carboxamide-tripeptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2, namely CHO-D-Phe-D-Leu-D-Met-NH2 and CHO-L-Phe-L-Leu-L-Met-NH2, respectively, have been synthesized in solution by classical methods and fully characterized. The tetrapeptide CHO-L-Phe-Gly-L-Leu-L-Met-NH2, representing the C-terminal portion of the tachykinin, Substance P, and resembling the sequence of the retro isomer, has also been synthesized and characterized. The three N alpha-formylated tripeptide amides, prepared in order to obtain a deeper insight into the model of binding at the formyl peptide chemotactic receptor on rabbit neutrophils, have been tested for their ability to induce granule enzyme secretion from rabbit peritoneal neutrophils. The retro isomer, CHO-L-Phe-L-Leu-L-Met-NH2 is approximately 100-fold less active, the retro-all-D analog, CHO-D-Phe-D-Leu-D-Met-NH2 approximately 10,000-fold less active and the Substance P analog CHO-L-Phe-Gly-L-Leu-L-Met-NH2 1000-fold less active than the parent formyl peptide chemoattractant, CHO-L-Met-L-Leu-L-Phe-NH2. We interpret these results to indicate that a precise alignment of amino acid side chains as well as backbone amide bonds is an important factor involved in the receptor recognition of the formyl tripeptide chemoattractant.

N alpha-formylated and tert-butyloxycarbonylated Phe-(Leu-Phe)n and (Leu-Phe)n peptides as agonists and antagonists of the chemotactic formylpeptide receptor of the rabbit peritoneal neutrophil.

The various diastereomers of the N alpha-formylated(CHO) and tert-butyloxycarbonylated (t-Boc) Phe-(Leu-Phe)n and (Leu-Phe)n methyl esters, where n = 1-2 and 1-3, respectively, have been newly synthesized and their physical properties described. The CHO-blocked peptides are all able to release beta-glucosaminidase from rabbit peritoneal neutrophils in a concentration-dependent manner. There is a strong effect of primary structure and of chirality on their biology activity; lengthening the peptide chain distinctly increases activity in each series and within a series the activity decreases in the order: all-L greater than D-L much greater than all-D. Of the t-Boc protected synthetic precursors, the all-L isomers have definite but weak agonist activity; the agonist activity of the other isomers is equivocal or not detectable. All the t-Boc peptides, however, are capable of acting as weak, specific antagonists. There is a dependence of antagonist activity on primary structure, but this is variable and contingent on the nature of the peptide. Similarly, an effect of chirality on antagonist activity, although present, also depends on the structure of the peptide. In the one instance directly tested, t-Boc-L-Phe-(D-Leu-L-Phe)2-OMe (OMe, methoxy) was found to be distinctly less active than the corresponding free acid.

Characteristics of binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine, to the receptors on rabbit neutrophils.

Binding of a potent chemotactic formyl tetrapeptide, formylmethionyl-leucyl-phenylalanyl-phenylalanine (fMet-Leu-Phe-Phe), to the formyl peptide receptors on the rabbit neutrophil was assessed by two approaches. A tritiated preparation of fMet-Leu-Phe-Phe was used for direct binding studies, whereas indirect studies comprised an assessment of the ability of the formyl tetrapeptide to competitively inhibit the binding of 35S-labeled formylmethionyl-leucyl-phenylalanine. These two approaches yielded analogous results. The formyl tetrapeptide fMet-Leu-Phe-Phe showed rapid and saturable binding to the same chemotactic receptors as the less potent formyl tripeptides with which it was compared. Its equilibrium-binding pattern, however, was different: fMet-Leu-Phe-Phe showed a homogeneous binding pattern, in contrast to the heterogeneity seen with the less potent compounds. The relative potencies for high-affinity binding of the two standard formyl tripeptides and fMet-Leu-Phe-Phe correlated well with their relative potencies for stimulating the biological response of degranulation; the relative potencies for low-affinity binding correlated less well.

Thermal regulation of FMLP receptors on human neutrophils.

Polymorphonuclear leukocytes (PMNs) from subjects diagnosed as having juvenile periodontitis (JP) have been categorized on the basis of their chemotactic (CTX) response to f-met-leu-phe (FMLP) when assayed concurrently with PMNs from periodontally healthy subjects (HP). When PMNs from JP groups demonstrating depressed CTX were assayed for lysosomal enzyme secretion (LES) in response to FMLP, there were no significant differences with respect to rate or amount. Significant differences were observed between HP and chemotactically depressed JP cells when assessed for FMLP receptor ligand binding at 23 degrees C, but not at 4 degrees C. Receptor differences observed at 23 degrees C in HP cells included an increase in amount of total binding, number of receptors, and available displaceable binding sites, compared with the chemotactically depressed JP PMNs, whereas the receptor affinities were similar. These data suggest that differences in FMLP receptor density in JP PMN that are chemotactically depressed may be related to processes that modulate receptor mobility and/or expression.

Isolation and reconstitution of the N-formylpeptide receptor from HL-60 derived neutrophils.

A multifunctional receptor for N-formylpeptides exists on the membranes of neutrophils. This receptor has now been isolated from neutrophils derived from HL-60 promyelocytic leukemia cells. After solubilization by Nonidet-P40 and purification by affinity chromatography and HPLC the isolated receptor was reconstituted into egg phosphatidylcholine vesicles by SM-2 Bio-Bead removal of the Nonidet-P40. Analysis of the affinity and selectivity of the receptor was done by direct binding of two high-affinity ligands, formyl-Met-Leu-[3H]Phe-OH and formyl-Nle-Leu-Phe-[3H]Tyr-OH. The data suggest that the receptor can be isolated and reconstituted without apparent alteration of its binding affinity and selectivity, and that there appear to be no co-factors or subunits upon which these binding characteristics are dependent.

Conformationally constrained chemotactic peptide analogs of high biological activity.

The stereochemically constrained chemotactic peptide analogs, formylmethionyl-alpha-aminoisobutyryl-phenylalanine (formyl-Met-Aib-Phe-OH) and formylmethionylcycloleucinylphenylalanine (formyl-Met-Cyl-Phe-OH) are highly effective in inducing lysosomal enzyme release from rabbit neutrophils. NMR studies of the Aib2 analog in (CD3)2SO favor a folded conformation in which the Phe NH group is inaccessible to solvent. Intramolecularly hydrogen-bonded conformations involving a Met-Aib-beta-turn or a gamma-turn centered at Aib2 are considered. The results suggest that folded conformations may allow highly active interactions with the neutrophil formylpeptide receptor.

Synthesis and pharmacological properties of [5-isoleucine]-, [8-isoleucine]-, and [5,8-diisoleucine]bradykinin.

Three bradykinin analogues have been synthesized in which the phenylalanine residue(s) at positions 5 and/or 8 have been substituted by isoleucine. All these analogues have weak bradykinin-like activity in isolated rat uterine smooth muscle or in rat blood pressure assay. No antagonistic activity was observed with any of these analogues. The importance of phenylalanine at positions 5 and 8 is discussed.

Angiotensin-induced steroidogenesis in rabbit adrenal: effects of pH and calcium.

The effect of variations in pH and Ca2+ on angiotensin II (A-II)-induced steroidogenesis was tested on isolated adrenal glomerulosa cell suspensions. The results show that a reduction in pH from 7.4 to 6.5 produces both a shift to the left of the A-II dose-response curve as well as an increase in maximum steroid production. In contrast, removal of Ca2+ from the incubation medium virtually abolished steroidogenesis to A-II (5 X 10(-9)M(, KCl(10mM) and ACTH (250 microU/ml). The Ca2+ antagonist D-600, however, was less effective than simple removal of Ca2+ as 10(-4) M was required to block the steroidogenic response to these same agonists. The results indicate that the response characteristics of this system to A-II resemble most closely those seen with isolated arterial smooth muscle - especially rabbit aortic strips.

Synthetic formyl-methionyl chemoattractants: a conformation-activity study of oxidized tripeptides.

The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED50s from 1-2 x 10(-9) M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and 1H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C = O...H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S = O...H-N interactions are postulated to tentatively explain the 1H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl3 solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.

Organ selective angiotensin antagonists: sarcosyl1-cysteinyl(S-methyl)8-angiotensin I.

An angiotensin antagonist, Sarcosyl1-Cysteinyl(S-Methyl)8-angiotensin I [Sar1-Cys(Me)8-ANG I] was synthesized and its pharmacological properties evaluated in vivo (rat blood pressure assay) and in vitro (rabbit aortic strips, guinea-pig ileum and rat uterus assays). It was found to be an extremely potent angiotensin II (ANG II) antagonist in the rat pressor assay (dose ratio for ANG II of 1300 during infusion of 5.0 micrograms/kg/min Sar1-Cys(Me)8-ANG I) and a moderately effective antagonist in guinea-pig ileum (pA2 congruent to 8.2). Moderate antagonism was also seen in the rabbit aortic strip preparation (pA2 congruent to 8.1) while the analog was inactive in the rat uterus assay. In each of the preparations where antagonist activity was observed there was evidence of non-competitive antagonism. Most striking was the inability of extremely high doses ( up to 125 micrograms ANG II/kg) of ANG II to overcome the Sar1-Cys(Me)8-ANG I blockade. In both the rat pressor and guinea-pig ileum assays the Sar1-Cys(Me)8-ANG I antagonism is completely abolished in the presence of the converting enzyme inhibitor SQ14225 (Captopril-Squibb). Organ selectivity of this analog is discussed in terms of the inherent activity of the active principle (i.e. the Sar1-Cys(Me)8-angiotensin II [Sar1-Cys(Me)8-ANG II] released by the action of converting enzyme) and the availability of converting enzyme in each bioassay.

Synthesis and pharmacology of a prohormone angiotensin antagonist.

A prohormone angiotensin antagonist, Sarcosyl1-Alanyl8-angiotensin I (Sar1-Ala8-A-I) was synthesized and its pharmacological properties were evaluated in three biological systems. It was found to be a good inhibitor in vivo in the rat blood pressure assay, somewhat less active in guinea pig ileum and a relatively weak antagonist in rat uterus. In vivo the inhibitory effect was greatly attenuated by the presence of the converting enzyme inhibitor BPP5alpha.

Acetylcholine potentiation of field stimulated rat vas deferens: a pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED200 (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 X 10(-6)M with the maximum twitch response being increased by more than 3 fold (325 +/- 30%). Carbachol (ED200 = 8.5 X 10(-7)M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10(-7) - 10(-5)M). Physostigmine 10(-8) - 10(-6)M) both enhanced the basal twitch response (215 +/- 8% of control at 10(-5)M) and the sensitivity of the RVD to ACh (ED200 = 3.3 X 10(-7)M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 +/- 3% at 10(-5)M. Hemicholinium (10(-4)M) also reduced the basal twitch responses by 23 +/- 5%. ACh (10(-7)M - 10(-5)M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.