Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs

The objective of this study was to determine whether the rates of deamidation of Asn⁸ in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu²⁷]GRF(1–32)NH₂ (both having Gly at position 15), [Ala¹⁵ Leu²⁷]bGRF(1–32)NH₂ and [Pro¹⁵ Leu²⁷]bGRF(1–32)NH₂ were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro¹⁵ Leu²⁷ bGRF(1–32)NH₂, [Leu²⁷]hGRF(1–32)NH₂, and Ala¹⁵ Leu²⁷ bGRF(1–32)NH₂, respectively. 2D NMR studies done in the presence of 40% CD₃OH indicated more helical structure for the Ala¹⁵ analog as compared to [Len²⁷]hGRF(1–32)NH₂. In both these peptides Asn⁸ was included in the helical region. In contrast, the lack of conformational information for the Pro¹⁵ analog indicated little helical structure around Asn⁸. The peptides’ deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro¹⁵ bGRF analog (t_1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly¹⁵ (t_1/2= 15.74 h) and Ala¹⁵ (t_1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn⁸ in GRF makes this residue less prone to deamidation.     

Conformational study of a synthetic analogue of alamethicin Influence of the conformation on ion-channel lifetimes

Alamethicin, a 20-residue peptaibol, induces voltage-dependent ion channels in lipid bilayers according to the barrel-stave model. A synthetic analogue (L2) in which all Aib were replaced by Leu shows a conductance behaviour similar to alamethicin, but channel lifetimes are drastically reduced. Among several hypotheses, a different conformation for L2 might be responsible for this phenomenon by increasing the α-helical content (alamethicin presents some 3.0₁₀-helical parts) and thus decreasing the length of the transmembrane part. A conformational study of L2 was undertaken using FTIR, CD and NMR spectroscopy, and the secondary structure was compared with alamethicin. These techniques showed an enhanced predominant helical structure as compared to alamethicin. Moreover, the NOE pattern showed an exclusively α-helical conformation, resulting in a smaller length of the L2 peptide. This shortening somewhat impedes the complete crossing of the membrane, and could then explain the reduction of its ion-channel lifetimes. © Munksgaard 1995.     

Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments

Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed μ/δ-agonist, and one related to DAMGO, a highly selective μ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its β-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human μ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55 °C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

Abstract: The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well‐known helix‐turn‐helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water‐mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well‐defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α‐helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α‐helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α‐helical conformation even if separated from the rest of the molecule.     

Characterisation of the solution conformation of a cyclic RGD peptide analogue by NMR spectroscopy allied with a genetic algorithm approach and constrained molecular dynamics

The solution conformation of a cyclic RGD peptide analogue, cyclo-(S,S)-2-merrcaptobenzoate-arginine-glycine-aspartate -2-mercaptoanilide, has been determined via two independent approaches for the searching of conformational space and identification of conformations consistent with NMR and CD spectroscopic data: (i) the use of a binary genetic algorithm and (ii) a molecular dynamics simulation. Inter-proton distances were obtained via analysis of cross-peak volumes from a two-dimensional ROESY NMR spectroscopy experiment at 600 MHz and were used as constraints for the computational calculations. The mercaptoanilide amide proton resonance chemical shift had a very small temperature coefficient, indicating that this proton was hydrogen-bonded. Circular diehroism data showed that, in solution, the torsion angle about the disulfide bond was negative, consistent with one of the distinct conformations around this bond in the 200 ps molecular dynamics simulation. The backbone conformations of the structures resulting from the two different approaches were very similar.     

VI. Synthesis,conformation, and ion-binding of two bicyclic nonapeptides *

Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (lγ→ 5?), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.     

Effect of protamine on the solubility and deamidation of human growth hormone

The effect of protamine on the solubility and deamidation of human growth hormone (hGH) was investigated. Protamine is an extremely basic peptide. It is isolated from sperm cells of salmon where it can build a complex with DNA due to electrostatic interactions. We hypothesize a similar electrostatic effect between negatively charged hGH and positively charged protamine residues. Arising cationic complexes are stabilized by electrostatic repulsion. This stabilizing complexation allows solubilization of hGH down to a pH of 5.4 (pI 5.3) at concentrations of 3.4 mg/ml. The minimal solubilizing molar ratio between hGH and protamine was found to be at least 1:23 (hGH:protamine) by turbidity and dynamic light scattering (DLS) measurements. Complexation was characterized by small-angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC). Electrostatic binding of protamine to hGH was also observed by a reversal in surface charge, as shown by zeta potential measurements. The presence of protamine did not alter the conformational structure of hGH which was determined by circular dichroism (CD) spectroscopy. A pH of 5.4 is known to slow deamidation of hGH and consequently retardation of hGH deamidation could be detected after 3 months storing by reversed-phase high-performance liquid chromatography (RP-HPLC).     

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. CySl82-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (T^v_{18 + 19}) and the corresponding tryptic peptide from rhGH (T_{18 + 19}) were identical. Electrospray mass spectrometry (ES/MS) of T^v_{18 + 19} isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T_{18 + 19} from rhGH. A synthetic T_{18 + 19} peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragments in ES/MS compared to T^v_{18 + 19} isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cysl82-SSS-Cys189) bridge not found in the corresponding T_{18 + 19} disulfide peptide. Furthermore, the synthetic T^v_{18 + 19} co-eluted in RP-HPLC with T_{18 + 19} isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T_{18 + 19} and T^v_{18 + 19} peptides were performed. Using these data all protons were assigned. The major chemical shift changes (δ§>0.05 ppm) observed were for the β-protons of Cys182 and Cys189 in T^v_{18 + 19} as compared to T_{18 + 19}. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data, rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore^TM. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties. © Munksgaard 1996.     

Conformational investigation of α,β-dehydropeptides VII*. Conformation of Ac-Pro-ΔAla-NHCH3 and Ac-Pro-(E)-ΔAbu-NHCH3: comparison with (Z)-substituted α,β-dehydropeptides†

The crystal structure and solution conformation of Ac-Pro-ΔAla-NHCH₃ and the solution conformation of Ac-Pro-(E)-ΔAbu-NHCH₃ were investigated by X-ray diffraction method and NMR, FTIR and CD spectroscopies. Ac-Pro-ΔAla-NHCH, adopts an extended-coil conformation in the crystalline state, with all-trans peptide bonds and the ΔAla residue being in a C₅ form, φ₁=– 71.4(4), ψ₁=– 16.8(4), φ₂=– 178.4(3) and ψ₂= 172.4(3)°. In inert solvents the peptide also assumes the C₅ conformation, but a γ-turn on the Pro residue cannot be ruled out. In these solvents Ac-Pro-(E)- ΔAbu-NHCH₃ accommodates a βII-turn, but a minor conformer with a nearly planar disposition of the CO—NH and C=C bonds (φ₂～0°) is also present. Previous spectroscopic studies of the (Z)-substituted dehydropeptides Ac-Pro-(Z)- ΔAbu-NHCH, and Ac-Pro-ΔVal-NHCH₃ reveal that both peptides prefer a βII-turn in solution. Comparison of conformations in the family of four Ac-Pro-ΔXaa-NHCH₃ peptides let us formulate the following order of their tendency to adopt a β-turn in solution: (Z)- ΔAbu > (E)- δAbu > ΔVal; ΔAla does not. None of the folded structures formed by the four compounds is stable in strongly solvating media. © Munksgaard 1996.     

The effects of sucrose on stability of human brain natriuretic peptide [hBNP (1–32)] and human parathyroid hormone [hPTH (1–34)]

Abstract: Although the effect of sucrose on the physical stability of proteins has been well documented, its impact on their chemical stability is largely unknown. The aim of this study was to investigate the potential effects of sucrose on the structural conformation of human brain natriuretic peptide [hBNP (1–32)] and the synthetic human parathyroid hormone [hPTH (1–34)], and link these effects to chemical degradation pathways of these peptides. The stability of hBNP (1–32) and hPTH (1–34) was studied at pH 5.5. Aggregation was monitored using size exclusion high‐performance liquid chromatography (SE‐HPLC), whereas oxidation and deamidation products were measured by reversed phase (RP) HPLC. Fourier transform infrared (FT‐IR) spectroscopy was used to study the peptides’ conformation. Sucrose retarded aggregation, deamidation, and oxidation of hBNP (1–32) and hPTH (1–34), with a maximum effect at relatively high concentrations (as much as 1 m ). FT‐IR spectroscopy indicated that sucrose maintained the native conformation of hBNP (1–32) and induced small conformation changes in the hPTH (1–34) structure. Sucrose enhanced the stability of hBNP (1–32) and hPTH (1–34) in liquid formulations. The stabilizing effect of sucrose was due to a large extent to retardation of oxidation and deamidation of hBNP (1–32) and hPTH (1–34).     

Peptides from chiral Cα,α-disubstituted glycines Synthesis and characterization,conformational energy computations and solution conformational analysis of Cα-methyl,Cα-isopropylglycine [(αMe)Val] derivatives and model peptides*

Conformational energy computations on Ac-l -(αMe)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral Cα-methyl, Cα-alkylglycyl residue. We have synthesized and characterized a variety of l -(αMe)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, ¹H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the l -(αMe)Val residue turns out to be a strong β-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other Cα-methyl, Cα-alkylglycyl residues.     

Multiple aspects of the interaction of biomacromolecules with inorganic surfaces

The understanding of the mechanisms involved in the interaction of biological systems with inorganic materials is of interest in both fundamental and applied disciplines. The adsorption of proteins modulates the formation of biofilms onto surfaces, a process important in infections associated to medical implants, in dental caries, in environmental technologies. The interaction with biomacromolecules is crucial to determine the beneficial/adverse response of cells to foreign inorganic materials as implants, engineered or accidentally produced inorganic nanoparticles. A detailed knowledge of the surface/biological fluids interface processes is needed for the design of new biocompatible materials. Researchers involved in the different disciplines face up with similar difficulties in describing and predicting phenomena occurring at the interface between solid phases and biological fluids. This review represents an attempt to integrate the knowledge from different research areas by focussing on the search for determinants driving the interaction of inorganic surfaces with biological matter.     

A review of near infrared spectroscopy and chemometrics in pharmaceutical technologies

Near-infrared spectroscopy (NIRS) is a fast and non-destructive analytical method. Associated with chemometrics, it becomes a powerful tool for the pharmaceutical industry. Indeed, NIRS is suitable for analysis of solid, liquid and biotechnological pharmaceutical forms. Moreover, NIRS can be implemented during pharmaceutical development, in production for process monitoring or in quality control laboratories.This review focuses on chemometric techniques and pharmaceutical NIRS applications. The following topics are covered: qualitative analyses, quantitative methods and on-line applications. Theoretical and practical aspects are described with pharmaceutical examples of NIRS applications.     

Galactosylated Chitosan Coated Liposomes of Ledipasvir for Liver Targeting: Chemical Synthesis,Statistical Optimization,In-vitro and In-vivo evaluation

Ledipasvir is a novel antiviral agent used in the treatment of hepatitis C. We aim in our study to increase its delivery to hepatocytes and prolong its retention within liver. Several formulae of ledipasvir loaded liposomes were prepared and the best formula regarding particle size, zeta potential, polydispersity index and entrapment efficiency was selected. On the other hand, galactosylated chitosan was synthesized in a chemical reaction. Then the best liposomes formula was coated with the galactosylated chitosan. Having galactose residues on their surface, the coated liposomes can bind to the asialoglycoprotein receptors on the targeted hepatocytes enhancing ledipasvir uptake into them. The galactosylated chitosan coated liposomes had particle size of 218.2 nm ± 7.21, zeta potential of 27.15 mV ± 1.76, polydispersity index of 0.278 ± 0.055 and entrapment efficiency % of 54.63% ± 0.05 respectively. The pharmacokinetic study revealed a significant increase in the liver peak concentration (C_max) and the area under liver concentration versus time curve AUC_(0–72 h) and significant prolongation in the liver terminal half life (t½) and mean residence time (MRT) in comparison to the oral dispersion of ledipasvir with values of 11,400 ng/g, 88,855 ng1h/g, 32.00 h and 18.11 h respectively.     

Amino-functionalized poloxamer 407 with both mucoadhesive and thermosensitive properties: preparation,characterization and application in a vaginal drug delivery system

Lack of mucoadhesive properties is the major drawback to poloxamer 407 (F127)-based in situ hydrogels for mucosal administration. The objective of the present study was to construct a novel mucoadhesive and thermosensitive in situ hydrogel drug delivery system based on an amino-functionalized poloxamer for vaginal administration. First, amino-functionalized poloxamer 407 (F127-NH₂) was synthesized and characterized with respect to its micellization behavior and interaction with mucin. Then using acetate gossypol (AG) as model drug, AG-loaded F127-NH₂-based in situ hydrogels (NFGs) were evaluated with respect to rheology, drug release, ex vivo vaginal mucosal adhesion, in vivo intravaginal retention and local irritation after vaginal administration to healthy female mice. The results show that F127-NH₂ is capable of forming a thermosensitive in situ hydrogel with sustained drug release properties. An interaction between positively charged F127-NH₂ and negatively charged mucin was revealed by changes in the particle size and zeta potential of mucin particles as well as an increase in the complex modulus of NFG caused by mucin. Ex vivo and in vivo fluorescence imaging and quantitative analysis of the amount of AG remaining in mouse vaginal lavage all demonstrated greater intravaginal retention of NFG than that of an unmodified F127-based in situ hydrogel. In conclusion, amino group functionalization confers valuable mucoadhesive properties on poloxamer 407.