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1.
Biomedicines ; 10(8)2022 Aug 14.
Article in English | MEDLINE | ID: mdl-36009516

ABSTRACT

This work aimed to test the effect of 7-day exposure of rats to multifunctional enkephalin analogs LYS739 and LYS744 at doses of 3 mg/kg and 10 mg/kg on the protein composition of rat spleen lymphocytes, brain cortex, and hippocampus. Alterations of proteome induced by LYS739 and LYS744 were compared with those elicited by morphine. The changes in rat proteome profiles were analyzed by label-free quantification (MaxLFQ). Proteomic analysis indicated that the treatment with 3 mg/kg of LYS744 caused significant alterations in protein expression levels in spleen lymphocytes (45), rat brain cortex (31), and hippocampus (42). The identified proteins were primarily involved in RNA processing and the regulation of cytoskeletal dynamics. In spleen lymphocytes, the administration of the higher 10 mg/kg dose of both enkephalin analogs caused major, extensive modifications in protein expression levels: LYS739 (119) and LYS744 (182). Among these changes, the number of proteins associated with immune responses and apoptotic processes was increased. LYS739 treatment resulted in the highest number of alterations in the rat brain cortex (152) and hippocampus (45). The altered proteins were functionally related to the regulation of transcription and cytoskeletal reorganization, which plays an essential role in neuronal plasticity. Administration with LYS744 did not increase the number of altered proteins in the brain cortex (26) and hippocampus (26). Our findings demonstrate that the effect of κ-OR full antagonism of LYS744 is opposite in the central nervous system and the peripheral region (spleen lymphocytes).

2.
Naunyn Schmiedebergs Arch Pharmacol ; 394(5): 955-965, 2021 05.
Article in English | MEDLINE | ID: mdl-33404687

ABSTRACT

Lithium (Li) represents a first choice mood stabilizer for bipolar disorder (BD). Despite extensive clinical use, questions regarding its mechanism of action and pathological mechanism of renal function impairment by Li remain open. The present study aimed to improve our knowledge in this area paying special attention to the relationship between the length of Li action, lipid peroxidation (LP), and Na+/K+-ATPase properties. The effects of therapeutic Li doses, administered daily to male Wistar rats for 1 (acute), 7 (short term) and 28 days (chronic), were studied. For this purpose, Na+/K+-ATPase activity measurements, [3H]ouabain binding and immunoblot analysis of α-Na+/K+-ATPase were performed. Li-induced LP was evaluated by determining the malondialdehyde concentration by HPLC. Sleep deprivation (SD) was used as an experimental approach to model the manic phase of BD. Results obtained from the kidney were compared to those obtained from erythrocytes and different brain regions in the same tested animals. Whereas treatment with therapeutic Li concentration did not bring any LP damage nor significant changes of Na+/K+-ATPase expression and [3H]ouabain binding in the kidney, it conferred strong protection against this type of damage in the forebrain cortex. Importantly, the observed changes in erythrocytes indicated changes in forebrain cortices. Thus, different resistance to SD-induced changes of LP and Na+/K+-ATPase was detected in the kidney, erythrocytes and the brain of Li-treated rats. Our study revealed the tissue-specific protective properties of Li against LP and Na+/K+-ATPase regulation.


Subject(s)
Antimanic Agents/pharmacology , Bipolar Disorder/drug therapy , Lipid Peroxidation/drug effects , Lithium Carbonate/pharmacology , Animals , Antimanic Agents/administration & dosage , Brain/drug effects , Brain/metabolism , Disease Models, Animal , Erythrocytes/drug effects , Erythrocytes/metabolism , Kidney/drug effects , Kidney/metabolism , Lithium Carbonate/administration & dosage , Male , Rats , Rats, Wistar , Sleep Deprivation/psychology , Sodium-Potassium-Exchanging ATPase/metabolism , Time Factors
3.
J Nat Prod ; 79(4): 1073-83, 2016 Apr 22.
Article in English | MEDLINE | ID: mdl-26998557

ABSTRACT

Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-Leu-Met-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure-activity relationship for the effect of amino acid residue substitution in HYL.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Bee Venoms/pharmacology , Bees/chemistry , Animals , Antimicrobial Cationic Peptides/chemistry , Humans , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity Relationship
4.
Biopolymers ; 106(3): 245-59, 2016 May.
Article in English | MEDLINE | ID: mdl-26916937

ABSTRACT

Deamination of vasopressin (AVP) enhances its antidiuretic activity. Moreover, introduction of D-Arg8 instead of its L enantiomer in deamino-vasopressin (dAVP) results in an extremely potent and selective antidiuretic agonist - desmopressin (dDAVP). In this study we describe the synthesis, pharmacological properties and structures of these two potent antidiuretic agonists, and their inverso analogs. The structures of the peptides are studied in micellar and liposomic models of cell membrane using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy supported by molecular dynamics simulations. Our conformational studies have shown that desmopressin in a membrane mimicking environment adopts one of the characteristic for vasopressin-like peptides ß-turn - in position 3,4. Furthermore, dDAVP shows the tendency to create a ß-turn in the Cys6-Gly9 C-tail, considered to be important for the antidiuretic activity, and also some tendency to adopt a 5,6 ß-turn. In desmopressin, in contrast to the native vasopressin, deamino-vasopressin and [D-Arg8]-vasopressin (DAVP), the Arg8 side chain, crucial for the pressor and antidiuretic activities, is very well exposed for interaction with the receptor, whereas Gly9, crucial for the pressor and uterotonic activities, is situated together with the C-terminal amide group very close to the tocin ring. The arrangements of the Gln4 and Asn5 side chains, being crucial for OT activity, also differ in desmopressin as compared to those of AVP, dAVP and DAVP. These differences in arrangement of the important for activities side chains are likely to explain extremely potent and selective antidiuretic activities of desmopressin. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 245-259, 2016.


Subject(s)
Antidiuretic Agents/chemical synthesis , Deamino Arginine Vasopressin/chemical synthesis , Liposomes/chemistry , Oxytocics/chemical synthesis , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Animals , Antidiuretic Agents/pharmacology , Cyclization , Deamino Arginine Vasopressin/pharmacology , Female , Fluorenes/chemistry , Hydrogen Bonding , Micelles , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Oxytocics/pharmacology , Phosphatidylglycerols/chemistry , Protein Structure, Secondary , Rats, Wistar , Solid-Phase Synthesis Techniques/methods , Uterus/drug effects , Uterus/physiology
5.
Eur Biophys J ; 44(8): 727-43, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26290060

ABSTRACT

We describe the synthesis, pharmacological properties, and structures of antidiuretic agonists, arginine vasopressin (AVP) and [D-Arg(8)]-vasopressin (DAVP), and their inverso analogues. The structures of the peptides are studied based on micellar and liposomic models of cell membranes using CD spectroscopy. Additionally, three-dimensional structures in mixed anionic-zwitterionic micelles are obtained using NMR spectroscopy and molecular dynamics simulations. NMR data have shown that AVP and DAVP tend to adopt typical of vasopressin-like peptides ß-turns: in the 2-5 and 3-6 fragments. The inverso-analogues also adopt ß-turns in the 3-6 fragments. For this reason, their inactivity seems to be due to the difference in side chains orientations of Tyr(2), Phe(3), and Arg(8), important for interactions with the receptors. Again, the potent antidiuretic activity of DAVP can be explained by CD data suggesting differences in mutual arrangement of the aromatic side chains of Tyr(2) and Phe(3) in this peptide in liposomes rather than of native AVP. In the presence of liposomes, the smallest conformational changes of the peptides are noticed with DPPC and the largest with DPPG liposomes. This suggests that electrostatic interactions are crucial for the peptide-membrane interactions. We obtained similar, probably active, conformations of the antidiuretic agonists in the mixed DPC/SDS micelles (5:1) and in the mixed DPPC/DPPG (7:3) liposomes. Thus it can be speculated that the anionic-zwitterionic liposomes as well as the anionic-zwitterionic micelles, mimicking the eukaryotic cell membrane environment, partially restrict conformational freedom of the peptides and probably induce conformations resembling those of biologically relevant ones.


Subject(s)
Antidiuretic Agents/chemistry , Arginine Vasopressin/analogs & derivatives , Cell Membrane/chemistry , Liposomes/chemistry , Micelles , Molecular Dynamics Simulation , Amino Acid Sequence , Animals , Antidiuretic Agents/chemical synthesis , Antidiuretic Agents/pharmacology , Arginine Vasopressin/chemical synthesis , Arginine Vasopressin/chemistry , Arginine Vasopressin/pharmacology , Cell Membrane/drug effects , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Wistar
6.
J Pept Sci ; 20(11): 885-95, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25123582

ABSTRACT

The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic ß-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bee Venoms/chemistry , Escherichia coli/drug effects , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/isolation & purification , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/isolation & purification , Bee Venoms/genetics , Bee Venoms/isolation & purification , Bee Venoms/pharmacology , Bees/genetics , Cell Membrane Permeability/drug effects , Drug Design , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Fluorescence , Hemolysis/drug effects , Hydrophobic and Hydrophilic Interactions , Liposomes/metabolism , Microscopy, Electron, Transmission , Molecular Sequence Data , Phospholipids/metabolism , Protein Structure, Secondary , Tryptophan/chemistry
7.
J Pept Sci ; 20(9): 725-35, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24920043

ABSTRACT

The antimicrobial 40-amino-acid-peptide lucifensin was synthesized by native chemical ligation (NCL) using N-acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid-phase synthesis is imcompatible. Two models of ligation were developed: [15+25] Ala-Cys and [19+21] His-Cys. The [19+21] His-Cys method gives lower yield because of the lower stability of 18-peptide-His-Nbz-CONH2 peptide, as suggested by density functional theory calculation. Acetamidomethyl-deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1-6], [C3-5], and [C2-4], which limits its ability to form α-helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one-pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium.


Subject(s)
Anti-Infective Agents , Antimicrobial Cationic Peptides , Defensins , Gram-Positive Bacteria/growth & development , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Defensins/chemical synthesis , Defensins/chemistry , Defensins/pharmacology , Protein Structure, Secondary
8.
Dev Comp Immunol ; 46(2): 165-70, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24747431

ABSTRACT

Expression of the previously reported defensin of the tick Dermacentor marginatus (defDM) was analysed in different organs by RT-PCR. mRNA of the defDM gene was detected in the hemolymph, midgut and salivary glands. Moreover defDM was isolated from the tick hemolymph using RP-HPLC and its sequence was determined by mass spectrometry and Edman degradation. Synthetic peptide was used for determining biological activities. The results showed an anti-Gram-positive bacterial role for the defensin. As D. marginatus ticks appear not to be vectors of the Lyme disease agent of the complex Borrelia burgdorferi sensu lato, we tested the influence of defDM on Borrelia afzelii. There is a very clear borrelicidal activity of the defensin, which is concentration dependent and suggests a possible role in the clearing of Borrelia ingested by D. marginatus ticks.


Subject(s)
Anti-Bacterial Agents/pharmacology , Arthropod Proteins/pharmacology , Borrelia burgdorferi Group/drug effects , Defensins/pharmacology , Dermacentor/chemistry , Animals , Anti-Bacterial Agents/isolation & purification , Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Arthropod Proteins/isolation & purification , Defensins/isolation & purification , Female , Microbial Sensitivity Tests
9.
J Pept Sci ; 20(6): 375-84, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24616110

ABSTRACT

A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Bee Venoms/chemistry , Bees/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Antimicrobial Cationic Peptides/isolation & purification , Bacillus subtilis/drug effects , Bee Venoms/isolation & purification , Bee Venoms/pharmacology , Dose-Response Relationship, Drug , Erythrocytes/drug effects , Escherichia coli/drug effects , Microbial Sensitivity Tests , Micrococcus luteus/drug effects , Models, Molecular , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Structure-Activity Relationship
10.
Amino Acids ; 45(1): 143-57, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23483218

ABSTRACT

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH2 (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bee Venoms/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Bee Venoms/chemistry , Bee Venoms/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Hymenoptera/metabolism , Microbial Sensitivity Tests , Sequence Analysis, Protein , Structure-Activity Relationship , Surface-Active Agents , Unilamellar Liposomes/metabolism
11.
Eur J Med Chem ; 62: 352-70, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23376252

ABSTRACT

A series of symmetrically bis-substituted imidazole analogs bearing at the N-1 and N-3 two biphenyl moieties ortho substituted either with tetrazole or carboxylate functional groups was designed based on docking studies and utilizing for the first time an extra hydrophobic binding cleft of AT1 receptor. The synthesized analogs were evaluated for their in vitro antagonistic activities (pA2 values) and binding affinities (-logIC50 values) to the Angiotensin II AT1 receptor. Among them, the potassium (-logIC50 = 9.04) and the sodium (-logIC50 = 8.54) salts of 4-butyl-N,N'-bis{[2'-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (12a and 12b, respectively) as well as its free acid 11 (-logIC50 = 9.46) and the 4-butyl-2-hydroxymethyl-N,N'-bis{[2'-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (14) (-logIC50 = 8.37, pA2 = 8.58) showed high binding affinity to the AT1 receptor and high antagonistic activity (potency). The potency was similar or even superior to that of Losartan (-logIC50 = 8.25, pA2 = 8.25). On the contrary, 2-butyl-N,N'-bis{[2'-[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (27) (-logIC50 = 5.77) and 2-butyl-4-chloro-5-hydroxymethyl-N,N'-bis{[2'-[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (30) (-logIC50 = 6.38) displayed very low binding affinity indicating that the orientation of the n-butyl group is of primary importance. Docking studies of the representative highly active 12b clearly showed that this molecule has an extra hydrophobic binding feature compared to prototype drug Losartan and it fits to the extra hydrophobic cavity. These results may contribute to the discovery and development of a new class of biologically active molecules through bis-alkylation of the imidazole ring by a convenient and cost effective synthetic strategy.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Drug Design , Imidazoles/pharmacology , Angiotensin II Type 1 Receptor Blockers/chemical synthesis , Angiotensin II Type 1 Receptor Blockers/chemistry , Dose-Response Relationship, Drug , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Models, Molecular , Molecular Structure , Quantum Theory , Structure-Activity Relationship
12.
Chem Biol Drug Des ; 81(3): 420-8, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23205571

ABSTRACT

In this study, we present the synthesis and pharmacological properties of new analogues of arginine vasopressin modified in the N-terminal part of the molecule with proline derivatives: indoline-2-carboxylic acid (Ica) and (2S,4R)-4-(naphthalene-2-ylmethyl)pyrrolidine-2-carboxylic acid. All the peptides were tested for pressor, antidiuretic and in vitro uterotonic activities. We also determined their binding affinity to the human oxytocin receptor. The Ica(2) substitution resulted in two moderately potent and selective antioxytocic agents: [Mpa(1), Ica(2), D-Arg(8)]VP and [Mpa(1),Ica(2),Val(4),D-Arg(8)]VP (pA(2) = 7.09 and 7.50, respectively). On the other hand, peptides modified with (2S,4R)-4-(naphthalene-2-ylmethyl)pyrrolidine-2-carboxylic acid, apart from their moderate antioxytocic activity, turned out to be weak antagonists of the pressor response to arginine vasopressin. The results of this study provide useful information about the structure-activity relationship of arginine vasopressin analogues and can help to design compounds with desired biological properties.


Subject(s)
Antidiuretic Agents/chemical synthesis , Arginine Vasopressin/analogs & derivatives , Drug Design , Indoles/chemistry , Proline/analogs & derivatives , Animals , Antidiuretic Agents/chemistry , Antidiuretic Agents/metabolism , Arginine Vasopressin/chemical synthesis , Arginine Vasopressin/metabolism , Humans , Kinetics , Proline/chemistry , Protein Binding , Rats , Rats, Wistar , Receptors, Oxytocin/chemistry , Receptors, Oxytocin/metabolism , Structure-Activity Relationship
13.
Eur J Med Chem ; 55: 358-74, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22889560

ABSTRACT

A convenient and facile synthesis, in silico docking studies and in vitro biological evaluation of N-substituted 5-butylimidazole derivatives as potent Angiotensin II (ANG II) receptor type 1 (AT1) blockers (ARBs) has been reported in the current study. Our efforts have been directed towards the development of an efficient synthetic route allowing the facile introduction of substituents on the imidazole ring. In particular, a series of imidazole based compounds bearing the biphenyl moiety at the N - 1 position, a halogen atom at the C-4 and polar substituents such as hydroxymethyl, aldo or carboxy group at the C-2 position were designed and synthesized. These compounds were evaluated for binding to human AT1 receptor and for ANG II antagonism in vitro on isolated rat uterus. Among them, 5-butyl-1-[[2'-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-2-carboxylic acid (30) exhibited higher binding affinity compared to the other analogues tested (-log IC(50) = 8.46). The latter analogue was also found to be the most active in the rat uterotonic test (pA(2) = 7.83). Importantly, the binding affinity was higher to that of losartan (-log IC(50) = 8.25) indicating the importance of carboxy group at the C-2 position. Experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/AT1 receptor interactions.


Subject(s)
Angiotensin II Type 1 Receptor Blockers/chemical synthesis , Angiotensin II Type 1 Receptor Blockers/pharmacology , Drug Discovery , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Molecular Docking Simulation , Receptor, Angiotensin, Type 1/metabolism , Angiotensin II Type 1 Receptor Blockers/chemistry , Angiotensin II Type 1 Receptor Blockers/metabolism , Animals , Chemistry Techniques, Synthetic , Female , HEK293 Cells , Humans , Imidazoles/chemistry , Imidazoles/metabolism , In Vitro Techniques , Inhibitory Concentration 50 , Protein Conformation , Rats , Rats, Wistar , Receptor, Angiotensin, Type 1/chemistry , Uterine Contraction/drug effects , Uterus/drug effects , Uterus/physiology
14.
Int J Pept Res Ther ; 18(2): 117-124, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22593719

ABSTRACT

In the current work we present some pharmacological characteristics of ten new analogues of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) modified in the N-terminal part of the molecule with a variety of acyl substituents. Of the many acylating agents used previously with B(2) receptor antagonists, the following residues were chosen: 1-adamantaneacetic acid (Aaa), 1-adamantanecarboxylic acid (Aca), 4-tert-butylbenzoic acid (t-Bba), 4-aminobenzoic acid (Aba), 12-aminododecanoic acid (Adc), succinic acid (Sua), 4-hydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3-(4-hydroxyphenyl)propionic acid and 6-hydroxy-2-naphthoic acid. Biological activity of the compounds was assessed in the in vivo rat blood pressure test and the in vitro rat uterus test. Surprisingly, N-terminal substitution of the bradykinin peptide chain itself with aforementioned groups resulted in antagonists of bradykinin in the pressor test and suppressed agonistic potency in the uterotonic test. These interesting findings need further studies as they can be helpful for designing more potent B(2) receptor blockers.

15.
Amino Acids ; 43(5): 2047-58, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22526241

ABSTRACT

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4'-pentenyl) alanine residues (S (5)) incorporated at i and i + 4 positions or between R-2-(7'-octenyl) alanine (R (8)) and S (5) incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.


Subject(s)
Alkenes/chemistry , Antimicrobial Cationic Peptides/chemical synthesis , Bee Venoms/chemistry , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/pharmacology , Bacteria/drug effects , Bacteria/growth & development , Bees/chemistry , Candida albicans/drug effects , Candida albicans/growth & development , Chromatography, Reverse-Phase , Circular Dichroism , Erythrocytes/drug effects , Hemolysis/drug effects , Humans , Magnetic Resonance Spectroscopy , Microbial Sensitivity Tests , Molecular Sequence Data , Protein Structure, Secondary , Proteolysis
16.
Cell Mol Life Sci ; 69(17): 2951-66, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22527714

ABSTRACT

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.


Subject(s)
Cell Membrane Permeability/drug effects , Cell-Penetrating Peptides/pharmacology , Cell-Penetrating Peptides/pharmacokinetics , Drug Delivery Systems , Endocytosis , Animals , Aorta/cytology , Aorta/drug effects , Aorta/metabolism , Astrocytoma/drug therapy , Astrocytoma/metabolism , Astrocytoma/pathology , Brain/metabolism , Cattle , Cells, Cultured , Chemotaxis , Chorioallantoic Membrane , Cyclic AMP/metabolism , Dermis/cytology , Dermis/drug effects , Dermis/metabolism , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Female , Hepacivirus , Hepatitis C/drug therapy , Hepatitis C/metabolism , Hepatitis C/virology , Humans , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Male , Neovascularization, Physiologic/drug effects , Protein Transport , Quantitative Structure-Activity Relationship , Rats , Rats, Wistar , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , Uterine Contraction/drug effects
17.
Peptides ; 33(1): 18-26, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22100226

ABSTRACT

Recently, we have isolated and characterized remarkable antimicrobial peptides (AMPs) from the venom reservoirs of wild bees. These peptides (melectin, lasioglossins, halictines and macropin) and their analogs display high antimicrobial activity against Gram-positive and -negative bacteria, antifungal activity and low or moderate hemolytic activity. Here we describe cytotoxicity of the above-mentioned AMPs and some of their analogs toward two normal cell lines (human umbilical vein endothelial cells, HUVEC, and rat intestinal epithelial cells, IEC) and three cancer cell lines (HeLa S3, CRC SW 480 and CCRF-CEM T). HeLa S3 cells were the most sensitive ones (concentration causing 50% cell death in the case of the most toxic analogs was 2.5-10 µM) followed by CEM cells. For the other cell lines to be killed, the concentrations had to be four to twenty times higher. These results bring promising outlooks of finding medically applicable drugs on the basis of AMPs. Experiments using fluorescently labeled lasioglossin III (Fl-VNWKKILGKIIKVVK-NH(2)) as a tracer confirmed that the peptides entered the mammalian cells in higher quantities only after they reached the toxic concentration. After entering the cells, their concentration was the highest in the vicinity of the nucleus, in the nucleolus and in granules which were situated at very similar places as mitochondria. Experiments performed using cells with tetramethylrhodamine labeled mitochondria showed that mitochondria were fragmented and lost their membrane potential in parallel with the entrance of the peptides into the cell and the disturbance of the cell membrane.


Subject(s)
Anti-Infective Agents/chemistry , Bee Venoms/chemistry , Peptides/chemistry , Peptides/pharmacology , Amino Acid Sequence , Animals , Anti-Infective Agents/toxicity , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Drug Screening Assays, Antitumor , Epithelial Cells/drug effects , HeLa Cells , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Intestines/cytology , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Sequence Data , Peptides/pharmacokinetics , Peptides/toxicity , Rats , Toxicity Tests
18.
Amino Acids ; 42(5): 1715-25, 2012 May.
Article in English | MEDLINE | ID: mdl-21416381

ABSTRACT

Analogs of the H-Tyr-Asp-Pro-Ala-Pro-OH pentapeptide with D-amino acid residues either in differing or in all of the positions of the sequences were prepared and their oostatic potency was compared with that of the parent pentapeptide. The D-amino acid residue containing analogs exhibited an equal or even higher oostatic effect in the flesh fly Neobellieria bullata than the parent peptide. Contrary to the rapid incorporation of radioactivity from the labeled H-Tyr-Asp-[3H]Pro-Ala-Pro-OH pentapeptide into the ovaries of N. bullata in vitro, the radioactivity incorporation from the labeled pentapeptides with either D-aspartic acid or D-alanine was significantly delayed. As compared to the parent pentapeptide, also the degradation of both the D-amino acid-containing analogs mentioned above proceeded at a significantly lower rate. The decreased intake of radioactivity, the lower degradation and finally also the high oostatic effect may be ascribed to the decreased enzymatic degradation of the peptide bonds neighboring the D-amino acid residues in the corresponding peptides. The introduction of the non-coded D: -amino acids thus enhances the oostatic effect in N. bullata owing to the prolonged half-life of the corresponding pentapeptides, which can thus affect more ovarian cells.


Subject(s)
Alanine/chemistry , D-Aspartic Acid/chemistry , Ovary/cytology , Peptides/chemistry , Alanine/metabolism , Amino Acids/biosynthesis , Amino Acids/chemistry , Animals , D-Aspartic Acid/metabolism , Female , Magnetic Resonance Spectroscopy , Ovary/growth & development , Proteolysis , Sarcophagidae/chemistry , Sarcophagidae/growth & development , Tritium/chemistry
19.
Amino Acids ; 43(2): 617-27, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22038179

ABSTRACT

In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26).


Subject(s)
Arginine Vasopressin/analogs & derivatives , Arginine Vasopressin/pharmacology , Oxytocin/analogs & derivatives , Oxytocin/pharmacology , Receptors, Oxytocin/antagonists & inhibitors , Amino Acid Sequence , Animals , Antidiuretic Agents , Arginine Vasopressin/chemical synthesis , Carboxylic Acids/chemistry , Cyclohexanes/chemistry , Drug Design , Female , HEK293 Cells , Humans , In Vitro Techniques , Male , Oxytocin/chemical synthesis , Protein Binding , Protein Structure, Secondary , Rats , Rats, Wistar , Receptors, Oxytocin/metabolism , Vasoconstrictor Agents
20.
Amino Acids ; 43(2): 751-61, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22038181

ABSTRACT

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.


Subject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Bee Venoms/chemistry , Bees/chemistry , Peptides, Cyclic/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Bee Venoms/chemical synthesis , Bee Venoms/pharmacology , Candida albicans/drug effects , Cystine/chemical synthesis , Cystine/chemistry , Erythrocytes/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/ultrastructure , Hemolysis , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Protein Structure, Secondary , Sequence Analysis, Protein
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