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.     

Inhibition of the activation of the first component of complement, C1, by various amino acids or peptides

The effects of various amino acids (or their analogues) and peptides on the activation or consumption of human complement by erythrocytes bound with hemolysin or heat-aggregated immunoglobulin G (aggIgG) were studied by using the hemolysis of hemolysin-bound erythrocytes, the consumption of complement by aggIgG in the serum, the hydrolysis of acetyl tyrosine ethyl ester by activated Cl (Cls), Cl hemolysis and a newly developed enzyme immunoassay, which directly measures interaction between Clq and aggIgG. Amino acids or peptides which were proposed to comprise Clq binding sites of the C2 region of IgG or their analogues were used. CH50 was inhibited by lysine or arginine to the largest extent, but other amino acids, including tranexamic acid and epsilon-amino caproic acid were not inhibitory up to 60 mM. The consumption of serum complement by aggIgG was prevented by arginine or lysine (about 60% inhibition at 60 mM) and by histidine to a lesser extent. The activation of Cls in the Cl complex by aggIgG precipitated at pH 5.5 was most inhibited by lysine, and to a lesser extent by tranexamic acid, arginine and epsilon-aminocaproic acid, but not glutamic acid or glycine. The results of Cl hemolysis indicated that, of all amino acids soluble at neutral pH, lysine and arginine were most effective in the inhibition of Cl hemolysis. Tranexamic acid and epsilon-aminocaproic acid were less effective, and glycine and norleucine were hardly effective. Among the dipeptides used, those that are composed of aromatic amino acids were very effective in the inhibition of Cl hemolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measuring and increasing protein solubility

High concentration protein delivery is difficult to achieve for several protein pharmaceuticals due to low solubility. In this review, we discuss different types of low protein solubility, including low in vitro solubility, which is relevant to the formulation of protein pharmaceuticals. We also discuss different methods of measuring protein solubility with an emphasis on the method of inducing amorphous precipitation using ammonium sulfate. Finally, we discuss strategies for increasing protein solubility, including site-directed mutagenesis. Evidence from solubility-changing mutations in the literature indicate that some hydrophilic residues (aspartic acid, glutamic acid, and serine) contribute significantly more favorably to protein solubility than other hydrophilic residues (asparagine, glutamine, threonine, lysine, and arginine). These findings should prove useful especially in cases where protein structure is not known. In these cases, instead of targeting hydrophobic residues that are often buried, one could target hydrophilic residues that do not contribute favorably to protein solubility and replace them with hydrophilic residues that contribute more favorably.     

Binding of amino beta-lactam antibiotics to soluble protein from rat intestinal mucosa--II. Mutual inhibition of binding among amino beta-lactam antibiotics and binding characteristics

The characteristics of binding of amino beta-lactam antibiotics including ampicillin, amoxicillin, cephalexin and cephradine to fraction b obtained from the 105,000 g supernatant of rat small intestinal mucosa was investigated. The mutual inhibition of binding among these antibiotics was observed, and these were dependent on the concentration of inhibitors. It was found that dipeptides such as L-carnosine and glycylglycine significantly reduced the binding of cephalexin to fraction b, but the binding of cephradine was only slightly decreased by these dipeptides. Furthermore, the binding of cephalexin and cephradine was not influenced by amino acids (L-phenylalanine, glycine). Although ANS(1-anilino-8-naphthalenesulfonic acid magnesium salt), which is a hydrophobic probe, bound to the fraction b, there was no competitive inhibition in binding between ANS and amino beta-lactam antibiotics. The Scatchard plot of binding data of cephalexin gave two dissociation constant (Kd) values (1.37 and 15.7 microM). On the other hand, one Kd value (11.2 microM) was obtained for ampicillin.     

Analysis of tryptophan, tyrosine and related dipeptides in mouse brain by isocratic high-performance liquid chromatography with switchable wavelength fluorescence detection

Tryptophan (Trp) and tyrosine (Tyr) are pharmacologically active compounds which, after administration of adequate doses, are increased in level in the brain, and stimulate neurotransmitter synthesis. Trp and Tyr containing dipeptides were tested as possible substitutes with regard to the effect on precursor level in the brain. Glycyltryptophan, alanyl-tryptophan and glycyl-tyrosine were intravenously applied to young female mice and the brain levels of dipeptides, Trp and Tyr measured 30 min after application. Neurotransmitter precursor levels in the brain increased similarly in all cases. The results suggest that the dipeptides are as effective as the single amino acids and may be superior because of their better solubility.     

Dog bites man or man bites dog? The enigma of the amino acid conjugations

The proposition posed is that the value of amino acid conjugation to the organism is not, as in the traditional view, to use amino acids for the detoxication of aromatic acids. Rather, the converse is more likely, to use aromatic acids that originate from the diet and gut microbiota to assist in the regulation of body stores of amino acids, such as glycine, glutamate, and, in certain invertebrates, arginine, that are key neurotransmitters in the central nervous system (CNS). As such, the amino acid conjugations are not so much detoxication reactions, rather they are homeostatic and neuroregulatory processes. Experimental data have been culled in support of this hypothesis from a broad range of scientific and clinical literature. Such data include the low detoxication value of amino acid conjugations and the Janus nature of certain amino acids that are both neurotransmitters and apparent conjugating agents. Amino acid scavenging mechanisms in blood deplete brain amino acids. Amino acids glutamate and glycine when trafficked from brain are metabolized to conjugates of aromatic acids in hepatic mitochondria and then irreversibly excreted into urine. This process is used clinically to deplete excess nitrogen in cases of urea cycle enzymopathies through excretion of glycine or glutamine as their aromatic acid conjugates. Untoward effects of high-dose phenylacetic acid surround CNS toxicity. There appears to be a relationship between extent of glycine scavenging by benzoic acid and psychomotor function. Glycine and glutamine scavenging by conjugation with aromatic acids may have important psychosomatic consequences that link diet to health, wellbeing, and disease.     

Novel L-amino acid ligases catalyzing oligopeptide synthesis

L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.     

4-Toluenesulfonylureido derivatives of amines, amino acids and dipeptides: a novel class of potential antitumor agents

The screening of a series of arylsulfonylureido derivatives of amines (such as histamine, or dopamine), aliphatic/aromatic amino acids (such as Gly, β-Ala, Val, Lys, Arg, Phe, Tyr, DOPA, etc.) and dipeptides (such as GlyGly, β-AlaHis) led to the identification of three derivatives that possess tumor growth inhibitory properties against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, and breast cancer cell lines in vitro. The new derivatives were prepared by reaction of 4-toluenesulfonyl isocyanate with (protected) amines, amino acids or dipeptides. The mechanism of antitumor action with these new derivatives is not known at the moment but it may imply uncoupling of mitochondria, as for the structurally related diarylsulfonylurea sulofenur, an investigational anticancer agent.     

Aromatic amino acid metabolites as potential protein binding inhibitors in human uremic plasma

Decreased binding of aromatic acidic drugs and endogenous metabolites to plasma proteins of patients with severe renal failure appears to be due to accumulation of unknown solutes. Both the warfarin and indole binding sites of albumin, the principal binding protein for these ligands, are affected. We used a large number of endogenous aromatic acids and synthetic congeners as displacers (a) better to characterize the chemical requirements for binding to each site and (b) to derive clues to the chemical structure of the undefined binding inhibitors in uremic plasma. 14C-tryptophan, 14C-warfarin and 14C-salicylate were used as bound ligands. Numerous indoles, quinolines and phenyl derivatives were moderate to strong displacers with several structural correlates. Increasing apolar side chain length enhanced displacing potency. A hydroxyl group at the 5 position of indoles and at the para position of phenyl derivatives severely reduced activity. The two ends of amphophilic molecules showed opposite requirements for displacement of tryptophan: the greater the polarity at the hydrophilic end, the greater the tryptophan displacing potency. Conversely, the greater the total hydrophobic mass of the remainder of the molecule, the more potent the inhibition of binding. The dipeptides l-tryptophyl-l-tryptophan and l-tryptophyl-l-phenylalanine were potent displacers. Computer-assisted analysis of warfarin binding in the presence of xanthurenic acid revealed inhibition by a mechanism other than simple competition, probably via a third albumin binding locus. We conclude that decreased binding in uremic plasma is most likely the summation effect of a number of retained aromatic acids, peptides, or both types of ligands.     

Carbonic anhydrase inhibitors: water-soluble 4-sulfamoylphenylthioureas as topical intraocular pressure-lowering agents with long-lasting effects

A series of sulfonamides has been obtained by reaction of 4-isothiocyanatobenzenesulfonamide with amines, amino acids, and oligopeptides. The new thiourea derivatives showed strong affinities toward isozymes I, II, and IV of carbonic anhydrase (CA, EC 4.2.1.1). In vitro inhibitory power was good (in the low-nanomolar range) for the derivatives of beta-phenylserine and alpha-phenylglycine, for those incorporating hydroxy and mercapto amino acids (Ser, Thr, Cys, Met), hydrophobic amino acids (Val, Leu, Ile), aromatic amino acids (Phe, His, Trp, Tyr, DOPA), and dicarboxylic amino acids as well as di/tri/tetrapeptides among others. Such CA inhibitors displayed very good water solubility (in the range of 2-3%) mainly as sodium (carboxylate) salts, with pH values of the obtained solutions being 6.5-7.0. Some of these preparations (such as the derivatives of Ser, beta-Ph-Ser, Leu, Asn, etc.) strongly lowered intraocular pressure (IOP) when applied topically, directly into the normotensive/glaucomatous rabbit eye, as 2% water solutions. It is interesting to note that not all the powerful CA inhibitors designed in the present study showed topical IOP-lowering effects (such as, for instance, the Cys and Lys derivatives, devoid of such properties) whereas the Pro, Arg, and oligopeptidyl thiourea derivatives showed reduced efficacy when administered topically. This may be due to the very hydrophilic nature of some of these compounds, whereas inhibitors with balanced hydro- and liposolubility also showed optimal in vivo effects. The interesting pharmacological properties of this new type of CA inhibitors, correlated with the neutral pH of their solutions used in ophthalmologic applications, make them attractive candidates for developing novel antiglaucoma drugs devoid of major ocular side effects.     

Inhibition of the release of endothelium-derived relaxing factor in vitro and in vivo by dipeptides containing NG-nitro-L-arginine.

1. We have shown that dipeptides containing NG-nitro-L-arginine (NO2Arg) inhibit the biosynthesis of endothelium-derived relaxing factor (EDRF) in vitro and in vivo. 2. In anaesthetized rats, intravenous administration at 1-30 mg kg-1 of the methyl ester of NO2Arg, NO2-Arg-L-phenylalanine (NO2Arg-Phe), L-alanyl-NO2Arg (Ala-NO2Arg) or NO2Arg-L-arginine (NO2Arg-Arg) produced dose-related increases in mean arterial blood pressure (MABP) which were unaffected by D-arginine (D-Arg; 20 mg kg-1 min-1 for 15 min), but prevented by co-infusions of L-arginine (L-Arg; 20 mg kg-1 min-1 for 15 min) or by their parent dipeptides. 3. NO2Arg methyl ester, NO2Arg-Phe methyl ester or Ala-NO2Arg methyl ester (10 mg kg-1, i.v.) also inhibited the reduction in MABP caused by the endothelium-dependent vasodilator, acetylcholine (30 micrograms kg-1 min-1 for 3 min), but not those induced by glycerly trinitrate (20 micrograms kg-1 min-1 for 3 min) or iloprost (6 micrograms kg-1 min-1 for 3 min) which act directly on the vascular smooth muscle. 4. Moreover, NO2Arg methyl ester, NO2Arg-Phe methyl ester or NO2Arg-Arg methyl ester (100 microM) inhibited the acetylcholine-induced relaxation of rabbit aortic strips, and NO2Arg-Phe methyl ester (30 microM) blocked the stimulated (bradykinin, 30 pmol) release of EDRF from bovine aortic endothelial cells grown on microcarrier beads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein

Purpose

Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability.

Methods

The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram.

Results

The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA.

Conclusions

These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

     

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity

Abstract: Porcine pepsin in water solutions containing 15–28% of dimethylformamide at pH 5 and 20–37°C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S′₁ of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.     

Solution X-ray scattering as a probe of hydration-dependent structuring of aqueous solutions

We report on new X-ray solution scattering experiments and molecular dynamics simulations conducted for increasing solute concentrations of N-acetyl-amino acid-amides and -methylamides in water, for the amino acids leucine, glutamine, and glycine. As the concentration increases, the main diffraction peak of pure water at Q = 2.0 Å^-1 shifts to smaller angle for the larger leucine and glutamine amino acids, and a new diffraction peak grows in at Q 0.8 Å^-1 for only the hydrophobic amino acid leucine. The unaltered value of the peak position at Q 0.8 Å^-1 over a large concentration range suggests that a stable and ordered leucine solute–solute distribution is sustained. Simulations of the distributions of leucines in water that reproduce the experimental observable show that mono-dispersed to small molecular aggregates of two to six hydrophobic amino acids are formed, as opposed to complete segregation of the hydrophobic solutes into one large cluster. The scattering results for the hydrophobic leucine amino acid are contrasted with experiments and simulations of the model hydrophilic side chain glutamine and the model backbone glycine. The self-assembly process of protein folding modeled with these experiments, in particular the condensation to a hydrophobic core, shares similar issues with the desolvation phenomena that are important in drug discovery.     

Extraction and activity of regulatory peptides from sea urchin eggs

Experiments were performed to extract low molecular weight peptides from the eggs of the sea urchin Strongylocentrotus pallidus using sterilizing microfiltration and solid-phase extraction, along with analytical gel chromatography and spectroscopy. The peptides extracted had molecular weights of 360 – 1200 Dal and were divided into two fractions: acidic peptides contained a predominance of glutamic and aspartic acid residues, along with aromatic amino acid residues; alkaline peptides contained mainly lysine and arginine residues and lacked aromatic amino acids. Assessment of the biological activity of these extracts demonstrated high specificity in stimulating the development of explants in organotypic cultures of spleen, testicle, myocardium, and cerebral cortex tissues.     

Influence of the hydrophilic face on the folding ability and stability of α‐helix bundles: relevance to the peptide catalytic activity

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self‐aggregating 18‐mer peptides designed previously to adopt a high level of α‐helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four α‐helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far‐ and near‐UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well‐defined tertiary structure acting as true catalysts.     

On the role of salt formation and structural similarity of co-formers in co-amorphous drug delivery systems

Co-amorphous drug delivery systems based on amino acids as co-formers have shown promising potential to improve the solubility and bioavailability of poorly water-soluble drugs. Potential salt formation is assumed to be a key molecular interaction responsible for amorphous stability and increased solubility. However, little is known about the importance of the overall structure of the co-former. In this study, the structurally related amino acids arginine (basic) and citrulline (neutral) were chosen together with four model drugs (acidic furosemide and nitrofurantoin; basic cimetidine and mebendazole) to investigate the importance of salt formation versus structural similarity of co-formers. Drug-amino acid mixtures were ball milled at a molar ratio of 1:1. Generally, arginine showed a higher tendency to successfully form co-amorphous systems with the model drugs compared with citrulline, irrespective of assumed salt formation. Salt forming mixtures showed much higher T_gs, faster dissolution rates, higher solubility and physical stability compared to the corresponding non-salt forming mixtures. In conclusion, structural similarity of the co-formers does not lead to similar co-former performance for a given drug. Salt formation is not a prerequisite for the formation of a co-amorphous system, but if a co-amorphous salt system is formed, improved dissolution rate and physical stability are observed.     

Spectroscopic Evidence of Tertiary Structural Differences Between Insulin Molecules in Fibrils

Protein fibrils are of great interest due to their involvement in several pathologies and their roles in the degradation of many therapeutic protein products. Fibrils share highly similar secondary structural motifs across different proteins and applied stress conditions. However, fibril morphology differs according to the surrounding conditions, with aromatic and hydrophobic amino acids playing important roles in mature fibril formation. In this study, we use Raman microscopy, by means of the aromatic amino acids in insulin molecules as markers, to probe for tertiary structure differences within fibrils. We compared 2 different fibril types, linear fibril bundles and spherulites. Generation of linear fibril bundles was undertaken in an acetic acid–containing formulation, whereas spherulites were generated in a hydrochloric acid–containing formulation. The Raman intensities of tyrosine and phenylalanine side chains suggest that there are significant differences between the fibril bundles. The findings suggest that the insulin components of the fibril strands are not arranged identically in the 2 fibril types and that this gives rise to differences in their tertiary structures. Overall, the work indicates that the physicochemical properties of fibril structures can be altered by changing the formulation and that these alterations can be monitored by Raman spectroscopy.