In this pilot study, a series of new 3,4-dihydroquinolin-2(1H)-one derivatives as potential dopamine receptor D2 (D2R) modulators were synthesized and evaluated in vitro. The preliminary structure-activity relationship disclosed that compound 5e exhibited the highest D2R affinity among the newly synthesized compounds. In addition, 5e showed a very low cytotoxic profile and a high probability to cross the blood-brain barrier, which is important considering the observed affinity. However, molecular modelling simulation revealed completely different binding mode of 5e compared to USC-D301, which might be the culprit of the reduced affinity of 5e toward D2R in comparison with USC-D301.

• MeSH
• Aripiprazole chemical synthesis   pharmacology   MeSH
• Cell Death MeSH
• Central Nervous System drug effects   MeSH
• Quinolones chemical synthesis   chemistry   pharmacology   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Blood-Brain Barrier drug effects   pathology   MeSH
• Ligands MeSH
• Models, Molecular MeSH
• Drug Design MeSH
• Receptors, Dopamine D2 chemistry   metabolism   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

• MeSH
• Allosteric Regulation drug effects   physiology   MeSH
• Androstanes metabolism   pharmacology   MeSH
• Androstenes metabolism   pharmacology   MeSH
• Benzimidazoles metabolism   pharmacology   MeSH
• CHO Cells MeSH
• Cholesterol metabolism   MeSH
• Cricetulus MeSH
• Cricetinae MeSH
• Humans MeSH
• Neuromuscular Nondepolarizing Agents metabolism   pharmacology   MeSH
• Neurosteroids metabolism   MeSH
• Receptors, Muscarinic metabolism   MeSH
• Gallamine Triethiodide metabolism   pharmacology   MeSH
• Binding Sites drug effects   physiology   MeSH
• Vecuronium Bromide analogs & derivatives   metabolism   pharmacology   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

(4-Oxo-2-thioxothiazolidin-3-yl)acetic acids exhibit a wide range of pharmacological activities. Among them, the only derivative used in clinical practice is the aldose reductase inhibitor epalrestat. Structurally related compounds, [(5Z)-(5-arylalkylidene-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)]acetic acid derivatives were prepared previously as potential antifungal agents. This study was aimed at the determination of aldose reductase inhibitory action of the compounds in comparison with epalrestat and evaluation of structure-activity relationships (SAR). The aldose reductase (ALR2) enzyme was isolated from the rat eye lenses, while aldehyde reductase (ALR1) was obtained from the kidneys. The compounds studied were found to be potent inhibitors of ALR2 with submicromolar IC50 values. (Z)-2-(5-(1-(5-butylpyrazin-2-yl)ethylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (3) was identified as the most efficacious inhibitor (over five times more potent than epalrestat) with mixed-type inhibition. All the compounds also exhibited low antiproliferative (cytotoxic) activity to the HepG2 cell line. Molecular docking simulations of 3 into the binding site of the aldose reductase enzyme identified His110, Trp111, Tyr48, and Leu300 as the crucial interaction counterparts responsible for the high-affinity binding. The selectivity factor for 3 in relation to the structurally related ALR1 was comparable to that for epalrestat. SAR conclusions suggest possible modifications to improve further inhibition efficacy, selectivity, and biological availability in the group of rhodanine carboxylic acids.

• MeSH
• Aldehyde Reductase antagonists & inhibitors   metabolism   MeSH
• Hep G2 Cells MeSH
• Enzyme Inhibitors chemical synthesis   chemistry   pharmacology   MeSH
• Acetic Acid chemical synthesis   chemistry   pharmacology   MeSH
• Humans MeSH
• Ligands MeSH
• Lens, Crystalline drug effects   enzymology   MeSH
• Rats, Wistar MeSH
• Rhodanine analogs & derivatives   chemistry   pharmacology   MeSH
• Thiazolidines chemistry   pharmacology   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Allergy and Immunology MeSH
• Immune System MeSH
• Publication type
• Textbook MeSH

• Conspectus
• Patologie. Klinická medicína
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• alergologie a imunologie

[11C]-PK11195 (PK11195) has been widely used with positron emission tomography (PET) to assess levels of the translocator protein 18 kDa (TSPO) as a marker of neuroinflammation. Recent ligands, such as [11C]-PBR28 and [11C]-DPA713, have improved signal-to-noise ratio and specificity for TSPO over PK11195. However, these second generation radiotracers exhibit binding differences due to a single polymorphism (rs6971) that leads to three genotypes: C/C, C/T and T/T associated with high, mixed and low binding affinities, respectively. Here we report that [3H]-DPA-713 in the presence of cholesterol or PK11195 has an accelerated dissociation rate from TSPO in platelets isolated from individuals with the T/T genotype. This allosteric interaction was not observed in platelets isolated from individuals with the C/C or C/T genotype. The results provide a molecular rationale for low binding affinity of T/T TSPO and further support the exclusion of these subjects from PET imaging studies using second generation TSPO ligands.

• MeSH
• Genotype MeSH
• Humans MeSH
• Neuroimaging methods   MeSH
• Positron-Emission Tomography methods   MeSH
• Radiopharmaceuticals chemistry   MeSH
• Receptors, GABA analysis   chemistry   genetics   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Amino Acid Motifs MeSH
• Phosphorylation genetics   MeSH
• Histone-Lysine N-Methyltransferase genetics   metabolism   MeSH
• HIV Integrase genetics   metabolism   MeSH
• HIV enzymology   genetics   MeSH
• Humans MeSH
• Mediator Complex Subunit 1 genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Myeloid-Lymphoid Leukemia Protein genetics   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

BACKGROUND: Neurotensin receptors are overexpressed in several cancer types including pancreatic ductal adenocarcinoma. Three NTR subtypes have been cloned: NTR-1, NTR-2 and NTR-3. The most expressed NTR-1 is not present in normal pancreatic tissue and has a low expression in chronic pancreatitis. OBJECTIVE: Objective of this study was to test in vitro affinity of the new 68Ga labelled neurotensin analogue DOTA-NT-20.3 (fragment 6-13, Ac-Lys(DOTA)-Pro-Arg(N-CH3)-Arg-Pro-Tyr-Tle-Leu) on the human pancreatic ductal adenocarcinoma cell line AsPC-1. METHOD: For the preparation of 68Ga-DOTA-NT-20.3, 68GaCl3 solution (eluted from 68Ge/68Ga generator) and 50 μg of precursor (Iason, Graz, Austria) water dissolved were used in an automatic synthesis module. The labeled compound was added to cell culture flask and incubated at 37°C. At various time points after tracer addition up to 80min, cells were recovered, rinsed and counted for radioactivity. Results were expressed as percent binding normalized to 200000 cells and affinity parameters were calculated. RESULTS: Labeling yield was ≥98 %. The molar ratio between labelled and total peptide was about 1/400. AsPC-1 cell line showed rapid uptake of the tracer including surface and internalized binding, tending to a plateau phase 80 min after tracer addition (11%/200.000 cells). The Kd (7.335 pmol) and Bmax (90.52 kBq) value indicated high tracer affinity for AsPC-1cell line especially if compared with the literature data regarding other malignancies (e.g. colonic cancer cell line). Binding sites were 1.09x106 sites per cell. CONCLUSION: New tracer 68Ga-DOTA-NT-20.3 can be a suitable candidate for the clinical use in patients with pancreatic ductal adenocarcinoma.

• MeSH
• Adenocarcinoma metabolism   pathology   MeSH
• Heterocyclic Compounds, 1-Ring chemistry   metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Pancreatic Neoplasms metabolism   pathology   MeSH
• Neurotensin agonists   chemistry   metabolism   MeSH
• Peptide Fragments chemistry   metabolism   MeSH
• Gallium Radioisotopes chemistry   metabolism   MeSH
• Receptors, Neurotensin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The cyclooxygenase-2 is a pro-inflammatory and cancer marker, whose mRNA stability and translation is regulated by the CUG-binding protein 2 interacting with AU-rich sequences in the 3' untranslated region. Here, we present the solution NMR structure of CUG-binding protein 2 RRM3 in complex with 5'-UUUAA-3' originating from the COX-2 3'-UTR. We show that RRM3 uses the same binding surface and protein moieties to interact with AU- and UG-rich RNA motifs, binding with low and high affinity, respectively. Using NMR spectroscopy, isothermal titration calorimetry and molecular dynamics simulations, we demonstrate that distinct sub-states characterized by different aromatic side-chain conformations at the RNA-binding surface allow for high- or low-affinity binding with functional implications. This study highlights a mechanism for RNA discrimination possibly common to multiple RRMs as several prominent members display a similar rearrangement of aromatic residues upon binding their targets.The RNA Recognition Motif (RRM) is the most ubiquitous RNA binding domain. Here the authors combined NMR and molecular dynamics simulations and show that the RRM RNA binding surface exists in different states and that a conformational switch of aromatic side-chains fine-tunes sequence specific binding affinities.

• MeSH
• 3' Untranslated Regions MeSH
• Amino Acid Motifs MeSH
• CELF Proteins chemistry   genetics   metabolism   MeSH
• Cyclooxygenase 2 genetics   MeSH
• Phenylalanine chemistry   metabolism   MeSH
• Protein Conformation MeSH
• Magnetic Resonance Spectroscopy MeSH
• RNA, Messenger chemistry   metabolism   MeSH
• Nerve Tissue Proteins chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Amino Acid Substitution MeSH
• AU Rich Elements MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (3H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations,3H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that3H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd73.8 nM) compared to pH 7.4 (Kd2312 nM), as previously reported for other GHB radioligands but similar Bmaxvalues. Using3H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that3H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand3H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

• MeSH
• Autoradiography methods   MeSH
• Cyclopentanes pharmacology   MeSH
• Rodentia MeSH
• Hydroxybutyrates pharmacology   MeSH
• Binding, Competitive MeSH
• Carboxylic Acids pharmacology   MeSH
• Brain drug effects   metabolism   MeSH
• Mice MeSH
• Radioligand Assay methods   MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Immunity MeSH
• Immune System physiology   physiopathology   MeSH
• Immunotherapy MeSH

• Conspectus
• Patologie. Klinická medicína
• NML Fields
• alergologie a imunologie
• biologie
• fyziologie
• NML Publication type
• kolektivní monografie