Lens epithelium-derived growth factor p75 (LEDGF/p75), member of the hepatoma-derived growth-factor-related protein (HRP) family, is a transcriptional co-activator and involved in several pathologies including HIV infection and malignancies such as MLL-rearranged leukemia. LEDGF/p75 acts by tethering proteins to the chromatin through its integrase binding domain. This chromatin interaction occurs between the PWWP domain of LEDGF/p75 and nucleosomes carrying a di- or trimethylation mark on histone H3 Lys36 (H3K36me2/3). Our aim is to rationally devise small molecule drugs capable of inhibiting such interaction. To bootstrap this development, we resorted to X-ray crystallography-based fragment screening (FBS-X). Given that the LEDGF PWWP domain crystals were not suitable for FBS-X, we employed crystals of the closely related PWWP domain of paralog HRP-2. As a result, as many as 68 diverse fragment hits were identified, providing a detailed sampling of the H3K36me2/3 pocket pharmacophore. Subsequent structure-guided fragment expansion in three directions yielded multiple compound series binding to the pocket, as verified through X-ray crystallography, nuclear magnetic resonance and differential scanning fluorimetry. Our best compounds have double-digit micromolar affinity and optimally sample the interactions available in the pocket, judging by the Kd-based ligand efficiency exceeding 0.5 kcal/mol per non-hydrogen atom. Beyond π-stacking within the aromatic cage of the pocket and hydrogen bonding, the best compounds engage in a σ-hole interaction between a halogen atom and a conserved water buried deep in the pocket. Notably, the binding pocket in LEDGF PWWP is considerably smaller compared to the related PWWP1 domains of NSD2 and NSD3 which feature an additional subpocket and for which nanomolar affinity compounds have been developed recently. The absence of this subpocket in LEDGF PWWP limits the attainable affinity. Additionally, these structural differences in the H3K36me2/3 pocket across the PWWP domain family translate into a distinct selectivity of the compounds we developed. Our top-ranked compounds are interacting with both homologous LEDGF and HRP-2 PWWP domains, yet they showed no affinity for the NSD2 PWWP1 and BRPF2 PWWP domains which belong to other PWWP domain subfamilies. Nevertheless, our developed compound series provide a strong foundation for future drug discovery targeting the LEDGF PWWP domain as they can further be explored through combinatorial chemistry. Given that the affinity of H3K36me2/3 nucleosomes to LEDGF/p75 is driven by interactions within the pocket as well as with the DNA-binding residues, we suggest that future compound development should target the latter region as well. Beyond drug discovery, our compounds can be employed to devise tool compounds to investigate the mechanism of LEDGF/p75 in epigenetic regulation.
- MeSH
- Small Molecule Libraries chemistry pharmacology chemical synthesis MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Intercellular Signaling Peptides and Proteins metabolism chemistry MeSH
- Models, Molecular MeSH
- Molecular Structure MeSH
- Protein Domains MeSH
- Drug Design * MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
PURPOSE: The aim of this study is to design a method of myocardial T1 quantification in small laboratory animals and to investigate the effects of spatiotemporal regularization and the needed acquisition duration. METHODS: We propose a compressed-sensing approach to T1 quantification based on self-gated inversion-recovery radial two/three-dimensional (2D/3D) golden-angle stack-of-stars acquisition with image reconstruction performed using total-variation spatiotemporal regularization. The method was tested on a phantom and on a healthy rat, as well as on rats in a small myocardium-remodeling study. RESULTS: The results showed a good match of the T1 estimates with the results obtained using the ground-truth method on a phantom and with the literature values for rats myocardium. The proposed 2D and 3D methods showed significant differences between normal and remodeling myocardium groups for acquisition lengths down to approximately 5 and 15 min, respectively. CONCLUSIONS: A new 2D and 3D method for quantification of myocardial T1 in rats was proposed. We have shown the capability of both techniques to distinguish between normal and remodeling myocardial tissue. We have shown the effects of image-reconstruction regularization weights and acquisition length on the T1 estimates.
- MeSH
- Phantoms, Imaging MeSH
- Rats MeSH
- Magnetic Resonance Imaging methods MeSH
- Myocardium * MeSH
- Image Processing, Computer-Assisted methods MeSH
- Reproducibility of Results MeSH
- Imaging, Three-Dimensional * methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Multidrug resistance (MDR) mechanisms in cancer cells are greatly influenced by glutathione transferase P1-1 (hGSTP1-1). The use of synthetic or natural compounds as hGSTP1-1 inhibitors is considered an effective approach to overcome MDR. Nine compounds consisting of coumarin-6-sulfonamide linked to chalcone derivatives were synthesized and evaluated for their ability to inhibit hGSTP1-1. Among the synthetic derivatives, compounds 5g, 5f, and 5a displayed the most potent inhibitory effect, with IC50 values of 12.2 ± 0.5 μΜ, 12.7 ± 0.7 and 16.3 ± 0.6, respectively. Kinetic inhibition analysis of the most potent molecule, 5g, showed that it behaves as a mixed-type inhibitor of the target enzyme. An in vitro cytotoxicity assessment of 5a, 5f, and 5g against the human prostate cancer cell lines DU-145 and PC3, as well as the breast cancer cell line MCF-7, demonstrated that compound 5g exhibited the most pronounced cytotoxic effect on all tested cell lines. Molecular docking studies were performed to predict the structural and molecular determinants of 5g, 5f, and 5a binding to hGSTP1-1. In agreement with the experimental data, the results revealed that 5g exhibited the lowest docking score among the three studied inhibitors as a consequence of shape complementarity, governed by van der Waals, hydrogen bonds and a π-π stacking interaction. These findings suggest that coumarin-chalcone hybrids offer new perspectives for the development of safe and efficient natural product-based sensitizers that can target hGSTP1-1 for anticancer purposes.
- MeSH
- Chalcone chemistry pharmacology MeSH
- Chalcones chemistry pharmacology MeSH
- Glutathione S-Transferase pi * antagonists & inhibitors metabolism MeSH
- Enzyme Inhibitors pharmacology chemistry MeSH
- Coumarins * chemistry pharmacology MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Cell Line, Tumor MeSH
- Antineoplastic Agents pharmacology chemistry MeSH
- Molecular Docking Simulation * MeSH
- Sulfonamides * chemistry pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Odloučení placenty může být katastrofální událostí s následky pro matku i plod. Prezentujeme masivní odloučení placenty, ke kterému došlo u mladé asymptomatické matky v 30. týdnu těhotenství. Ačkoli elektronický monitoring plodu a ultrazvuk umožnily rychlou diagnózu retroplacentárního hematomu o velikosti 8 × 5 cm, plod během akutního císařského řezu odumřel. Plod byl intubován a resuscitován, ale neúspěšně. Histologické vyšetření placenty prokázalo ztenčené a na sebe navrstvené choriové klky se zvýšeným množstvím kapilár a syncitiálními hnízdy a ložisky fibrinoidní nekrózy za přítomnosti hyalinních pruhů jak na straně matky, tak na straně plodu.
Abruptio placenta can be a catastrophic event with a high association with adverse maternal and fetal outcomes. We present a case of massive abruptio placenta occurring in a young asymptomatic mother at 30 weeks‘ gestation. Although electronic fetal monitoring and ultrasound allowed a prompt diagnosis of an 8 × 5 cm retroplacental hematoma, the fetus died at the time of emergency cesarean section. The fetus was intubated, but could not be resuscitated. Histologic examination of the placenta documented thinning and stacked hypercapillarized villi, with syncytial buds and foci of fibrinoid necrosis in the presence of hyaline streaks on both the maternal and fetal sides.
- MeSH
- Back Pain etiology MeSH
- Cesarean Section methods MeSH
- Uterine Hemorrhage diagnostic imaging etiology pathology MeSH
- Fetal Distress diagnosis etiology MeSH
- Hematoma diagnostic imaging etiology pathology MeSH
- Histological Techniques MeSH
- Young Adult MeSH
- Fetal Monitoring MeSH
- Stillbirth MeSH
- Abruptio Placentae * diagnostic imaging pathology MeSH
- Pregnancy MeSH
- Ultrasonography MeSH
- Check Tag
- Young Adult MeSH
- Pregnancy MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013-2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC β-lactamase, and 4.1 % extended spectrum β-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Escherichia coli * genetics isolation & purification pathogenicity classification MeSH
- Virulence Factors genetics MeSH
- Gastrointestinal Diseases microbiology MeSH
- Genome, Bacterial MeSH
- Escherichia coli Infections * microbiology epidemiology MeSH
- Infant MeSH
- Humans MeSH
- Multilocus Sequence Typing MeSH
- Infant, Newborn MeSH
- Child, Preschool MeSH
- Escherichia coli Proteins genetics MeSH
- Diarrhea * microbiology MeSH
- Whole Genome Sequencing * MeSH
- Serogroup MeSH
- Trans-Activators MeSH
- Check Tag
- Infant MeSH
- Humans MeSH
- Male MeSH
- Infant, Newborn MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
Accurate estimates of intermolecular interaction energy, ΔE, are crucial for modeling the properties of organic electronic materials and many other systems. For a diverse set of 50 dimers comprising up to 50 atoms (Set50-50, with 7 of its members being models of single-stacking junctions), benchmark ΔE data were compiled. They were obtained by the focal-point strategy, which involves computations using the canonical variant of the coupled cluster theory with singles, doubles, and perturbative triples [CCSD(T)] performed while applying a large basis set, along with extrapolations of the respective energy components to the complete basis set (CBS) limit. The resulting ΔE data were used to gauge the performance for the Set50-50 of several density-functional theory (DFT)-based approaches, and of one of the localized variants of the CCSD(T) method. This evaluation revealed that (1) the proposed "silver standard" approach, which employs the localized CCSD(T) method and CBS extrapolations, can be expected to provide accuracy better than two kJ/mol for absolute values of ΔE, and (2) from among the DFT techniques, computationally by far the cheapest approach (termed "ωB97X-3c/vDZP" by its authors) performed remarkably well. These findings are directly applicable in cost-effective yet reliable searches of the potential energy surfaces of noncovalent complexes.
- MeSH
- Benchmarking * MeSH
- Dimerization MeSH
- Electronics * MeSH
- Physical Phenomena MeSH
- Polymers MeSH
- Publication type
- Journal Article MeSH
Spruce wood and Typha (wetland plant) derived biochars pyrolyzed at 350 °C and 600 °C were tested for their sorption affinity for organic pollutants (diclofenac, methylparaben, benzotriazole and sodium 1-decanesulfonate) and nutrients (nitrate, ammonium, phosphate and boron) commonly found in greywater. Batch and column studies combined with molecular dynamics modelling determined the sorption capacity, kinetics, and described the underlying mechanisms. The spruce biochar (600 °C) exhibited the highest sorption capacity mainly for the tested organics. The dynamic test performed for spruce biochar (600 °C) showed that the magnitude of desorption was low, and the desorbed amount ranged between 3 and 11 %. Molecular dynamics modelling (a computational tool for elucidating molecular-level interactions) indicated that the increased sorption of nitrate and boron on spruce biochar (600 °C) could be attributed to hydrophobic interactions. The molecular dynamics shows that predominant adsorption of organic pollutants was governed by π-π stacking, with a minor role of hydrogen-bonding on the biochar surface. In summary, higher pyrolysis temperature biochar yielded greater adsorption capacity greywater borne contaminants and the reaction temperature (10-34 °C) and presence of anionic surfactant had a limited effect on the adsorption of organic pollutants, suggesting efficacious application of biochar in general for greywater treatment in nature-based systems.
Bacteriophages of Borrelia burgdorferi are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. No virulent borrelial viruses have been identified, but all B. burgdorferi isolates carry a prophage φBB1 as resident circular plasmids. Like its host, the φBB1 phage is quite distinctive and shares little sequence similarity with other known bacteriophages. We expressed φBB1 head morphogenesis proteins in Escherichia coli which resulted in assembly of homogeneous prolate procapsid structures and used cryo-electron microscopy to determine the three-dimensional structure of these particles. The φBB1 procapsids consist of 415 copies of the major capsid protein and an equal combined number of three homologous capsid decoration proteins that form trimeric knobs on the outside of the particle. One of the end vertices of the particle is occupied by a portal assembled from twelve copies of the portal protein. The φBB1 scaffolding protein is entirely α-helical and has an elongated shape with a small globular domain in the middle. Within the tubular section of the procapsid, the internal scaffold is built of stacked rings, each composed of 32 scaffolding protein molecules, which run in opposite directions from both caps with a heterogeneous part in the middle. Inside the portal-containing cap, the scaffold is organized asymmetrically with ten scaffolding protein molecules bound to the portal. The φBB1 procapsid structure provides better insight into the vast structural diversity of bacteriophages and presents clues of how elongated bacteriophage particles might be assembled.
Mpox is a zoonotic disease caused by the mpox virus (MPXV), which has gained attention due to its rapid and widespread transmission, with reports from more than 100 countries. The virus belongs to the Orthopoxvirus genus, which also includes variola virus and vaccinia virus. In poxviruses, the RNA cap is crucial for the translation and stability of viral mRNAs and also for immune evasion. This study presents the crystal structure of the mpox 2'-O-methyltransfarase VP39 in complex with a short cap-0 RNA. The RNA substrate binds to the protein without causing any significant changes to its overall fold and is held in place by a combination of electrostatic interactions, π-π stacking and hydrogen bonding. The structure also explains the mpox VP39 preference for a guanine base at the first position; it reveals that guanine forms a hydrogen bond that an adenine would not be able to form.
- MeSH
- Humans MeSH
- Methyltransferases chemistry MeSH
- Methylation MeSH
- Mpox, Monkeypox * MeSH
- RNA Caps * metabolism MeSH
- Binding Sites MeSH
- Viral Proteins genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Capillary electrophoresis connected with tandem mass spectrometry was employed for the development of a method for determination of various tyrosine kinase inhibitors in plasma samples. A stacking online preconcentration with a 120 cm-long capillary was used for the determination of bosutinib, dasatinib, canertinib, and erlotinib at physiologically relevant concentrations. The optimization included both capillary electrophoresis and mass spectrometry steps. Under optimal conditions, 50 mM formic acid pH 2.5, an injection time of 120 s, and an optimized mass spectrometry set-up (as sheath liquid composition 75:24.9:0.1 (v/v) methanol, water, formic acid, and appropriate conditions for ion transitions), LODs in a range of 3.9-23.0 nmol·L-1 were observed. The method was validated in terms of linearity, limit of detection, limit of quantification, repeatability of migration times and peak area, and recovery using plasma as a matrix for analytes. The results showed that this method has great promise for use in many analytical tasks, e.g., therapeutic drug monitoring.
- Publication type
- Journal Article MeSH
