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

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

Protein crystallogenesis represents a key step in X-ray crystallography studies that employ co-crystallization and ligand soaking for investigating ligand binding to proteins. Co-crystallization is a method that enables the precise determination of binding positions, although it necessitates a significant degree of optimization. The utilization of microseeding can facilitate a reduction in sample requirements and accelerate the co-crystallization process. Ligand soaking is the preferred method due to its simplicity; however, it requires careful control of soaking conditions to ensure the successful integration of the ligands. This research protocol details the procedures for co-crystallization and soaking to achieve protein-ligand complex formation, which is essential for advancing drug discovery. Additionally, a simple protocol for demonstrating soaking for educational purposes is described.

• Klíčová slova
• advanced crystallization, co‐crystallization, crystal soaking, crystallization protocol, microseeding,
• MeSH
• konformace proteinů MeSH
• krystalizace metody   MeSH
• krystalografie rentgenová metody   MeSH
• ligandy MeSH
• proteiny * chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ligandy MeSH
• proteiny * MeSH

Galectins are a family of galactoside-binding proteins involved in various pathophysiological processes, which makes them attractive targets for drug discovery. The derivatization of d-galactose at C3 and C1 positions has been shown to increase the affinity of synthetic galectin antagonists. In this study, two small libraries of d-galactose derivatives have been designed and synthesized. The first series involved the development of novel aromatic 3-azolyl-3-deoxy-d-galactopyranoses. The second series consisted of epimeric analogs of glyceryl β-S-d-galactopyranosides, which were also derivatized. Binding-affinity evaluations for galectin-1 and galectin-3 have revealed that galactose analogs from both series have potential for further optimization. Notably, a combination of modifications at the C3 position of the galactose ring and on the aglycone has led to the identification of promising galectin inhibitors, specifically the compounds 29R and 32S.

• Klíčová slova
• Azoles, Carbohydrates, Fluorescence polarization, Galectins, S-glycosides,
• MeSH
• galektin 1 * metabolismus   antagonisté a inhibitory   MeSH
• galektin 3 * metabolismus   antagonisté a inhibitory   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• thiogalaktosidy * chemie   chemická syntéza   farmakologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• galektin 1 * MeSH
• galektin 3 * MeSH
• ligandy MeSH
• thiogalaktosidy * MeSH

The novel diiron amine complexes [Fe2Cp2(CO)(NH2R')(μ-CO){μ-CN(Me)(Cy)}]CF3SO3 [R' = H, 3; Cy, 4; CH2CH2NH2, 5; CH2CH2NMe2, 6; CH2CH2(4-C6H4OMe), 7; CH2CH2(4-C6H4OH), 8; Cp = η5-C5H5, Cy = C6H11 = cyclohexyl] were synthesized in 49-92 % yields from [Fe2Cp2(CO)2(μ-CO){μ-CN(Me)(Cy)}]CF3SO3, 1a, using a straightforward two-step procedure. They were characterized by IR and multinuclear NMR spectroscopy, and the structure of 7 was confirmed through X-ray diffraction analysis. Complexes 3-8 and the acetonitrile adducts [Fe2Cp2(CO)(NCMe)(μ-CO){μ-CN(Me)(R)}]CF3SO3 (R = Cy, 2a; Me, 2b; Xyl = 2,6-C6H3Me2, 2c) were assessed for their water solubility, octanol-water partition coefficient and stability in physiological-like solutions. The in vitro antiproliferative activity of 2a-c and 3-8 was tested on seven human cancer cell lines (A2780, A2780R, PC3, A549, MCF7, HOS and HT-29), while the selectivity was evaluated using normal MRC-5 cells. Overall, the complexes exhibited variable cytotoxicity, with IC50 values reaching the low micromolar range for 3, 7 and 8 in A2780 and A2780R cells, along with significant selectivity. Targeted experiments covered cell cycle modification, induction of cell death, mitochondrial membrane potential, ROS production and interaction with DNA and bovine serum albumin (BSA) as a model protein. The interaction of 3 with BSA was further investigated through computational studies. Results showed a negligible increase in intracellular ROS levels (except for 2b) and insignificant changes in mitochondrial membrane potential.

• Klíčová slova
• Bioorganometallic chemistry, Cellular effects, Diiron complexes, In vitro cytotoxicity, Labile ligand,
• MeSH
• aminy chemie   farmakologie   MeSH
• komplexní sloučeniny chemie   farmakologie   chemická syntéza   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• proliferace buněk * účinky léků   MeSH
• protinádorové látky * farmakologie   chemie   chemická syntéza   MeSH
• screeningové testy protinádorových léčiv * MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• železo chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminy MeSH
• komplexní sloučeniny MeSH
• ligandy MeSH
• protinádorové látky * MeSH
• železo MeSH

In this study, three dinuclear copper(II) complexes of ligand 2,6-bis[(N-methyl-piperazine-1-yl)methyl]-4-formyl phenol (L1) and one of 2,6-bis[(N-methylpiperazine-1-yl)methyl]-4-formyl phenol dimethylacetal (L2) with copper(II) ions have been investigated as new types of biomimetic catalysts for the oxidative transformation of different aminophenols and phenyldiamines. All the complexes of interest were newly synthesized and further characterized by IR spectroscopy, UV-Vis and mass spectrometry, X-ray diffraction, and selected electrochemical measurements. Crystal structures of these dinuclear copper(II) complexes have revealed that the coordination-shell geometry of copper atoms is close to a tetragonal pyramid. Catecholase, phenoxazinone synthase, and horseradish peroxidase-like activities were observed in pure methanol and water-methanol mixtures in the presence of molecular oxygen. The potential applicability of the complexes under study is discussed with respect to their possibilities and limitations in the replacement of natural copper-containing oxidoreductases in the oxidative degradation of water-insoluble chlorinated aminophenols in the dye industry or in the production of phenoxazine-based drugs.

• Klíčová slova
• biomimetic catalysis, dinuclear copper(II) complexes, organic dyes, oxidative transformation, pharmaceuticals,
• MeSH
• barvicí látky chemie   MeSH
• biomimetické materiály chemie   chemická syntéza   MeSH
• katalýza MeSH
• katecholoxidasa metabolismus   chemie   MeSH
• komplexní sloučeniny * chemie   chemická syntéza   MeSH
• křenová peroxidasa metabolismus   chemie   MeSH
• krystalografie rentgenová MeSH
• ligandy MeSH
• měď * chemie   MeSH
• oxidace-redukce MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• katecholoxidasa MeSH
• komplexní sloučeniny * MeSH
• křenová peroxidasa MeSH
• ligandy MeSH
• měď * MeSH

The literature documenting the value of drug-like molecules found in natural products is vast. Although many dietary and herbal remedies have been found to be effective for treating intestinal inflammation, the identification of their active components has lagged behind. In this study, we find that a major ginger component, furanodienone (FDN), is a selective pregnane X receptor (PXR) ligand with agonistic transcriptional outcomes. We show that FDN binds within a sub-pocket of the PXR ligand binding domain (LBD), with subsequent alterations in LBD structure. Using male mice, we show that orally provided FDN has potent PXR-dependant anti-inflammatory outcomes that are colon-specific. Increased affinity and target gene activation in the presence of synergistically acting agonists indicates further opportunities for augmenting FDN activity, efficacy and safety. Collectively, these results support the translational potential of FDN as a therapeutic agent for the treatment and prevention of colonic diseases.

• MeSH
• antiflogistika farmakologie   chemie   MeSH
• furany farmakologie   MeSH
• kolitida farmakoterapie   chemicky indukované   metabolismus   patologie   MeSH
• kolon účinky léků   patologie   metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• pregnanový X receptor * metabolismus   genetika   MeSH
• zánět farmakoterapie   metabolismus   MeSH
• zázvor lékařský * chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiflogistika MeSH
• furany MeSH
• ligandy MeSH
• pregnanový X receptor * 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 use of quantum mechanical potentials in protein-ligand affinity prediction is becoming increasingly feasible with growing computational power. To move forward, validation of such potentials on real-world challenges is necessary. To this end, we have collated an extensive set of over a thousand galectin inhibitors with known affinities and docked them into galectin-3. The docked poses were then used to systematically evaluate several modern force fields and semiempirical quantum mechanical (SQM) methods up to the tight-binding level under consistent computational workflow. Implicit solvation models available with the tested methods were used to simulate solvation effects. Overall, the best methods in this study achieved a Pearson correlation of 0.7-0.8 between the computed and experimental affinities. There were differences between the tested methods in their ability to rank ligands across the entire ligand set as well as within subsets of structurally similar ligands. A major discrepancy was observed for a subset of ligands that bind to the protein via a halogen bond, which was clearly challenging for all the tested methods. The inclusion of an entropic term calculated by the rigid-rotor-harmonic-oscillator approximation at SQM level slightly worsened correlation with experiment but brought the calculated affinities closer to experimental values. We also found that the success of the prediction strongly depended on the solvation model. Furthermore, we provide an in-depth analysis of the individual energy terms and their effect on the overall prediction accuracy.

• MeSH
• galektiny * chemie   metabolismus   MeSH
• konformace proteinů MeSH
• kvantová teorie * MeSH
• ligandy MeSH
• simulace molekulového dockingu MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• galektiny * MeSH
• ligandy MeSH

The new diiron complexes [Fe2Cp2(CO)(L)(μ-CO){μ-CN(Me)(Cy)}]CF3SO3 (L = pyridine, 3a; 4-aminopyridine, 3b; 4-dimethylaminopyridine, 3c; 4-trifluoromethylpyridine, 3d; nicotinic acid, 4; Cp = η5-C5H5, Cy = C6H11 = cyclohexyl) were synthesized in moderate to high yields using two distinct synthetic routes from the precursors 1 (L = CO, for 4) and 2 (L = NCMe, for 3a-d), respectively. All products were characterized by IR and multinuclear NMR spectroscopy, and the structures of 3b and 3d were ascertained by X-ray diffraction studies. The behavior of the complexes in aqueous solutions (solubility, Log Pow, stability) was assessed using NMR and UV-Vis methods. The in vitro antiproliferative activity of 3a-c and 4 was evaluated against seven human cancer cell lines (A2780, A2780R, A549, MCF-7, PC3, HOS and HT-29) and one normal cell line (MRC-5), following 24 h of incubation (MTT test). Overall, 3-4 demonstrated stronger cytotoxicity than cisplatin, with 3c emerging as the most potent compound. The activity seems primarily linked to the inhibition of metabolic processes in the cancer cells, including depletion of reactive oxygen species (ROS) levels. However, subtle differences have been observed between the complexes, with 4 exerting its cytotoxicity through a distinct multimodal mechanism.

• Klíčová slova
• Bioorganometallic chemistry, Cellular effects, Diiron complexes, In vitro cytotoxicity, Metals in medicine, Pyridine ligand,
• MeSH
• komplexní sloučeniny * chemie   farmakologie   chemická syntéza   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• ligandy MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky * farmakologie   chemie   chemická syntéza   MeSH
• pyridiny * chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplexní sloučeniny * MeSH
• ligandy MeSH
• protinádorové látky * MeSH
• pyridine MeSH Prohlížeč
• pyridiny * MeSH