Bacterial β sliding clamp (β-clamp) is an emerging drug target currently lacking small-molecule inhibitors with good in vivo activity. Thus, there is a need for fast and simple screening methods for identifying inhibitor candidates. Here we demonstrate the use of nuclear magnetic resonance spectroscopy (NMR) for evaluating compound binding to the E. coli β-clamp. To identify suitable molecular probes, a series of tetrahydrocarbazoles were synthesized, some of which contain fluorine. Key challenges in the synthesis were formation of regioisomers during the Fischer indole reaction and reducing racemization at the stereogenic center. The tetrahydrocarbazoles were assayed against the E. coli β-clamp by saturation-transfer difference (STD) NMR, waterLOGSY and T1ρ. Analysis by isothermal titration calorimetry gave KD-values of 1.7-14 μM for three fluorinated probe candidates, and NMR chemical shift perturbation experiments confirmed these molecules to directly interact with the β-clamp binding pocket. Binding of the fluorinated molecules to β-clamp was easily observed with 19F-observed T2-based binding experiments, and proof of concept for a fluorine-based binding assay for E. coli β-clamp binders is provided.

• Klíčová slova
• (19)F NMR displacement assay, E. coli, Racemization, STD NMR, Tetrahydrocarbazole, β sliding clamp,
• MeSH
• Escherichia coli * účinky léků   MeSH
• halogenace MeSH
• karbazoly * chemie   chemická syntéza   farmakologie   metabolismus   MeSH
• magnetická rezonanční spektroskopie * MeSH
• molekulární sondy chemie   chemická syntéza   metabolismus   MeSH
• molekulární struktura MeSH
• proteiny z Escherichia coli metabolismus   antagonisté a inhibitory   chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karbazoly * MeSH
• molekulární sondy MeSH
• proteiny z Escherichia coli MeSH

The main role of dimeric 14-3-3 proteins is to modulate the activity of several hundred binding partners by interacting with phosphorylated residues of the partner proteins, often located in disordered regions. The inherent flexibility or large size of 14-3-3 complexes hampers their structural characterization by X-ray crystallography, cryo-electron microscopy (EM) and traditional solution nuclear magnetic resonance (NMR) spectroscopy. Here, we employ solution 1D 19F-Trp NMR spectroscopy to characterize substrate binding and dimerization of 14-3-3 proteins, focusing on 14-3-3ζ - an abundant human isoform as an example. Both conserved Trp residues are located in distinct functionally important sites - the dimeric interface and the ligand-binding groove. We substituted them by 5F-Trp, thereby introducing a convenient NMR probe. Fluorination of the two Trp did not impact the stability and interaction properties of 14-3-3ζ in a substantive manner, permitting to carry out 19F NMR experiments to assess 14-3-3's structure and behavior. Importantly, 5F-Trp228 reports on binding of substrates in the amphipathic binding groove of 14-3-3ζ and permitted to distinguish distinct recognition modes. Thus, we established that 19F NMR is a powerful approach to evaluate the binding of partner proteins to 14-3-3 and to characterize the properties of the resulting complexes.

• Klíčová slova
• (19)F NMR, 14-3-3 monomer, 14-3-3 proteins, Quaternary structure, Substrate binding,
• MeSH
• lidé MeSH
• ligandy MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• proteiny 14-3-3 * chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny 14-3-3 * MeSH

Electric fields generated by protein scaffolds are crucial in enzymatic catalysis. This review surveys theoretical approaches for detecting, analyzing, and comparing electric fields, electrostatic potentials, and their effects on the charge density within enzyme active sites. Pioneering methods like the empirical valence bond approach rely on evaluating ionic and covalent resonance forms influenced by the field. Strategies employing polarizable force fields also facilitate field detection. The vibrational Stark effect connects computational simulations to experimental Stark spectroscopy, enabling direct comparisons. We highlight how protein dynamics induce fluctuations in local fields, influencing enzyme activity. Recent techniques assess electric fields throughout the active site volume rather than only at specific bonds, and machine learning helps relate these global fields to reactivity. Quantum theory of atoms in molecules captures the entire electron density landscape, providing a chemically intuitive perspective on field-driven catalysis. Overall, these methodologies show protein-generated fields are highly dynamic and heterogeneous, and understanding both aspects is critical for elucidating enzyme mechanisms. This holistic view empowers rational enzyme engineering by tuning electric fields, promising new avenues in drug design, biocatalysis, and industrial applications. Future directions include incorporating electric fields as explicit design targets to enhance catalytic performance and biochemical functionalities.

• MeSH
• biokatalýza MeSH
• enzymy * chemie   metabolismus   MeSH
• katalytická doména MeSH
• kvantová teorie MeSH
• proteiny * chemie   metabolismus   MeSH
• statická elektřina MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• enzymy * MeSH
• proteiny * MeSH

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

• Klíčová slova
• 14‐3‐3 proteins, extracellular signal‐regulated kinase 2, microtubule‐associated protein, nuclear magnetic resonance, protein kinase A,
• MeSH
• fosforylace MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• mitogenem aktivovaná proteinkinasa 1 metabolismus   genetika   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   genetika   MeSH
• proteiny 14-3-3 * metabolismus   chemie   genetika   MeSH
• proteiny asociované s mikrotubuly * metabolismus   chemie   genetika   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• MAPK1 protein, human MeSH Prohlížeč
• mitogenem aktivovaná proteinkinasa 1 MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny 14-3-3 * MeSH
• proteiny asociované s mikrotubuly * MeSH
• YWHAZ protein, human MeSH Prohlížeč

The development of small molecule drugs that target protein binders is the central goal in medicinal chemistry. During the lead compound development process, hundreds or even thousands of compounds are synthesized, with the primary focus on their binding affinity to protein targets. Typically, IC50 or EC50 values are used to rank these compounds. While thermodynamic values, such as the dissociation constant (KD), would be more informative, they are experimentally less accessible. In this study, we compare isothermal calorimetry (ITC) with surface plasmon resonance (SPR) using human STING, a key protein of innate immunity, and several cyclic dinucleotides (CDNs) that serve as its ligands. We demonstrate that SPR, with recent technological advancements, provides KDs that are sufficiently accurate for drug development purposes. To illustrate the versatility of our approach, we also used SPR to estimate the KD of poxin binding to cyclic GMP-AMP (cGAMP) that serves as a second messenger in the innate immune system. In conclusion, SPR offers a high benefit-to-cost ratio, making it an effective tool in the drug design process.

• Klíčová slova
• Cyclic dinucleotide, ITC, Poxin, SPR, STING,
• MeSH
• kalorimetrie MeSH
• lidé MeSH
• ligandy MeSH
• membránové proteiny * metabolismus   chemie   MeSH
• nukleotidy cyklické * chemie   metabolismus   MeSH
• povrchová plasmonová rezonance * MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cyclic guanosine monophosphate-adenosine monophosphate MeSH Prohlížeč
• ligandy MeSH
• membránové proteiny * MeSH
• nukleotidy cyklické * MeSH
• STING1 protein, human MeSH Prohlížeč

Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcription. CDK7 plays a pivotal role in cell division and proliferation, and the CDK7 gene often exhibits mutations or copy number loss in cancer. Pharmacological targeting of CDK7 has been proposed as a cancer treatment strategy and several inhibitors are currently in clinical trials. As opposed to CDK2, the use of structure-assisted drug design for CDK7 has been limited. We present here CDK2m7, a CDK2-based CDK7 mimic created by mutagenesis of the CDK2 active site pocket. CDK2m7 can be produced in E. coli in a fully active complex with cyclin A2 in high yield and purity. CDK2m7 exhibits a shift in inhibitor selectivity from CDK2 to CDK7 and readily crystallizes. Therefore, it can be used in structure-assisted design of CDK7 inhibitors, as demonstrated by the crystal structure of the complex with inhibitor SY5609. CDK2m7 thus represents a simple and affordable platform for CDK7 rational drug development.

• Klíčová slova
• Cyclin-dependent kinase, Inhibitor, SY5609, Selectivity, Structure-assisted inhibitor design, X-ray crystallography,
• MeSH
• cyklin-dependentní kinasa 2 * chemie   genetika   metabolismus   antagonisté a inhibitory   MeSH
• cyklin-dependentní kinasy * chemie   antagonisté a inhibitory   metabolismus   genetika   MeSH
• inhibitory proteinkinas * farmakologie   chemie   MeSH
• katalytická doména MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární mimikry MeSH
• molekulární modely MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CDK2 protein, human MeSH Prohlížeč
• CDK7 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 * MeSH
• cyklin-dependentní kinasy * MeSH
• inhibitory proteinkinas * MeSH
• kinasa aktivující cyklin dependentní kinasy * MeSH

AT-9010 (2'-methyl-2'-fluoro guanosine triphosphate) is a GTP analog whose prodrug, AT-752 is under consideration in human medicine as a potential antiviral drug against certain flaviviruses. It was previously believed to inhibit viral replication by acting primarily as a chain terminator. However, it was discovered recently that it also binds the GTP binding site of the methyltransferase (MTase) domain of the orthoflavivirus polymerase, thus interfering with RNA capping. Here, we investigated the binding of AT-9010 to Ntaya and Zika virus MTases. Structural analysis using X-ray crystallography revealed similar interactions between the base and sugar moieties of AT-9010 and key residues in both MTases, although differences in hydrogen bonding were observed. Our analysis also suggested that the triphosphate part of AT-9010 is flexible. Despite minor variations, the overall binding mode of AT-9010 was found to be the same for all of the flaviviral MTases examined, suggesting a structural basis for the efficacy of AT-9010 against multiple orthoflavivirus MTases.

• MeSH
• adenosin analogy a deriváty   MeSH
• antivirové látky * farmakologie   metabolismus   chemie   MeSH
• Flavivirus enzymologie   MeSH
• guanosintrifosfát metabolismus   MeSH
• krystalografie rentgenová MeSH
• methyltransferasy * metabolismus   chemie   MeSH
• molekulární modely MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• virové proteiny metabolismus   chemie   genetika   MeSH
• virus zika enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosin MeSH
• antivirové látky * MeSH
• guanosintrifosfát MeSH
• methyltransferasy * MeSH
• sinefungin MeSH Prohlížeč
• virové proteiny MeSH

The 5'-3' exoribonuclease Xrn2, known as Rat1 in yeasts, terminates mRNA transcription by RNA polymerase II (RNAPII). In the torpedo model of termination, the activity of Xrn2/Rat1 is enhanced by Rai1, which is recruited to the termination site by Rtt103, an adaptor protein binding to the RNAPII C-terminal domain (CTD). The overall architecture of the Xrn2/Rat1-Rai1-Rtt103 complex remains unknown. We combined structural biology methods to characterize the torpedo complex from Saccharomyces cerevisiae and Chaetomium thermophilum. Comparison of the structures from these organisms revealed a conserved protein core fold of the subunits, but significant variability in their interaction interfaces. We found that in the mesophile, Rtt103 utilizes an unstructured region to augment a Rai1 β-sheet, while in the thermophile Rtt103 binds to a C-terminal helix of Rai1 via its CTD-interacting domain with an α-helical fold. These different torpedo complex assemblies reflect adaptations to the environment and impact complex recruitment to RNAPII.

• Klíčová slova
• NMR, RNAPII, cryo-EM, exonuclease, structure, termination, thermophiles, torpedo complex,
• MeSH
• Chaetomium * metabolismus   chemie   MeSH
• exoribonukleasy * chemie   metabolismus   genetika   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• RNA-polymerasa II metabolismus   chemie   MeSH
• Saccharomyces cerevisiae - proteiny * chemie   metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• exoribonukleasy * MeSH
• RAT1 protein, S cerevisiae MeSH Prohlížeč
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny * MeSH

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which plays numerous and pivotal roles in human physiology and pathophysiology. Therefore, pharmacotherapeutic targeting of the AhR is a highly pertinent issue. The identification of new AhR ligands and the characterization of the interactions between the AhR ligands and AhR protein requires appropriate methodology. In spite the AhR is monomeric intracellular soluble receptor, the full-length human AhR protein has not been crystallized so far, and its isolation in a form applicable in the binding assays is highly challenging. Recent advances, including crystallization of AhR fragments, recombinant protein technologies, and cryogenic electron microscopy, allowed for exploitation of diverse experimental techniques for studying interactions between ligands and the AhR. In the current paper, we review existing AhR ligand binding assays, including their description, applicability and limitations.

• Klíčová slova
• aryl hydrocarbon receptor, interactions, ligands, protein binding,
• MeSH
• lidé MeSH
• ligandy MeSH
• receptory aromatických uhlovodíků * metabolismus   MeSH
• transkripční faktory bHLH metabolismus   chemie   MeSH
• vazba proteinů * MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• AHR protein, human MeSH Prohlížeč
• ligandy MeSH
• receptory aromatických uhlovodíků * MeSH
• transkripční faktory bHLH 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.

• Klíčová slova
• AlphaScreen, Cap snatching, DNA-linked Inhibitor Antibody assay, Influenza A polymerase, Inhibitor, Pimodivir,
• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• DNA MeSH
• lidé MeSH
• RNA čepičky * metabolismus   MeSH
• RNA-dependentní RNA-polymerasa * antagonisté a inhibitory   metabolismus   MeSH
• rychlé screeningové testy * metody   MeSH
• vazebná místa MeSH
• virové proteiny * antagonisté a inhibitory   metabolismus   MeSH
• virus chřipky A * účinky léků   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky * MeSH
• DNA MeSH
• RNA čepičky * MeSH
• RNA-dependentní RNA-polymerasa * MeSH
• virové proteiny * MeSH