In this contribution, four new compounds synthesized from 4-hydroxycoumarin and tyramine/octopamine/norepinephrine/3-methoxytyramine are characterized spectroscopically (IR and NMR), chromatographically (UHPLC-DAD), and structurally at the B3LYP/6-311++G*(d,p) level of theory. The crystal structure of the 4-hydroxycoumarin-octopamine derivative was solved and used as a starting geometry for structural optimization. Along with the previously obtained 4-hydroxycoumarin-dopamine derivative, the intramolecular interactions governing the stability of these compounds were quantified by NBO and QTAIM analyses. Condensed Fukui functions and the HOMO-LUMO gap were calculated and correlated with the number and position of OH groups in the structures. In vitro cytotoxicity experiments were performed to elucidate the possible antitumor activity of the tested substances. For this purpose, four cell lines were selected, namely human colon cancer (HCT-116), human adenocarcinoma (HeLa), human breast cancer (MDA-MB-231), and healthy human lung fibroblast (MRC-5) lines. A significant selectivity towards colorectal carcinoma cells was observed. Molecular docking and molecular dynamics studies with carbonic anhydrase, a prognostic factor in several cancers, complemented the experimental results. The calculated MD binding energies coincided well with the experimental activity, and indicated 4-hydroxycoumarin-dopamine and 4-hydroxycoumarin-3-methoxytyramine as the most active compounds. The ecotoxicology assessment proved that the obtained compounds have a low impact on the daphnia, fish, and green algae population.
- MeSH
- 4-hydroxykumariny chemická syntéza chemie farmakologie MeSH
- antitumorózní látky chemická syntéza chemie farmakologie MeSH
- difrakce rentgenového záření MeSH
- HCT116 buňky MeSH
- HeLa buňky MeSH
- karboanhydrasy chemie metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie enzymologie MeSH
- neurotransmiterové látky chemie MeSH
- oktopamin chemie MeSH
- proliferace buněk účinky léků MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In the present study, we report the design and synthesis of novel CAN508 sulfonamide-based analogues (4, 8a-e, 9a-h and 10a-e) as novel carbonic anhydrase (CA) inhibitors with potential CDK inhibitory activity. A bioisosteric replacement approach was adopted to replace the phenolic OH of CAN508 with a sulfamoyl group to afford compound 4. Thereafter, a ring-fusion approach was utilized to furnish the 5/5 fused imidazopyrazoles 8a-e which were subsequently expanded to 6/5 pyrazolopyrimidines 9a-h and 10a-e. All the synthesized analogues were evaluated for their inhibitory activity toward isoforms hCA I, II, IX and XII. The target tumor-associated isoforms hCA IX and XII were effectively inhibited with KIs ranges 6-67.6 and 10.1-88.6 nM, respectively. Furthermore, all compounds were evaluated for their potential CDK2 and 9 inhibitory activities. Pyrazolopyrimidines 9d, 9e and 10b displayed weak CDK2 inhibitory activity (IC50 = 6.4, 8.0 and 11.6 μM, respectively), along with abolished CDK9 inhibitory activity. This trend suggested that pyrazolopyrimidine derivatives merit further optimization to furnish more effective CDK2 inhibitor lead. On account of their excellent activity and selectivity towards hCA IX and XII, pyrazolopyrimidines 10 were evaluated for their anti-proliferative activity toward breast cancer MCF-7 and MDA-MB-468 cell lines under normoxic and hypoxic conditions. The most potent anti-proliferative agents 10a, 10c and 10d significantly increased cell percentage at sub-G1 and G2-M phases with concomitant decrease in the S phase population in MCF-7 treated cells. Finally, a docking study was undertaken to investigate the binding mode for the most selective hCA IX and XII inhibitors 10a-e, within hCA II, IX and XII active sites.
- MeSH
- antitumorózní látky chemie farmakologie MeSH
- azosloučeniny chemie MeSH
- inhibitory karboanhydras chemie farmakologie MeSH
- karboanhydrasy chemie MeSH
- léky antitumorózní - screeningové testy MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádory farmakoterapie patologie MeSH
- proliferace buněk MeSH
- pyrazoly chemie MeSH
- racionální návrh léčiv * MeSH
- simulace molekulového dockingu MeSH
- sulfonamidy chemie MeSH
- techniky in vitro MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
An accurate description of solvation effects is of high importance in modeling biomolecular systems. Our main interest is to find an accurate yet efficient solvation model for semiempirical quantum-mechanical methods applicable to large protein-ligand complexes in the context of computer-aided drug design. We present a survey of readily available methods and a new reparametrization of the COSMO solvent model for PM6 and PM7 calculations in MOPAC. We have tested the reparametrized method on validation data sets of small drug-like molecules for which experimental solvation free energies are available as well as on a set of large model systems of the active site of carbonic anhydrase II interacting with a series of ligands for which experimental affinity values are known. In both cases, there is a significant improvement in accuracy after the reparametrization and the addition of a nonpolar term to the COSMO solvent model.
BACKGROUND: The pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the β-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants. RESULTS: Recombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I β-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of β-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases. CONCLUSIONS: CaNce103p is a β-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other β-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.
- MeSH
- Candida albicans enzymologie MeSH
- karboanhydrasy chemie MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Patogenní kvasinky rodu Candida představují nejrozšířenější příčinu mykotických onemocnění. Tyto druhy jsou úspěšnými patogeny díky tomu, že jsou schopné se rozmnožovat v nejrůznějších místech jako je například kůže, gastrointestinální trakt, krev nebo vaginální sliz- nice a také díky schopnosti adheze na abiotické povrchy. Jednou z klíčových strategií přežití těchto patogenů je jejich schopnost poliferovat v různých koncentracích oxidu uhličitého (CO2). K zvládnutí takového rozdílu používají kvasinky rodu Candida karbanické anhydrasy (CA) kódované homologem genu NCE103, které katalyzují reverzibilní hydrataci CO2 na bikarbonát. CA jsou postradatelné během infekce krevního řečiště, ale jsou nezbytné pro přežití kvasinek na kůži nebo na abiotických površích. Kvasinkové CA jsou strukturně odlišné od lidských a představují tak vhodný cíl pro vývoj profylaktických látek.
Pathogenic yeasts of the genus Candida represent the most prevalent cause of mycotic diseases worldwide. These species are successful pathogens due to their ability to proliferate under a wide variety of conditions, colonizing host niches as diverse as skin, blood, or vagina and also to adhere to abiotic surfaces. One of the key survival strategies of fungal pathogens is the ability to proliferate in different carbon dioxide (CO2) concentrations. To cope with such difference, Candida possesses carbonic anhydrase (CA), encoded by NCE103 gene, which catalyzes reversible hydratation of CO2 into bicarbonate. This enzyme is dispensable during the bloodstream infection, but it is essential for survival of the fungus on skin or abiotic surfaces. Fungal CAs are structurally unrelated to human CAs, which makes them an ideal target for prophylactic intervention.
- Klíčová slova
- NCE103,
- MeSH
- antifungální látky farmakologie klasifikace MeSH
- Candida enzymologie genetika patogenita MeSH
- fungální léková rezistence účinky léků MeSH
- houby enzymologie MeSH
- kandidóza invazivní patologie terapie MeSH
- kandidóza etiologie farmakoterapie klasifikace patologie MeSH
- karboanhydrasy * genetika chemie klasifikace účinky léků MeSH
- kvasinky * enzymologie klasifikace patogenita MeSH
- oxid uhličitý MeSH
- Publikační typ
- práce podpořená grantem MeSH
Hydrophobins are small proteins that play a role in a number of processes during the filamentous fungi growth and development. These proteins are characterized by the self-assembly of their molecules into an amphipathic membrane at hydrophilic-hydrophobic interfaces. Isolation and purification of hydrophobins generally present a challenge in their analysis. Hydrophobin SC3 from Schizophyllum commune was selected as a representative of class I hydrophobins in this work. A novel procedure for selective and effective isolation of hydrophobin SC3 based on solid-phase extraction with polytetrafluoroethylene microparticles loaded in a small self-made microcolumn is reported. The tailored binding of hydrophobins to polytetrafluoroethylene followed by harsh elution conditions resulted in a highly specific isolation of hydrophobin SC3 from the model mixture of ten proteins. The presented isolation protocol can have a positive impact on the analysis and utilization of these proteins including all class I hydrophobins. Hydrophobin SC3 was further subjected to reduction of its highly stable disulfide bonds and to chymotryptic digestion followed by mass spectrometric analysis. The isolation and digestion protocols presented in this work make the analysis of these highly hydrophobic and compact proteins possible.
- MeSH
- albuminy chemie MeSH
- ananasovník chemie MeSH
- bromelainy chemie MeSH
- Canavalia chemie MeSH
- chymotrypsin chemie MeSH
- cytochromy c chemie MeSH
- disulfidy chemie MeSH
- erytrocyty enzymologie MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie metody MeSH
- karboanhydrasy chemie MeSH
- kaseiny chemie MeSH
- koně MeSH
- konkanavalin A chemie MeSH
- kur domácí MeSH
- lidé MeSH
- mikrosféry * MeSH
- mléko enzymologie MeSH
- myokard metabolismus MeSH
- polytetrafluoroethylen chemie MeSH
- proteomika metody MeSH
- Schizophyllum chemie MeSH
- skot MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- tandemová hmotnostní spektrometrie MeSH
- thermolysin chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Carborane-based compounds are promising lead structures for development of inhibitors of carbonic anhydrases (CAs). Here, we report structural and computational analysis applicable to structure-based design of carborane compounds with selectivity toward the cancer-specific CAIX isoenzyme. We determined the crystal structure of CAII in complex with 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane at 1.0 Å resolution and used this structure to model the 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane interactions with CAIX. A virtual glycine scan revealed the contributions of individual residues to the energy of binding of 1-methylenesulfamide-1,2-dicarba-closo-dodecaborane to CAII and CAIX, respectively.
- MeSH
- glycin chemie MeSH
- inhibitory karboanhydras chemie farmakologie MeSH
- karboanhydrasy chemie MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- kvantová teorie * MeSH
- lidé MeSH
- molekulární modely * MeSH
- sloučeniny boru chemie farmakologie MeSH
- substrátová specifita účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer. 2007 Wiley-Liss, Inc.
- MeSH
- antigeny nádorové chemie imunologie MeSH
- epitopy chemie imunologie MeSH
- financování organizované MeSH
- imunoglobuliny - Fab fragmenty chemie MeSH
- izoenzymy chemie imunologie MeSH
- kalorimetrie MeSH
- karboanhydrasy chemie imunologie MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- monoklonální protilátky chemie MeSH
- nádorové buněčné linie MeSH
- počítačová simulace MeSH
- sekvence aminokyselin MeSH
- termodynamika MeSH
- vazebná místa protilátek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
