Inhibition by various peptides of the activation of C1, the first component of complement, and the interaction of C gamma 2 domain of IgG with C1q

Three groups of peptides were synthesized, each of which was proposed to be a part of the C1q binding sites of the C gamma 2 domain of IgG. They were: Trp(277)-Tyr-Val-Asp-Gly (WYVDG), Thr(289)-Lys-Pro-Arg (tuftsin) and Gly(316)-Lys-Glu-Tyr-Lys (GKEYK) or portions of these peptides. Assays included CH50, consumption of serum complement induced by heat-aggregated IgG, C1 hemolysis and an enzyme immunoassay that directly measures interaction between C1q and IgG. Peptides near Gly(316) such as GKEY, GKE or EYK inhibited CH50 and heat-aggregated IgG-induced consumption of serum complement. WYVDG also inhibited CH50, with 50% inhibition at 2.05 mM, which was more than the concentrations of peptides near Gly(316) at 50% inhibition. Tuftsin was only slightly inhibitory in both systems. Results of C1 hemolysis indicated that dipeptides composed of two aromatic amino acids, especially Trp-Tyr, were more inhibitory than dipeptides of which one residue was an aromatic amino acid. Peptides such as EYK, GKEY or GKE were very inhibitory, and tuftsin was far less inhibitory than these peptides in C1 hemolysis. Results of enzyme immunoassay also showed that dipeptides composed of two aromatic amino acids were more inhibitory than dipeptides of which one residue was aromatic amino acid. WYVDG was most inhibitory in enzyme immunoassay, but tuftsin, EYK, GKEY GKE and KE were less effective.     

Clionasterol: a potent inhibitor of complement component C1

Clionasterol (1a), clionasterol monoacetate (1b) and 5alpha,8alpha-epidioxy-24alpha-ethylcholest-6-en-3-ol (2), isolated from the marine sponge Xestospongia exigua, and beta-sitosterol (3) were tested for their influence on the classical (CP) and alternative (AP) pathways of activation of the human complement system in vitro. All the sterols inhibited the CP in a dose-dependent manner but no detectable effect was observed in the AP even at concentrations of 400 microM. Clionasterol was found to be a potent inhibitor of CP (IC50 = 4.1 microM) being ten-fold more active than beta-sitosterol. The presence of the epidioxy group on C-5 and C-8 of compound 2 caused a pronounced decrease of the inhibitory effect. Mechanistic studies on the anticomplementary effect of clionasterol revealed that it interferes with the complement component C1.     

Inhibition of protein kinase C by defensins, antibiotic peptides from human neutrophils

Defensins, human neutrophil peptide (HNP) antibiotics, potently inhibited phospholipid/Ca2+ protein kinase (protein kinase C, PKC) and phosphorylation of endogenous proteins from rat brains catalyzed by the enzyme. Of the three defensin peptides, HNP-2 appeared to be more potent than HNP-1 and HNP-3. Kinetic studies indicated that defensins inhibited PKC noncompetitively with respect to phosphatidylserine (a phospholipid cofactor), Ca2+ (an activator), ATP (a phosphoryl donor) and histone H1 (a substrate protein) with Ki values ranging from 1.2 to 1.7 microM. Defensins, unlike polymyxin B (another peptide inhibitor of PKC), did not inhibit the binding of [3H]phorbol 12,13-dibutyrate to PKC; however, defensins, like polymyxin B, inhibited the PKC activity stimulated by 12-O-tetradecanoylphorbol-13-acetate. Defensins had little or no effect on myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase) and the holoenzyme or catalytic subunit of cyclic AMP-dependent protein kinase, indicating a specificity of action of defensins. It is suggested that defensins, among the most potent peptide inhibitors of PKC so far identified, may have profound effects on functions of neutrophils and other mammalian cells, in addition to their well-recognized antimicrobial activities.     

Phosphorylation of the complement component, C9, by an ecto-protein kinase of human leukemic cells.

Ecto-protein kinases (ecto-PK) are surface constituents of many, if not all, animal cell types; normal, transformed or malignant. The occurrence of ecto-PK on the surface of human leukemia cell lines was described [Paas, Y., Fishelson, Z., 1995. Shedding of tyrosine and serine/threonine ecto-PK from human leukemic cells. Arch. Biochem. Biophys. 316 780-788.]. These ecto-PKs have been shown to phosphorylate several exogenous substrates, including the complement C9 protein, an essential component of the terminal complement system. C9 is phosphorylated by ecto-PK of K562 cells on serine residue(s). Phosphorylation occurs in the N-terminal C9a portion produced by cleavage of phosphorylated C9 with human alpha-thrombin. C9 polymers generated upon incubation of C9 with ZnCl2 do not serve as substrates for the K562 ecto-PK. In contrast, unfolded C9, obtained by reduction and alkylation, serves as a superior substrate for the K562 ecto-PK. Native C9 phosphorylation produced a rather low stoichiometry of incorporated phosphate (around 3%) per C9. Despite that, the phosphorylated C9 expressed reduced hemolytic activity. The complement-sensitive variant of K562 (K562/S) did not express the C9 phosphorylating activity. Various PK inhibitors tested failed to block C9 phosphorylation. Only heparin and 2,3-diphosphoglycerate (dpGA) prevented C9 phosphorylation, indicating that the ecto-PK is related to the casein kinase CK2. C9 can be phosphorylated by ecto-PK from other tumor cells, including Jurkat, SK-OV-3 and BT-474. These results suggest that extracellular phosphorylation of C9 may serve as a protective mechanism against complement in tumor cells.     

Advancements in the anti-diabetes chemotherapeutics based on amino acids, peptides, and peptidomimetics

Diabetes Mellitus (DM) is a highly prevalent chronic disease. Recent years have witnessed development of many new oral drugs; novel insulin analogs and their delivery systems for the treatment of patients with either type-1 or type-2 DM. The impetus for developing new antidiabetic drugs comes from the unmet need of pharmacological tools that allow diabetic patients to achieve recommended glucose control targets by precise, safe and effective ways. The number of people afflicted with DM worldwide has increased considerably in recent years and is projected to increase dramatically over the next decades. In the recent times, design and synthesis of bioactive peptides and peptidomimetics has undergone a paradigm shift. Non-proteinogenic amino acids, peptides and peptidomimetics are emerging as novel drug candidates for the treatment of various diseases and/or disorders. This review mainly discusses the advancements in the usage of unnatural amino acids, peptides and peptidomimetics as potential therapeutic agents for the treatment of DM.     

Effects of the cleavage peptides,C3a and C3ai,from the third component of hog complement on leukocyte accumulation and vascular permeability in vivo

Summary In vivo anti-platelet de-aggregatory activity of exogenous prostacyclin is enhanced after its passage through the pulmonary circulation of anaesthetized cats, probably because of a concomitant generation of endogenous prostacyclin by the lungs. Evidence is also presented that perfused lungs of guinea pigs and rats spontaneously release considerable amounts of prostacyclin. It is therefore postulated that a continuous biosynthesis of prostacyclin by pulmonary endothelium is a general physiological phenomenon, while the generation of thromboxane A₂ by lungs occurs in response to pathological stimuli. Coronary and cerebral arteries are supposed to benefit from this hormonal function of the lungs.     

De novo design of selective antibiotic peptides by incorporation of unnatural amino acids

The evolution of drug-resistant bacteria is one of the most critical problems facing modern medicine and requires the development of new drugs that exhibit their antibacterial activity via novel mechanisms of action. One potential source of new drugs could be the naturally occurring peptides that exhibit antimicrobial activity via membrane disruption. To develop antimicrobial peptides exhibiting increased potency and selectivity against Gram positive, Gram negative, and Mycobacterium bacteria coupled with reduced hemolytic activity, peptides containing unnatural amino acids have been designed, synthesized, and evaluated. These compounds were designed on the basis of the electrostatic surface potential maps derived from the NMR determined SDS and DPC micelle-bound conformations of (Ala8,13,18)magainin-2 amide. Unnatural amino acids were incorporated into the polypeptide backbone to control the structural and physicochemical properties of the peptides to introduce organism selectivity and potency. The methods and results of this investigation are described below.     

Quantitative structure-activity relationships of the bitter thresholds of amino acids, peptides, and their derivatives

Bitter thresholds of a total of 93 amino acids, peptides, and their derivatives were analyzed quantitatively by use of hydrophobicity parameters reported for amino acid side chains and those for the whole molecules estimated from partition coefficients obtained experimentally. We also explored the steric parameters that best explained the variation in the intensity of bitterness attributable to the molecular shape. The results showed that the total length along the zigzag peptide backbone chain of the molecule is an important factor. The bitterness of nonzwitterionic N-acyl and ester derivatives and that of neutral N-acyl ester derivatives were expressed by a single, common equation together with those of zwitterionic amino acids and peptides. Thus the interaction via the charge with the receptor site was probably not an indispensible factor for triggering of the bitter sensation. This study, together with earlier ones, may serve as a prototype of approaches toward unraveling structure-activity relationships of complex molecules like amino acids, peptides, and their derivatives that are of medicinal or agricultural importance.     

Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor

INTRODUCTION: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. METHODS: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. RESULTS: About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. DISCUSSION: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.     

Advances in the synthesis of bioactive unnatural amino acids and peptides

The key role of proteins and amino acids in the structure and function of living matter has stimulated extensive studies. Modified amino acids with enhanced biological activity, proteolitic stability and bioavailability are of increasing interest in protein design and engineering as drug candidates. In the last few years, several efforts have been devoted to the synthesis of amino acids having unusual side chains and unnatural chirality, commonly referred to as "nonproteinogenic" or "unnatural" amino acids, even though some of them can be isolated from natural sources. In this review we describe recent advances in the amino acid side-chain transformations and backbone modifications by oxidative and fluorination procedures.     

Effect of amino acids,peptides and related compounds on the autooxidation of ascorbic acid

The autooxidation rate of ascorbic acid (AA) at 10^-5 m under aerobic conditions at pH 7.4 was found to be 1.16 μmol/min/1. A number of compounds at low concentrations were found to inhibit this oxidation rate. These were in order of effectiveness: EDTA (10^-6 m ) > Mercaptoethanol > aminoethyl cysteine, oxidized glutathione > glycylglycylhistidine > glycylhistidyllysine > 3-methyl histidine ? histidine > histamine > hypertensin > cysteic acid > imidazole > glutamine > hydroxyproline, and lysine. All other amino acids and peptides examined had little or no effect on the autooxidation rate of ascorbic acid. Ascorbate solutions, treated with Chelex-100 (divalent chelating resin) or containing low concentrations of EDTA (10^-7 m ) did not show a significant reduction of the rate of autooxidation. Of particular interest was the finding that 3-methyl histidine had a significant inhibitory effect on ascorbic acid oxidation whereas 1-methyl histidine had no effect. These data suggest that ascorbic acid forms complexes with certain compounds and that this interaction stabilizes ascorbic acid against auto-oxidation.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Inhibition of complement components C3 and C4 by cadralazine and its active metabolite

Summary The effect of cadralazine and its active metabolite CGP 22639 on the covalent binding reaction of C4 and C3 has been studied. Trypsin-Sepharose was used to activate radio-labelled C3 and C4 and binding of the radio-labelled protein to the trypsin-Sepharose was measured.Cadralazine inhibited 50% of the binding of C3 and C4 at concentrations of 19 mmol/l and 15 mmol/l, respectively. Its active metabolite was more potent and inhibited 50% of the C3 and C4 binding at concentrations of 8 and 3.5 mmol/l, respectively. These concentrations are much higher than those found in plasma during therapy.This is consistent with the clinical observation that in patients with normal kidney function cadralazine is not an inducer of SLE.     

Inhibition of plasmin-mediated prostromelysin-1 activation by interaction of long chain unsaturated fatty acids with kringle 5

C18 unsaturated fatty acids were here found to inhibit proMMP (matrix metalloproteinase)-3 activation by plasmin. This effect was suppressed by lysine ligand competitors, indicating that it was mediated by binding to kringle domains. Surface plasmon resonance analysis demonstrated that oleic acid interacted to a similar extent with plasmin and kringle 5 (KD values of 3.4 x 10(-8) and 5.9 x 10(-8)M) while interaction with kringles 1-2-3 was 10-fold lower. Furthermore, oleic acid stimulated the amidolytic activity of plasmin and mini-plasmin, but not micro-plasmin. Oleic acid also enhanced u-PA (urokinase-type plasminogen activator)-mediated plasminogen activation over 50-fold. Taken together, these data indicate that inhibition of plasmin-induced proMMP-3 activation by unsaturated fatty acids was mediated through their preferential binding to kringle 5. The influence of elaidic acid on the plasmin/MMP-3/MMP-1 proteolytic cascade was assessed ex vivo. Exogenous addition of plasmin to dermal fibroblasts or supplementation of gingival fibroblast culture medium with plasminogen triggered this cascade. In both instances, elaidic acid totally abolished proMMP-3 and proMMP-1 activation. Additionally, a significant decrease in lattice retraction and collagen degradation in a range similar to that obtained with Batimastat was observed when human gingival fibroblasts were cultured in plasminogen-containing type I collagen gels, indicative of the dual influence of unsaturated fatty acids on MMP activation and activity. In conclusion, unsaturated fatty acids or molecules with similar structures could be attractive target for the development of natural pharmacological inhibitors directed against plasmin and/or MMPs in different pathological contexts such, skin UV irradiation, vascular diseases and tumour growth and invasion.     

Antibacterial halogenoacetyl derivatives of amino acids and simple peptides.

The vital role of D-alanine and L-lysine in the peptidoglycan crosslinking process in the bacterial cell wall prompted preparation of various small peptides incorporating these amino acids. N-Iodoacetyl or -bromoacetyl derivatives of the peptides were then prepared in the hope that they would serve as active-site-directed irreversible inhibitors of cell wall transpeptidases. Certain of the halogenoacetyl dipeptide esters, but not the corresponding free acids, showed slight antistaphylococcal activity. Subsequent structural variation showed that inclusion of C-alanine or L-lysine was not necessary, since antibacterial activity was at least as good when the dipeptide unite was replaced by glycylglycine or by an omega-aminoalkanoic acid. It was concluded that the observed antibacterial activity was probably not due to specific inhibition of a cell wall transpeptidase.     

Synthesis and biological properties of fullerene-containing amino acids and peptides

Organofullerene derivatives have shown a great potential in a wide variety of biological activities such as DNA photocleavage, HIV-protease inhibition, neuroprotection and apoptosis. Among the plethora of functionalized organofullerenes that have been synthesized, fullerene-based amino acids are particularly appealing for structural studies and biological applications. When the fullerene-framework is incorporated into peptides, its original properties can be substantially modified. In addition, the water-solubility of the fullerene derivatives is enhanced, which makes such molecules amenable to biological studies. In this review, recent advances in the growing field of medicinal chemistry of fullerene derivatives will be discussed. Emphasis will be given to the synthesis of the biggest unnatural amino acid 3,4-fulleroproline (Fpr) and its derivatives. For example, Fpr derivatives have been found to interact with different hydrolytic enzymes and selectively discriminate between rationally designed peptides. Fullerene-based peptides have been found to substantially activate enzymes involved in the oxidative deamination of biogenic amines. In addition, their membranotropic properties and effects on the structure and permeability of the lipid bilayer of phosphatidylcholine liposomes as well as the transmembrane transport of bivalent metal ions have been studied. Finally, applications in medicinal chemistry of such types of amino acids and peptides will be highlighted.     

Inhibition of receptor-coupled phosphoinositide hydrolysis by sulfur-containing amino acids in rat brain slices

Sulfur-containing amino acids were found to inhibit norepinephrine-stimulated [3H]phosphoinositide hydrolysis in rat cortical slices. Of the amino acids tested, L-cysteine was the most potent, inhibiting the response by 42 and 85% at concentrations of 50 and 500 microM respectively. L-Cystine and L-serine-O-sulfate also inhibited the response to norepinephrine, but to a lesser degree than did L-cysteine. L-Homocysteic acid slightly potentiated phosphoinositide hydrolysis at a concentration of 100 microM, but caused inhibition at 500 microM. L-Cysteine sulfinate produced effects intermediate to those of L-cysteine and L-homocysteic acid, having no effect on the response to norepinephrine at 50 microM, but causing 84% inhibition at 500 microM. The D-isomers of cysteine and homocysteic acid were much less potent than were the L-isomers. Examination of the time course of the inhibition of norepinephrine-stimulated [3H]phosphoinositide hydrolysis by L-cysteine showed that it was inhibited almost completely after 15, 30, 45 and 60 min of incubation. L-Cysteine and L-homocysteic acid caused similarly strong inhibitions of the production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. The hydrolysis of [3H]phosphoinositides stimulated by norepinephrine in slices from rat hippocampus and striatum were inhibited by L-cysteine to an extent similar to that occurring in cortical slices. These results demonstrate that several sulfur-containing amino acids, some of which have been proposed to be endogenous excitatory amino acid neurotransmitters, effectively modulate the response to norepinephrine of the phosphoinositide second messenger system in rat brain.