Pavlína Řezáčová
Academy of Sciences of the Czech Republic
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Featured researches published by Pavlína Řezáčová.
Proceedings of the National Academy of Sciences of the United States of America | 2005
Petr Cígler; Milan Kožíšek; Pavlína Řezáčová; Jiří Brynda; Zbyszek Otwinowski; Jana Pokorná; Jaromír Plešek; Bohumír Grüner; Lucie Dolečková-Marešová; Martin Máša; Juraj Sedláček; Jochen Bodem; Hans-Georg Kräusslich; Vladimír Král; Jan Konvalinka
HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.
Viruses | 2009
Jana Pokorná; Ladislav Machala; Pavlína Řezáčová; Jan Konvalinka
The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.
Angewandte Chemie | 2013
Jiří Brynda; Pavel Mader; Václav Šícha; Milan Fábry; Kristýna Poncová; Mario Bakardiev; Bohumír Grüner; Petr Cigler; Pavlína Řezáčová
CA inhibitors: Human carbonic anhydrases (CAs) are diagnostic and therapeutic targets. Various carborane cages are shown to act as active-site-directed inhibitors, and substitution with a sulfamide group and other substituents leads to compounds with high selectivity towards the cancer-specific isozyme IX. Crystal structures of the carboranes in the active site provide information that can be applied to the structure-based design of specific inhibitors.
Journal of Virology | 2008
Milan Kožíšek; Klára Grantz Šašková; Pavlína Řezáčová; Jiří Brynda; Noortje M. van Maarseveen; Dorien de Jong; Charles A. Boucher; Ron M. Kagan; Monique Nijhuis; Jan Konvalinka
ABSTRACT While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.
Biochemical Journal | 2010
Jiří Salát; Guido C. Paesen; Pavlína Řezáčová; Michalis Kotsyfakis; Zuzana Kovářová; Miloslav Šanda; Juraj Majtan; Lenka Grunclová; Helena Horká; John F. Andersen; Jiří Brynda; Martin Horn; Miles A. Nunn; Petr Kopáček; Jan Kopecký; Michael Mareš
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the hosts defence during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 A (1 A=0.1 nm) and was used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the hosts adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
Journal of Physical Chemistry B | 2013
Adam Pecina; Martin Lepšík; Jan Řezáč; Jiří Brynda; Pavel Mader; Pavlína Řezáčová; Pavel Hobza; Jindřich Fanfrlík
The crystal structures of two novel carborane-sulfamide inhibitors in the complex with human carbonic anhydrase II (hCAII) have been studied using QM/MM calculations. Even though both complexes possess the strongly interacting sulfamide···zinc ion motif, the calculations have revealed the different nature of binding of the carborane parts of the inhibitors. The neutral closo-carborane cage was bound to hCAII mainly via dispersion interactions and formed only very weak dihydrogen bonds. On the contrary, the monoanionic nido cage interacted with the protein mainly via electrostatic interactions. It formed short and strong dihydrogen bonds (stabilization of up to 4.2 kcal/mol; H···H distances of 1.7 Å) with the polar hydrogen of protein NH2 groups. This type of binding is unique among all of the classical organic and inorganic inhibitors of hCAII. Virtual glycine scanning allowed us to identify the amino-acid side chains, which made important contributions to ligand-binding energies. In summary, using QM/MM calculations, we have provided a detailed understanding of the differences between the interactions of two carborane sulfamides, identified the amino acids of hCAII with which they interact, and thus paved the way for the computer-aided rational design of selective boron-cluster-containing hCAII inhibitors.
Journal of Medicinal Chemistry | 2012
Jiří Schimer; Petr Cigler; Jan Veselý; Klára Grantz Šašková; Martin Lepšík; Jiří Brynda; Pavlína Řezáčová; Milan Kožíšek; Ivana Císařová; Heike Oberwinkler; Hans-Georg Kraeusslich; Jan Konvalinka
HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.
Antimicrobial Agents and Chemotherapy | 2012
Milan Kožíšek; Sandra Henke; Klára Grantz Šašková; Graeme Brendon Jacobs; Anita Schuch; Bernd Buchholz; Viktor Müller; Hans-Georg Kräusslich; Pavlína Řezáčová; Jan Konvalinka; Jochen Bodem
ABSTRACT During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1′ active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.
Molecular Microbiology | 2008
Pavlína Řezáčová; Milan Kožíšek; Shiu Moy; Irena Sieglová; Andrzej Joachimiak; Mischa Machius; Zbyszek Otwinowski
Expression of genes in the gapA operon encoding five enzymes for triose phosphate interconversion in Bacillus subtilis is negatively regulated by the Central glycolytic genes Regulator (CggR). CggR belongs to the large SorC/DeoR family of prokaryotic transcriptional regulators, characterized by an N‐terminal DNA‐binding domain and a large C‐terminal effector‐binding domain. When no glucose is present in growth media, CggR binds to its target DNA sequence and blocks the transcription of genes in the gapA operon. In the presence of glucose, binding of the known effector molecule fructose‐1,6‐bisphosphate abolishes this interaction. We have identified dihydroxyacetone phosphate, glucose‐6‐phosphate and fructose‐6‐phosphate as additional CggR ligands that can bind to the effector‐binding site. Crystal structures of C‐CggR, the C‐terminal effector‐binding domain of CggR, both unliganded as well as in complex with the four ligands at resolutions between 1.65 and 1.80 Å reveal unique ligand‐specific structural changes in the binding site that affect the dimer interface. Binding affinities of these ligands were determined by isothermal titration calorimetry. Chemical cross‐linking shows that CggR oligomerization is mediated through its effector‐binding domain, and that binding of the different ligands differentially affects the distribution of oligomers. Electrophoretic mobility shift assays (EMSAs) confirmed a destabilizing effect of fructose‐1,6‐bisphosphate on the CggR/DNA complex, and also showed similar effects for dihydroxyacetone phosphate. Our results suggest that CggR stability and function may be modulated by various effectors in a complex fashion.
Proteins | 2007
Pavlína Řezáčová; Dominika Borek; Shiu Moy; Andrzej Joachimiak; Zbyszek Otwinowski
The crystal structure of the Midwest Center for Structural Genomics target APC35832, a 14.7‐kDa cytosolic protein from Bacillus stearothermophilus, has been determined at 1.3 Å resolution by the single anomalous diffraction method from a mercury soaked crystal. The APC35832 protein is a representative of large group of bacterial and archeal proteins entirely consisting of the Toprim (topoisomerase‐primase) domain. This domain is found in the catalytic centers of many enzymes catalyzing phosphodiester bond formation or cleavage, but the function of small Toprim domain proteins remains unknown. Consistent with the sequence analysis, the APC35832 structure shows a conserved Toprim fold, with a central 4‐stranded parallel β‐sheet surrounded by four α‐helixes. Comparison of the APC35832 structure with its closest structural homolog, the catalytic core of bacteriophage T7 primase, revealed structural conservation of a metal binding site and isothermal titration calorimetry indicates that APC35832 binds Mg2+ with a sub‐millimolar dissociation constant (Kd). The APC35832–Mg2+ complex structure was determined at 1.65 Å and reveals the role of conserved acidic residues in Mg2+ ion coordination. The structural similarities to other Toprim domain containing proteins and potential function and substrates of APC35832 are discussed in this article. Proteins 2008.