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1.
Eur J Med Chem ; 174: 309-329, 2019 Jul 15.
Article in English | MEDLINE | ID: mdl-31055147

ABSTRACT

Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5'-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure-activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.


Subject(s)
Antitubercular Agents/pharmacology , Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Isoquinolines/pharmacology , Mycobacterium tuberculosis/drug effects , Piperazines/pharmacology , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , IMP Dehydrogenase/chemistry , Isoquinolines/chemical synthesis , Isoquinolines/chemistry , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Structure-Activity Relationship
2.
Molecules ; 23(9)2018 Aug 30.
Article in English | MEDLINE | ID: mdl-30200264

ABSTRACT

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.


Subject(s)
Light , Peptides, Cyclic/chemistry , Photochemical Processes/drug effects , Chromatography, Liquid , Dimethyl Sulfoxide/chemistry , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Dynamics Simulation , Molecular Structure , Photolysis , Spectrometry, Fluorescence , Ultraviolet Rays
3.
Bioorg Med Chem Lett ; 28(19): 3265-3270, 2018 10 15.
Article in English | MEDLINE | ID: mdl-30143423

ABSTRACT

Aurora kinases as regulators of cell division have become promising therapeutic targets recently. Here we report novel, low molecular weight benzothiophene-3-carboxamide derivatives designed and optimized for inhibiting Aurora kinases. The most effective compound 36 inhibits Aurora kinases in vitro in the nanomolar range and diminishes HCT 116 cell viability blocking cytokinesis and inducing apoptosis. According to western blot analysis, the lead molecule inhibits Aurora kinases equipotently to VX-680 (Tozasertib) and similarly synergizes with other targeted drugs.


Subject(s)
Amides/chemistry , Aurora Kinase A/antagonists & inhibitors , Aurora Kinase B/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Thiophenes/chemistry , HCT116 Cells , Humans , Inhibitory Concentration 50
4.
Bioorg Med Chem Lett ; 28(14): 2391-2398, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29935772

ABSTRACT

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Sunitinib, a multikinase inhibitor, was the first Fms-like tyrosine kinase 3 (FLT3) inhibitor clinically used against AML. Off-target effects are a major concern for multikinase inhibitors. As targeted delivery may reduce such undesired side effects, our goal was to develop novel amino acid substituted derivatives of sunitinib which are potent candidates to be used conjugated with antibodies and peptides. In the current paper we present the synthesis, physicochemical and in vitro characterization of sixty two Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutant kinase inhibitors, bearing amino acid moieties, fit to be conjugated with peptide-based delivery systems via their carboxyl group. We determined the solubility, pKa, CHI and LogP values of the compounds along with their inhibition potential against FLT3-ITD mutant kinase and on MV4-11 cell line. The ester derivatives of the compounds inhibit the growth of the MV4-11 leukemia cell line at submicromolar concentration.


Subject(s)
Amino Acids/pharmacology , Antineoplastic Agents/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Sunitinib/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Amino Acids/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leukemia, Myeloid, Acute/metabolism , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Solubility , Structure-Activity Relationship , Sunitinib/chemical synthesis , Sunitinib/chemistry , Tandem Repeat Sequences/drug effects , fms-Like Tyrosine Kinase 3/metabolism
5.
J Med Chem ; 61(14): 6277-6292, 2018 Jul 26.
Article in English | MEDLINE | ID: mdl-29928803

ABSTRACT

The overexpression of AXL kinase has been described in many types of cancer. Due to its role in proliferation, survival, migration, and resistance, AXL represents a promising target in the treatment of the disease. In this study we present a novel compound family that successfully targets the AXL kinase. Through optimization and detailed SAR studies we developed low nanomolar inhibitors, and after further biological characterization we identified a potent AXL kinase inhibitor with favorable pharmacokinetic profile. The antitumor activity was determined in xenograft models, and the lead compounds reduced the tumor size by 40% with no observed toxicity as well as lung metastasis formation by 66% when compared to vehicle control.


Subject(s)
Drug Design , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Animals , Caco-2 Cells , Humans , Mice , Protein Kinase Inhibitors/pharmacokinetics , Structure-Activity Relationship , Sulfonamides/pharmacokinetics , Tissue Distribution , Axl Receptor Tyrosine Kinase , Benzenesulfonamides
6.
Bioorg Med Chem Lett ; 28(4): 769-773, 2018 02 15.
Article in English | MEDLINE | ID: mdl-29329658

ABSTRACT

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).


Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity Relationship , Polo-Like Kinase 1
7.
Eur J Med Chem ; 108: 623-643, 2016 Jan 27.
Article in English | MEDLINE | ID: mdl-26724730

ABSTRACT

Melanoma is an aggressive form of skin cancer and it is generally associated with poor prognosis in patients with late-stage disease. Due to the increasing occurrence of melanoma, there is a need for the development of novel therapies. A new series of diarylamide and diarylurea derivatives containing imidazo[1,2-a]pyridine or imidazo[1,2-a]pyrazine scaffold was designed and synthesized to investigate their in vitro efficacy against the A375P human melanoma cell line. We found several compounds expressing submicromolar IC50 values against the A375P cells, from which 15d, 17e, 18c, 18h, 18i demonstrated the highest potencies with IC50 below 0.06 µM.


Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Drug Design , Imidazoles/pharmacology , Melanoma/drug therapy , Melanoma/pathology , Pyrazines/pharmacology , Pyridines/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Molecular Structure , Pyrazines/chemical synthesis , Pyrazines/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship
8.
J Mol Recognit ; 28(4): 253-60, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25683456

ABSTRACT

WSXWS motif is a conserved amino acid sequence that is present in type I cytokine receptors. This motif that can be found both in the ligand binding chains and signal transducer molecule of the receptors with different amino acids at the position "X" plays a role in the receptor folding, ligand binding and signal transduction as well. Structural analysis proved that WSEWS motif of IL-6R is located in a highly accessible location in the protein. Structural properties and chemotaxis of a tetrapeptide library with SXWS sequence, where X was the 19 proteinogenic amino acids except cystein were systematically studied earlier. It has been proved that C-terminal amidation and the identity of amino acid X had a pronounced influence on the chemotactic properties but less of the structure of the peptides. Here, we present our findings on the effect of a tetrapeptide and a pentapeptide library with the sequence of SXWS and WSXWS on the chemotaxis and adhesion of J774 murine macrophage cell line. We studied the effect of the presence/absence of N-terminal tryptophan and the different amino acids at the X position on these physiological responses. Results indicated that amino acid X had a marked influence on chemotaxis, adhesion as well as on proliferation induced by (W)SXWS peptides. Elongation of SXWS sequence with a tryptophan at the N terminus also altered pronouncedly all the physiological responses of the cells studied. A good correlation could be observed between the chemotaxis and the proliferation and physicochemical parameters of the amino acid X.


Subject(s)
Chemotaxis , Macrophages/cytology , Macrophages/metabolism , Peptides/metabolism , Animals , Cell Adhesion , Cell Line , Cytokine Receptor gp130/metabolism , Mice
9.
Acta Pharm Hung ; 84(3): 91-104, 2014.
Article in Hungarian | MEDLINE | ID: mdl-25470976

ABSTRACT

The EGFR inhibitor erlotinib possesses high anti-tumor effect but despite the good clinical responses in most of the cases recrudescence occures. This can be attributed to a secondary, acquired mutation causing resistance to tyrosine kinase inhibitors. In our work we were looking for small-molecule inhibitors, which simultaneously affect on the proliferation of erlotinib-sensitive PC9 cells and PC9-ER erlotinib-resistant cells. A set of molecules were selected from Vichem Chemie Research Ltd.'s kinase inhibitor compound library (Nested Chemical Library™). According to the results of medium throughput screening (MTS) of this set of compounds, novel structures with pyrido[2,3-b]pyrazine core were designed. These compounds were proved to be effective inhibitors of resistant cells in phenotypic screening. Based on these results structure-activity relationships were set up. The pyrido[2,3-b]pyrazine core was synthesized by a condensation reaction, which resulting two asymmetric products. In the reaction two regioisomer intermediates formed, and one of the products is the intermediate of the effective compounds. This condensation reaction was optimized, the regioisomers were identified by NMR analysis and X-ray crystallography. As a result of optimization we found that lower reaction temperature and replacement of dimethylformamide solvent with trifluoroacetic acid provided the undesired isomer in less than 2 % ratio.


Subject(s)
Antineoplastic Agents/pharmacology , Biochemistry/methods , Cell Line, Tumor/drug effects , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Quinazolines/pharmacology , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Humans , Protein Kinase Inhibitors/pharmacology , Structure-Activity Relationship
10.
J Mol Recognit ; 25(1): 24-31, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22213448

ABSTRACT

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position 'X' of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS-SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent-exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer-assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the 'X' position indicate that member 'X' of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane.


Subject(s)
Amino Acids/chemistry , Chemotaxis/physiology , Models, Molecular , Peptides/chemistry , Tetrahymena/physiology , Chemotactic Factors , Chromatography, High Pressure Liquid , Computational Biology/methods , Computer Simulation , Mass Spectrometry , Molecular Conformation , Oligopeptides/chemical synthesis , Oligopeptides/chemistry , Peptides/chemical synthesis , Tetrahymena/growth & development
11.
Biomaterials ; 30(36): 6803-13, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19765821

ABSTRACT

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide-lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys)4]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 +/- 5.83 pg Fe and 87.46 +/- 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys)4-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell-nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.


Subject(s)
Endocytosis/physiology , Liposomes , Magnetics , Nanoparticles/chemistry , Oxidative Stress , 3T3 Cells , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Cations/chemistry , Cell Proliferation , Cytoskeleton/metabolism , Ferric Compounds/chemistry , Lipid Bilayers/chemistry , Liposomes/chemistry , Liposomes/metabolism , Materials Testing , Mice , Neurons/cytology , Neurons/metabolism , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Stem Cells/cytology , Stem Cells/metabolism
12.
Bioconjug Chem ; 20(7): 1307-14, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19552459

ABSTRACT

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/pharmacokinetics , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Oligonucleotides, Antisense/pharmacokinetics , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , Amino Acid Sequence , Animals , Carrier Proteins/chemical synthesis , Carrier Proteins/genetics , Cell Line, Transformed , Cell-Penetrating Peptides , Circular Dichroism , Down-Regulation , Drug Carriers/chemical synthesis , Gene Transfer Techniques , Mice , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/genetics , Protein Conformation , Protein Transport , RNA-Binding Protein EWS
13.
Antiviral Res ; 82(1): 89-94, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19200809

ABSTRACT

Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound 8e displayed an antivirally effective concentration of 0.4 microM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index > or =50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound 8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Glycopeptides/chemistry , Glycopeptides/pharmacology , Orthomyxoviridae/drug effects , Animals , Cell Line , Dogs , Drug Evaluation, Preclinical , Humans , Hydrophobic and Hydrophilic Interactions , Influenza, Human/drug therapy , Influenza, Human/virology , Orthomyxoviridae/physiology , Structure-Activity Relationship
14.
Arch Biochem Biophys ; 454(2): 146-54, 2006 Oct 15.
Article in English | MEDLINE | ID: mdl-16987492

ABSTRACT

Octanoyl and palmitoyl groups were coupled to the N-terminus of an analog of the SV40 nuclear localization signal peptide, SV126-133(Ser128), to study the effect of the fatty acid chain length on the complex formation with a single-stranded antisense oligodeoxynucleotide (ODN) and on the cellular uptake of the complex. The strongest binding affinity was observed for the palmitoylated peptide, indicating the better accessibility of the positively charged lysyl and arginyl side-chains to the phosphate groups due to the turn structures stabilized by the palmitoyl group. On increase of the peptide to ODN molar ratio (rM), gradual unstacking of the bases was observed, the maximal rate being reached at rM=10. At rM>10 restacking of the nucleotide bases was detected and the ODN was completely encapsulated in a liposome-like structure made up of palmitoylated peptides. Cell translocation experiments revealed a highly efficient cell transport of the ODN by palmitoylated SV40 peptide at rM>10.


Subject(s)
Caprylates/chemistry , Oligodeoxyribonucleotides, Antisense/metabolism , Oligopeptides/chemistry , Palmitic Acids/chemistry , Acylation , Animals , Biological Transport , Cell Membrane Permeability , Circular Dichroism , Drug Carriers , Gene Transfer Techniques , Green Fluorescent Proteins/genetics , Mice , Microscopy, Atomic Force , Molecular Conformation , NIH 3T3 Cells , Oligodeoxyribonucleotides, Antisense/administration & dosage , Oligodeoxyribonucleotides, Antisense/pharmacology , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS , Spectroscopy, Fourier Transform Infrared
15.
Biochim Biophys Acta ; 1764(10): 1586-91, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16997641

ABSTRACT

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.


Subject(s)
Goats/metabolism , Lactalbumin/chemistry , Tryptophan/chemistry , Amino Acid Substitution , Animals , Calorimetry, Differential Scanning , Crystallography , Fluorescence , Lactalbumin/genetics , Mutation , Spectrometry, Fluorescence , Tryptophan/genetics
16.
J Biol Chem ; 280(44): 36873-82, 2005 Nov 04.
Article in English | MEDLINE | ID: mdl-16103112

ABSTRACT

For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin alpha2beta1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of alpha2beta1 because it only binds to the activated state of alpha2beta1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of alpha2beta1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of alpha2beta1 are dependent on the cell type where alpha2beta1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and alpha2beta1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of alpha2beta1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.


Subject(s)
Antibodies, Monoclonal/pharmacology , Blood Platelets/metabolism , Collagen Type I/metabolism , Integrin alpha2beta1/chemistry , Integrin alpha2beta1/metabolism , Actins/metabolism , Animals , Blood Platelets/immunology , CHO Cells , Cell Adhesion , Cricetinae , Humans , Integrin alpha2beta1/immunology , Jurkat Cells , Leukocytes, Mononuclear/metabolism , Peptide Fragments/chemical synthesis , Peptide Fragments/metabolism , Platelet Adhesiveness/immunology , Protein Conformation
17.
J Pept Sci ; 10(1): 56-65, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14959892

ABSTRACT

The mucin-2 (MUC2) glycoprotein secreted by the epithelial cells of human colon may be abnormally under-glycosylated in the case of cancer. Monoclonal antibody (mAb) 994 raised against the immunogenic part of the protein core, recognizes malignant human colon tissues as well as pentapeptides with TX1TX2T motif present in MUC2. Using a combinatorial approach and ELISA experiments it was found that mAb 994 is able to recognize peptides of the sub-library TQTX2T very strongly, and to some extent also peptides from TETX2T, TLTX2T and TVTX2T sub-libraries. Binding studies with peptides corresponding to the TQTX2T and TETX2T sub-libraries showed that mAb 994 recognized only six peptides (IC50 = 9-208 micromol dm(-3)) from the 19 compounds of the TQTX2T sub-library and only three peptides (IC50 = 3500-16700 micromol dm(-3)) from the 'second-best' TETX2T sub-library. The most pronounced mAb binding occurred when Gln was in position X1 and it was much weaker in the case of Glu, Val or Leu. As for X2 amino acids, the presence of Pro, Ala can provide a strong, while Tyr, Trp, Phe and Ser a weaker, peptide-antibody interaction. Data from this study suggest that pentapeptide TQTPT, whose sequence is present in the native protein, is bound most strongly. However, almost identical binding properties were observed with peptide TQTAT, whose sequence is not present in the protein. Apart from this, some other 'heteroclitic' peptides were found with a different rank in the binding-hierarchy. Based on these peptides artificial compounds can be prepared as potential candidates for vaccine development. Results of this study also provide a rationale for understanding the molecular background of the heteroclitic nature of the MUC2 protein core specific mAb 994.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunodominant Epitopes/chemistry , Mucins/chemistry , Peptides/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions , Chromatography, High Pressure Liquid , Humans , Molecular Sequence Data , Mucin-2 , Mucins/immunology , Peptide Library , Peptides/chemical synthesis , Peptides/immunology , Repetitive Sequences, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
18.
Cell Biol Int ; 27(8): 695-700, 2003.
Article in English | MEDLINE | ID: mdl-12867161

ABSTRACT

The chemotactic effects of six formylated, putatively bacterial peptides (fMLP, fMLPP, fMMM, fMP, fMV, and fMS) were studied. From the set of six peptides, only fMLP (one of the most effective chemoattractant peptides in mammals) elicited a significant positive chemotactic response in the eukaryotic ciliate Tetrahymena pyriformis, while the other formylated ligands, e.g. fMMM (which is also effective in mammals), had neutral or antagonistic effects in Tetrahymena. A study of their amino acid sequences points to an, as yet obscure, interaction between C-terminal f-Met and N-terminal aromatic Phe. Some optimal physicochemical characteristics (e.g. solvent exposed area, solubility) of the molecule may be responsible for this special feature of f-MLP at such a low level of phylogeny. This means that the unicellular Tetrahymena is able to select between related molecules, giving high priority to the molecule that is the most chemoattractive in mammals. The results call attention to the possible presence of f-Met receptors at a unicellular level and to the evolutionary conservation of chemotaxis-activating processes.


Subject(s)
Chemotaxis/drug effects , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , N-Formylmethionine/chemistry , Peptides/pharmacology , Tetrahymena pyriformis/drug effects , Animals , Chemotaxis/physiology , Dose-Response Relationship, Drug , Ligands , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides/chemical synthesis , Peptides/chemistry , Tetrahymena pyriformis/physiology
19.
Cell Biochem Funct ; 21(2): 113-20, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12736899

ABSTRACT

Chemotactic properties of 3-7-mer peptides containing an EWS motive and their peptide amides synthesized and characterized by us were investigated in Tetrahymena pyriformis GL model. Analysis of the peptide acids shows that SEWS possesses exceptionally strong (660%+/-21; 430%+/-18) chemoattractant ability at 10(-12) and 10(-11) m respectively. The shorter peptide (EWS) possesses chemorepellent activity, while longer peptides display neutral (WSEWS) or moderate chemoattractant (EWSEWS and GEWSEWS) chemotactic ability. Amidation of the C-terminus can significantly modify the character of peptides: it points to the conclusion that a free alpha-COOH group at this position is required for the high efficiency of SEWS, while in the shorter (EWS) and longer peptides (WSEWS and EWSEWS) amidation can result in chemoattractant ligands. Evaluation of the structure-function relationship of these compounds establishes the significance of Glu (E) with its high surface-exposed area and negatively-charged side chain. The high discriminative ability and good chemotactic responsiveness of Tetrahymena support the theory that a chemotactic signalling mechanism working in higher levels of phylogeny is a well conserved and inducible one even in protozoa.


Subject(s)
Chemotaxis/physiology , Oligopeptides/metabolism , Tetrahymena pyriformis/metabolism , Amides/metabolism , Amino Acid Motifs/physiology , Animals , Dose-Response Relationship, Drug
20.
J Pept Sci ; 8(1): 13-22, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11831559

ABSTRACT

In this paper, the solid phase synthesis and chemical characterization of members of an SXWS sub-library (SAWS, SDWS and SKWS) as well as the comparison of their chemotactic properties with those of SEWS, which exhibits a prominent effect at 10(-12) M on a ciliated protozoan, Tetrahymena pyriformis, are described. We found that the chemotaxis of cells induced with the SXWS peptides varied according to the nature of the amino acid residue (Ala, Asp, Lys) in position X. The chemotactic activity of SEWS was not surpassed by any of three new tetrapeptides, although SAWS was also chemoattractant. Interestingly, SDWS, with an acidic side chain at position X, could not elicit any chemotactic response. SKWS, however, showed mild but significant chemorepellent activity over a wide concentration range. Chemotactic selection studies showed that the two chemoattractant peptides (SAWS and SEWS) had an expressed ability to select high-responder offspring cell populations. Peptides with neutral (SDWS) or chemorepellent (SKWS) properties were not able to select such subpopulations from the mixed cultures of Tetrahymena, indicating that the chemotactic response elicited by SXWS peptides is ligand-specific. For ligand-binding experiments N-terminally labelled fluorescent derivatives of SXWS peptides were prepared. applying [4-[7-hydroxycoumaryl]]acetic acid (Hca-OH) or 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) as markers. Hca-OH was introduced using an active ester technique as the last step of SPPS, or after cleavage in solution. The oxazolone naOx-OEt reacted with the amino group of the peptide by liberation of EtOH. The binding characteristics of fixed Tetrahymena cells with the naOx-labelled peptides showed good correlation between binding profiles and chemotactic responsiveness (SEWS > SAWS > SDWS - SKWS). A similar binding pattern was observed in the case of Hca-peptides (SEWS > SAWS > SDWS). Hca-SKWS, however, bound remarkably to the cell surface. The binding activity of the Hca-peptides was less pronounced than that of the naOx-peptides, indicating the importance of the fluorophores applied.


Subject(s)
Oligopeptides/chemistry , Peptides/chemistry , Tetrahymena pyriformis/metabolism , Animals , Chemotaxis , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Mass Spectrometry , Models, Chemical , Peptide Biosynthesis , Peptides/metabolism , Protein Binding , Protein Structure, Tertiary , Time Factors
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