ORCID Profile
0000-0001-9215-9817
Current Organisation
Institute of Organic Chemistry and Biochemistry
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Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.EJMECH.2021.113798
Abstract: Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered. Herein, we designed and synthesized two series of new ANPs branched at the C1' position as mimics of adenosine monophosphate. The novel ANPs efficaciously inhibited Trypanosoma brucei adenine PRT (TbrAPRT1) activity in vitro and it was shown that the configuration on the C1' chiral centre strongly influenced their activity: the (R)-enantiomers proved to be more potent compared to the (S)-enantiomers. Two ANPs, with K
Publisher: Elsevier BV
Date: 03-2023
Publisher: Worldwide Protein Data Bank
Date: 18-06-2014
DOI: 10.2210/PDB4NGT/PDB
Publisher: Worldwide Protein Data Bank
Date: 10-09-2014
DOI: 10.2210/PDB4MWO/PDB
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.PEP.2015.01.006
Abstract: Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cell receptor-activated T cells. While in normal tissues LLT1:NKR-P1 interaction (representing an alternative "missing-self" recognition system) play an immunomodulatory role in regulation of crosstalk between NK and antigen presenting cells, LLT1 is upregulated in glioblastoma cells, one of the most lethal tumors, where it acts as a mediator of immune escape of glioma cells. Here we report transient expression and characterization of soluble His176Cys mutant of LLT1 ectodomain in an eukaryotic expression system of human suspension-adapted HEK293S GnTI(-) cell line with uniform N-glycans. The His176Cys mutation is critical for C-type lectin-like domain stability, leading to the reconstruction of third canonical disulfide bridge in LLT1, as shown by mass spectrometry. Purified soluble LLT1 is homogeneous, deglycosylatable and forms a non-covalent homodimer whose dimerization is not dependent on presence of its N-glycans. As a part of production of soluble LLT1, we have adapted HEK293S GnTI(-) cell line to growth in suspension in media facilitating transient transfection and optimized novel high cell density transfection protocol, greatly enhancing protein yields. This transfection protocol is generally applicable for protein production within this cell line, especially for protein crystallography.
Publisher: Worldwide Protein Data Bank
Date: 06-2016
DOI: 10.2210/PDB5F09/PDB
Publisher: Worldwide Protein Data Bank
Date: 25-03-2015
DOI: 10.2210/PDB4U7Q/PDB
Publisher: Worldwide Protein Data Bank
Date: 26-02-2014
DOI: 10.2210/PDB4MUM/PDB
Publisher: American Chemical Society (ACS)
Date: 11-03-2011
DOI: 10.1021/JM2000213
Abstract: Isoquinolinesulfonamides inhibit human carbonic anhydrases (hCAs) and display selectivity toward therapeutically relevant isozymes. The crystal structure of hCA II in complex with 6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ylsulfonamide revealed unusual inhibitor binding. Structural analyses allowed for discerning the fine details of the inhibitor binding mode to the active site, thus providing clues for the future design of even more selective inhibitors for druggable isoforms such as the cancer associated hCA IX and neuronal hCA VII.
Publisher: Elsevier BV
Date: 08-2014
DOI: 10.1016/J.BMC.2014.05.061
Abstract: Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure-activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in Ki value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.
Publisher: Worldwide Protein Data Bank
Date: 12-02-2014
DOI: 10.2210/PDB4L6A/PDB
Publisher: Wiley
Date: 26-07-2018
Publisher: International Union of Crystallography (IUCr)
Date: 12-02-2011
Publisher: Worldwide Protein Data Bank
Date: 25-03-2015
DOI: 10.2210/PDB4U7V/PDB
Publisher: Springer Science and Business Media LLC
Date: 15-11-2014
DOI: 10.1007/S12104-013-9531-1
Abstract: Cytosolic dNT-1 nucleotidase plays a key role in the homeostasis of pyrimidine deoxyribonucleotides in mammalian cells. The enzyme is responsible for the dephosphorylation of physiological substrates as well as nucleoside analogues that are used in antiviral and anticancer therapies, therefore selective inhibition of the dNT-1 nucleotidase activity may lead to an increase in efficacy of this type of therapeutic compounds. Here, we report the backbone ¹H, ¹³C and ¹⁵N assignments for the 47 kDa dNT-1 dimer, which will be used for structural characterisation of dNT-1 complexes with small molecule inhibitors obtained through modification of pyrimidine nucleotide scaffolds or optimisation of successful binders obtained from the screening of fragment libraries.
Publisher: Worldwide Protein Data Bank
Date: 10-09-2014
DOI: 10.2210/PDB4NFL/PDB
Publisher: Worldwide Protein Data Bank
Date: 06-04-2016
DOI: 10.2210/PDB5I67/PDB
Publisher: Portland Press Ltd.
Date: 10-12-2018
DOI: 10.1042/BCJ20180764
Abstract: Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
Publisher: Worldwide Protein Data Bank
Date: 21-09-2016
DOI: 10.2210/PDB5L50/PDB
Publisher: Cold Spring Harbor Laboratory
Date: 28-07-2021
DOI: 10.1101/2021.07.28.453943
Abstract: Tick-borne encephalitis virus (TBEV) causes about 5-6 thousand cases annually, while there is still no effective treatment for this virus. To fill this gap, a high-affinity chimeric anti-TBEV antibody ch14D5 has previously been constructed, and high protective activity in a murine TBEV model has been shown for this antibody. However, the mechanism of action of this antibody and the recognized epitope have not been known yet. In this study, it is shown by X-ray crystallography that this antibody recognizes a unique epitope on the lateral ridge of the D3 domain of glycoprotein E, which is readily accessible for binding. The orientation of this antibody relative to the virion surface makes bivalent binding possible, which facilitates the cross-linking of glycoprotein E molecules and thus blocking of surface rearrangements required for infection. Since the antibody tightly binds to this protein even at pH ∼ 5.0, it locks the virion in an acidic environment inside the late endosomes or phagosomes and, therefore, effectively blocks the fusion of the viral and endosomal hagosomal membranes. We believe that this is why the ch14D5 antibody does not induce an antibody-dependent enhancement of infection in vivo , which is critical in the development of antibody-based therapeutic agents. In addition, the structure of the antibody-glycoprotein E interface can be used for the rational design of this antibody for enhancing its properties.
Publisher: Elsevier BV
Date: 12-2017
Publisher: Worldwide Protein Data Bank
Date: 10-09-2014
DOI: 10.2210/PDB4L6C/PDB
Publisher: Springer Science and Business Media LLC
Date: 19-10-2016
Publisher: International Union of Crystallography (IUCr)
Date: 29-01-2014
DOI: 10.1107/S1399004713030502
Abstract: The human 5′(3′)-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.
Publisher: International Union of Crystallography (IUCr)
Date: 29-10-2018
DOI: 10.1107/S2059798318013049
Abstract: α-L-Rhamnosidases cleave terminal nonreducing α-L-rhamnosyl residues from many natural rhamnoglycosides. This makes them catalysts of interest for various biotechnological applications. The X-ray structure of the GH78 family α-L-rhamnosidase from Aspergillus terreus has been determined at 1.38 Å resolution using the sulfur single-wavelength anomalous dispersion phasing method. The protein was isolated from its natural source in the native glycosylated form, and the active site contained a glucose molecule, probably from the growth medium. In addition to its catalytic domain, the α-L-rhamnosidase from A. terreus contains four accessory domains of unknown function. The structural data suggest that two of these accessory domains, E and F, might play a role in stabilizing the aglycon portion of the bound substrate.
Publisher: Wiley
Date: 30-12-2017
DOI: 10.1111/FEBS.14360
Abstract: β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 Å revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of β-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering. Structural data are available in the PDB database under the accession number 5OAR. β-N-acetylhexosaminidase (EC 3.2.1.52).
Publisher: International Union of Crystallography (IUCr)
Date: 17-01-2012
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5MD00235D
Abstract: Based on previously known inhibitor–enzyme complex structures, we developed a promising inhibitor by mimicking the phosphate ion and achieved 50- and 100-fold increases in the inhibitory potency towards cdN and mdN, respectively.
Publisher: Worldwide Protein Data Bank
Date: 09-09-2015
DOI: 10.2210/PDB4YIK/PDB
Publisher: Springer Science and Business Media LLC
Date: 09-03-2015
DOI: 10.1038/NCOMMS7461
Abstract: HIV protease (PR) is required for proteolytic maturation in the late phase of HIV replication and represents a prime therapeutic target. The regulation and kinetics of viral polyprotein processing and maturation are currently not understood in detail. Here we design, synthesize, validate and apply a potent, photodegradable HIV PR inhibitor to achieve synchronized induction of proteolysis. The compound exhibits subnanomolar inhibition in vitro . Its photolabile moiety is released on light irradiation, reducing the inhibitory potential by 4 orders of magnitude. We determine the structure of the PR-inhibitor complex, analyze its photolytic products, and show that the enzymatic activity of inhibited PR can be fully restored on inhibitor photolysis. We also demonstrate that proteolysis of immature HIV particles produced in the presence of the inhibitor can be rapidly triggered by light enabling thus to analyze the timing, regulation and spatial requirements of viral processing in real time.
Publisher: Worldwide Protein Data Bank
Date: 21-09-2016
DOI: 10.2210/PDB5K7Y/PDB
Publisher: MDPI AG
Date: 21-06-2018
DOI: 10.3390/V10070339
Publisher: Worldwide Protein Data Bank
Date: 12-02-2014
DOI: 10.2210/PDB4L57/PDB
Publisher: International Union of Crystallography (IUCr)
Date: 30-07-2019
DOI: 10.1107/S2059798319009586
Abstract: The haloacid dehalogenase (HAD) superfamily is one of the largest known groups of enzymes and the majority of its members catalyze the hydrolysis of phosphoric acid monoesters into a phosphate ion and an alcohol. Despite the fact that sequence similarity between HAD phosphatases is generally very low, the members of the family possess some characteristic features, such as a Rossmann-like fold, HAD signature motifs or the requirement for Mg 2+ ion as an obligatory cofactor. This study focuses on a new hypothetical HAD phosphatase from Thermococcus thioreducens . The protein crystallized in space group P 2 1 2 1 2, with unit-cell parameters a = 66.3, b = 117.0, c = 33.8 Å, and the crystals contained one molecule in the asymmetric unit. The protein structure was determined by X-ray crystallography and was refined to 1.75 Å resolution. The structure revealed a putative active site common to all HAD members. Computational docking into the crystal structure was used to propose substrates of the enzyme. The activity of this thermophilic enzyme towards several of the selected substrates was confirmed at temperatures of 37°C as well as 60°C.
Publisher: Worldwide Protein Data Bank
Date: 21-09-2016
DOI: 10.2210/PDB5L4Z/PDB
Publisher: Worldwide Protein Data Bank
Date: 07-03-2012
DOI: 10.2210/PDB3TNE/PDB
Publisher: International Union of Crystallography (IUCr)
Date: 26-03-2011
No related grants have been discovered for Petr Pachl.