Antimalarika chlorochin a hydroxychlorochin jsou při dlouhodobém užívání toxická pro sítnici. Toxicita je závislá zejména na dosažené kumulativní dávce léčiva a může přetrvávat i po ukončení léčby. Senzitivní a specifická vyšetření očního pozadí v mydriáze, vyšetření centrální oblasti zorného pole do 10°, optická koherenční tomografie se spektrální doménou, autofluorescence fundu a multifokální elektroretinografie slouží k časnému rozpoznání funkčních změn. Okamžité přerušení léčby může snížit riziko poruchy vidění. Klíčová jsou pravidelná screeningová oční vyšetření, spolupráce revmatologa či jiného ošetřujícího lékaře s oftalmologem, stejně tak edukace pacienta.

Long-term usage of the antimalarial drugs chloroquine and hydroxychloroquine has a retinotoxic effect. Toxicity is mainly dependent on the cumulative dose of medication and may persist even after treatment has been concluded. Eye examination comprising dilated fundus evaluation, testing the central 10 degrees of the visual field, spectral domain optical coherence tomography, fundus autofluorescence and multifocal electroretinography help with early detection of functional changes. Immediate discontinuation of these medications may reduce the risk of visual impairment. Regular ophthalmologic evaluation, interdisciplinary cooperation and patient education are important to prevent late ocular complications.

• MeSH
• Medical History Taking MeSH
• Chloroquine * analogs & derivatives   pharmacology   adverse effects   toxicity   MeSH
• Humans MeSH
• Retinal Diseases * etiology   chemically induced   prevention & control   therapy   MeSH
• Aged MeSH
• Visual Field Tests MeSH
• Treatment Outcome MeSH
• Visual Acuity MeSH
• Check Tag
• Humans MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Case Reports MeSH

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.

• MeSH
• Actinobacteria classification   genetics   isolation & purification   MeSH
• DNA Primers * MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Consensus Sequence MeSH
• Peptide Synthases chemistry   genetics   MeSH
• Polymerase Chain Reaction MeSH
• Position-Specific Scoring Matrices MeSH
• Soil Microbiology MeSH
• High-Throughput Screening Assays * MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Human insulin-like growth factor 1 (IGF-1) is a 70 amino acid protein hormone, with key impact on growth, development, and lifespan. The physiological and clinical importance of IGF-1 prompted challenging chemical and biological trials toward the development of its analogs as molecular tools for the IGF-1 receptor (IGF1-R) studies and as new therapeutics. Here, we report a new method for the total chemical synthesis of IGF-1 analogs, which entails the solid-phase synthesis of two IGF-1 precursor chains that is followed by the CuI-catalyzed azide-alkyne cycloaddition ligation and by biomimetic formation of a native pattern of disulfides. The connection of the two IGF-1 precursor chains by the triazole-containing moieties, and variation of its neighboring sequences (Arg36 and Arg37), was tolerated in IGF-1R binding and its activation. These new synthetic IGF-1 analogs are unique examples of disulfide bonds' rich proteins with intra main-chain triazole links. The methodology reported here also presents a convenient synthetic platform for the design and production of new analogs of this important human hormone with non-standard protein modifications.

• MeSH
• Arginine chemistry   MeSH
• NIH 3T3 Cells drug effects   MeSH
• Click Chemistry MeSH
• Cycloaddition Reaction MeSH
• Disulfides chemistry   MeSH
• Fibroblasts MeSH
• Phosphorylation MeSH
• Insulin-Like Growth Factor I analogs & derivatives   chemical synthesis   chemistry   metabolism   pharmacology   MeSH
• Humans MeSH
• Copper chemistry   MeSH
• Methionine chemistry   MeSH
• Mice MeSH
• Drug Evaluation, Preclinical methods   MeSH
• Protein Domains MeSH
• Proto-Oncogene Proteins c-akt metabolism   MeSH
• Receptor, IGF Type 1 metabolism   MeSH
• Solid-Phase Synthesis Techniques MeSH
• Triazoles chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mutations in BCR-ABL kinase domain are associated with resistance to tyrosine kinase inhibitors in some patients with chronic myeloid leukemia. Therefore, mutation detection becomes essential in such patients. We aimed to apply high-resolution melt curve analysis (HRM) for a rapid screening prior to sequencing to select only mutation positive samples. One hundred and one samples with different mutations and mutational ratios were used for HRM testing. HRM results of 100/101 samples were concordant with sequencing data. We found HRM as a suitable and sensitive method for initial rapid screening of BCR-ABL KD mutations to sequence only positive samples.

• MeSH
• Fusion Proteins, bcr-abl MeSH
• Leukemia, Myelogenous, Chronic, BCR-ABL Positive genetics   MeSH
• Adult MeSH
• False Positive Reactions MeSH
• Financing, Government MeSH
• Middle Aged MeSH
• Humans MeSH
• Mutation MeSH
• DNA Mutational Analysis methods   MeSH
• Protein-Tyrosine Kinases genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Evaluation Study MeSH

The PB2 subunit of the influenza virus polymerase complex is essential for viral replication, primarily through a mechanism known as cap-snatching. In this process, PB2 binds to the 5' cap structure of host pre-mRNAs, enabling the viral polymerase to hijack the host transcriptional machinery. This binding facilitates the cleavage and integration of the capped RNA fragment into viral mRNA, thereby promoting efficient viral replication. Inhibiting the PB2-cap interaction is therefore crucial, as it directly disrupts the viral replication cycle. Consequently, targeting PB2 with specific inhibitors is a promising strategy for antiviral drug development against influenza. However, there are currently no available methods for the high-throughput screening of potential inhibitors. The development of new inhibitor screening methods of potential PB2 binders is the focus of this study. In this study, we present two novel methods, DIANA and AlphaScreen, for screening influenza PB2 cap-binding inhibitors and evaluate their effectiveness compared to the established differential scanning fluorimetry (DSF) technique. Using a diverse set of substrates and compounds based on the previously described PB2 binder pimodivir, we thoroughly assessed the capabilities of these new methods. Our findings demonstrate that both DIANA and AlphaScreen are highly effective for PB2 inhibitor screening, offering distinct advantages over traditional techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). These advantages include improved scalability, reduced sample requirements, and the capacity for label-free detection. Notably, DIANA's ability to determine Ki values from a single-well measurement significantly enhances its practicality and efficiency in inhibitor screening. This research represents a significant step forward in the development of more efficient and scalable screening strategies, helping advance efforts in the discovery of antiviral drugs against influenza.

• MeSH
• Antiviral Agents * pharmacology   chemistry   MeSH
• Fluorometry methods   MeSH
• Humans MeSH
• Piperidines pharmacology   MeSH
• Pyridines MeSH
• Pyrimidines MeSH
• Pyrroles MeSH
• RNA Caps metabolism   MeSH
• RNA-Dependent RNA Polymerase antagonists & inhibitors   metabolism   MeSH
• High-Throughput Screening Assays * methods   MeSH
• Viral Proteins * antagonists & inhibitors   metabolism   MeSH
• Influenza A virus drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA to compete with the known peptide inhibitor and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA and were found to block viral replication at low micromolar concentrations.

• MeSH
• Quinazolines chemical synthesis   chemistry   pharmacology   MeSH
• HIV-1 drug effects   metabolism   MeSH
• Capsid drug effects   metabolism   MeSH
• Small Molecule Libraries MeSH
• Anti-HIV Agents metabolism   pharmacology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Recombinant Proteins biosynthesis   MeSH
• Virus Replication drug effects   MeSH
• Reproducibility of Results MeSH
• High-Throughput Screening Assays MeSH
• Thermodynamics MeSH
• Cell Survival drug effects   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

• MeSH
• Cytokines antagonists & inhibitors   MeSH
• Furans pharmacology   MeSH
• HEK293 Cells MeSH
• Inflammasomes antagonists & inhibitors   MeSH
• Humans MeSH
• Lipopolysaccharides MeSH
• Macrophages drug effects   MeSH
• Disease Models, Animal MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cryopyrin-Associated Periodic Syndromes genetics   MeSH
• Drug Evaluation, Preclinical MeSH
• NLR Family, Pyrin Domain-Containing 3 Protein antagonists & inhibitors   genetics   MeSH
• Protein Domains MeSH
• Sulfonamides pharmacology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.

• MeSH
• Arabidopsis drug effects   genetics   metabolism   MeSH
• Brefeldin A pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Chromones chemistry   pharmacology   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Endocytosis drug effects   MeSH
• Plants, Genetically Modified MeSH
• Membrane Glycoproteins genetics   metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Mutation MeSH
• Protein Domains MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae drug effects   metabolism   MeSH
• Molecular Docking Simulation MeSH
• Transcription Factors genetics   metabolism   MeSH
• Protein Transport drug effects   MeSH
• Guanine Nucleotide Exchange Factors chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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

Alzheimer's disease (AD) is one of the most significant neurodegenerative disorders and its symptoms mostly appear in aged people. Catechol-o-methyltransferase (COMT) is one of the known target enzymes responsible for AD. With the use of 23 known inhibitors of COMT, a query has been generated and validated by screening against the database of 1500 decoys to obtain the GH score and enrichment value. The crucial features of the known inhibitors were evaluated by the online ZINC Pharmer to identify new leads from a ZINC database. Five hundred hits were retrieved from ZINC Pharmer and by ADMET (absorption, distribution, metabolism, excretion, and toxicity) filtering by using FAF-Drug-3 and 36 molecules were considered for molecular docking. From the COMT inhibitors, opicapone, fenoldopam, and quercetin were selected, while ZINC63625100_413 ZINC39411941_412, ZINC63234426_254, ZINC63637968_451, and ZINC64019452_303 were chosen for the molecular dynamics simulation analysis having high binding affinity and structural recognition. This study identified the potential COMT inhibitors through pharmacophore-based inhibitor screening leading to a more complete understanding of molecular-level interactions.

• MeSH
• Alzheimer Disease drug therapy   enzymology   physiopathology   MeSH
• Gene Expression MeSH
• Databases, Pharmaceutical MeSH
• Catechol O-Methyltransferase Inhibitors chemistry   pharmacology   MeSH
• Protein Interaction Domains and Motifs MeSH
• Catechol O-Methyltransferase chemistry   MeSH
• Kinetics MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation, beta-Strand MeSH
• Humans MeSH
• Ligands MeSH
• Nootropic Agents chemistry   pharmacology   MeSH
• High-Throughput Screening Assays * MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation MeSH
• Substrate Specificity MeSH
• Protein Structure, Tertiary MeSH
• Thermodynamics MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH