Balázs Varga

Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice.

Contrasting data were reported regarding the effects of cannabinoids on anxiety and social behaviour in both animals and humans. The cognitive effects of cannabinoids and their interactions with the HPA‐axis raise the possibility that cannabinoid effects are context but not behaviour specific. To assess this hypothesis, we submitted CB1 receptor knock‐out (CB1‐KO) and wild‐type (WT) mice to tests, which involved similar behaviours, but the behavioural context was different. The elevated plus‐maze test was performed under less and more anxiogenic conditions, i.e. under low and high light, respectively. We also compared the social behaviour of the two genotypes in the resident/intruder and social interaction tests. Both tests represent a social challenge and induce similar behaviours, but involve different contexts. The behaviour of CB1‐KO and WT mice was similar under low light, but CB1 gene disruption increased anxiety‐like behaviour under the high light condition. CB1 gene disruption promoted aggressive behaviour in the home‐cage, whereas it inhibited social behaviour in the unfamiliar cage. Thus, the anxiogenic‐like effect was restricted to the more stressful unfamiliar environment. These data suggest that the effects of CB1 gene disruption were context and not behaviour specific. Novelty stress resulted in higher ACTH levels in CB1‐KOs than in WTs, which suggests that context dependency occurred in conjunction with an altered HPA axis function. The present data at least partly explain contrasting effects of cannabinoids in different contexts as well as in different species and strains that show differential stress responses and coping strategies.

Correlated species differences in the effects of cannabinoid ligands on anxiety and on GABAergic and glutamatergic synaptic transmission

Cannabinoid ligands show therapeutic potential in a variety of disorders including anxiety. However, the anxiety‐related effects of cannabinoids remain controversial as agonists show opposite effects in mice and rats. Here we compared the effects of the cannabinoid agonist WIN‐55,212 and the CB1 antagonist AM‐251 in CD1 mice and Wistar rats. Special attention was paid to antagonist–agonist interactions, which had not yet been studied in rats. In mice, WIN‐55,212 decreased whereas AM‐251 increased anxiety. The antagonist abolished the effects of the agonist. In contrast, WIN‐55,212 increased anxiety in rats. Surprisingly, the antagonist potentiated this effect. Cannabinoids affect both GABAergic and glutamatergic functions, which play opposite roles in anxiety. We hypothesized that discrepant findings resulted from species differences in the relative responsiveness of the two transmitter systems to cannabinoids. We investigated this hypothesis by studying the effects of WIN‐55,212 on evoked hippocampal inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs). IPSCs were one order of magnitude more sensitive to WIN‐55,212 in mice than in rats. In mice, IPSCs were more sensitive than EPSCs to WIN‐55,212. This is the first study showing that the relative cannabinoid sensitivity of GABA and glutamate neurotransmission is species‐dependent. Based on behavioural and electrophysiological findings, we hypothesize that WIN‐55,212 reduced anxiety in mice by affecting GABA neurotransmission whereas it increased anxiety in rats via glutamatergic mechanisms. In rats, AM‐251 potentiated this anxiogenic effect by inhibiting the anxiolytic GABAergic mechanism. We suggest that the anxiety‐related effects of cannabinoids depend on the relative cannabinoid responsiveness of GABAergic and glutamatergic neurotransmission.

The effects of cannabinoids on contextual conditioned fear in CB1 knockout and CD1 mice

We studied the effects of cannabinoids on contextual conditioned fear responses. CB1 knockout and wild-type (CD1) mice were exposed to a brief session of electric shocks, and their behavior was studied in the same context 24 h later. In wild-type mice, shock exposure increased freezing and resting, and decreased locomotion and exploration. The genetic disruption of the CB1 receptor abolished the conditioned fear response. The CB1 antagonist AM-251 reduced the peak of the conditioned fear response when applied 30 min before behavioral testing (i.e. 24 h after shocks) in CD1 (wild-type) mice. The cannabinoid agonist WIN-55,212-2 markedly increased the conditioned fear response in CD1 mice, the effect of which was potently antagonized by AM-251. Thus, cannabinoid receptor activation appears to strongly promote the expression of contextual conditioned fear. In earlier experiments, cannabinoids did not interfere with the expression of cue-induced conditioned fear but strongly promoted its extinction. Considering the primordial role of the amygdala in simple associative learning (e.g. in cue-induced fear) and the role of the hippocampus in learning more complex stimulus relationships (e.g. in contextual fear), the present and earlier findings are not necessarily contradictory, but suggest that cannabinoid signaling plays different roles in the two structures. Data are interpreted in terms of the potential involvement of cannabinoids in trauma-induced behavioral changes.

A Non-Fluorinated Monobenzocyclooctyne for Rapid Copper-Free Click Reactions

We designed and synthesised carboxymethylmonobenzocyclooctyne (COMBO) through a four-step reaction pathway. COMBO is a new, structurally simple, non-fluorinated, and directly conjugable copper-free click reagent, which shows excellent reaction kinetics, as also evidenced by theoretical calculations. Additionally, the carboxylic acid appendage allows further conjugation to biomolecules or fluorescent labels. The utility of COMBO in bioorthogonal labelling schemes was demonstrated when a COMBO-containing fluorescent label was employed in glycan imaging of HeLa cells (metabolically modified to have azidosialic acid residues on their cell-surface glycans).

Active site of mycobacterial dUTPase: structural characteristics and a built-in sensor.

dUTPases are essential to eliminate dUTP for DNA integrity and provide dUMP for thymidylate biosynthesis. Mycobacterium tuberculosis apparently lacks any other thymidylate biosynthesis pathway, therefore dUTPase is a promising antituberculotic drug target. Crystal structure of the mycobacterial enzyme in complex with the isosteric substrate analog, alpha,beta-imido-dUTP and Mg(2+) at 1.5A resolution was determined that visualizes the full-length C-terminus, previously not localized. Interactions of a conserved motif important in catalysis, the Mycobacterium-specific five-residue-loop insert and C-terminal tetrapeptide could now be described in detail. Stacking of C-terminal histidine upon the uracil moiety prompted replacement with tryptophan. The resulting sensitive fluorescent sensor enables fast screening for binding of potential inhibitors to the active site. K(d) for alpha,beta-imido-dUTP binding to mycobacterial dUTPase is determined to be 10-fold less than for human dUTPase, which is to be considered in drug optimization. A robust continuous activity assay for kinetic screening is proposed.

Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize γ-32P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:α,β‐imino‐dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C‐terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the α‐phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C‐terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C‐terminus.

Cannabinoid CB1 receptor dependent effects of the NMDA antagonist phencyclidine in the social withdrawal model of schizophrenia

Clinical and laboratory findings suggest that cannabinoid signalling is implicated in schizophrenia. However, the interaction remains poorly understood, as data are often contradictory. Here we investigated wild-type (WT) and cannabinoid CB1 receptor-knockout (CB1-KO) mice in the phencyclidine-induced social withdrawal model of schizophrenia. N-methyl-D-aspartate (NMDA) antagonists (including phencyclidine) induce psychotic symptoms in humans, and are used to model schizophrenia in a variety of experimental conditions. In WTs, 5 mg/kg phencyclidine increased locomotion and stereotyped behaviours, and decreased social interactions. These changes are consistent with a schizophrenia-like effect. In CB1-KOs, phencyclidine decreased locomotion, enhanced ataxia and stereotypy more markedly than in WTs, but did not affect social interactions. Locomotion showed a significant negative correlation with both ataxia and stereotypy, suggesting that in CB1-KOs, the locomotor suppressive effect of phencyclidine was secondary to changes in these variables. Our findings demonstrate that CB1 gene disruption dramatically alters the behavioural effects of the NMDA antagonist phencyclidine, suggesting that the CB1 receptor is involved in schizophrenia. As social disruption and stereotypy respectively are believed to model negative and positive symptoms of schizophrenia, our findings tentatively suggest that cannabinoids are differentially involved in these two symptom categories. These findings require verification by experiments involving CB1 receptor blockers, as the genetic and pharmacological blockade of receptors may not always provide similar results.

Methylene substitution at the α-β bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species‐specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP‐analogous compounds will be required as a second step to invoke species‐specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, α,β‐methylene‐dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of α,β‐imino‐dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:α,β‐methylene‐dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the “trans” α‐phosphate conformation of α,β‐methylene‐dUDP differs from the catalytically competent “gauche” α‐phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the α‐phosphorus by more than 3 Å. The three‐dimensional structures determined in this work show that the binding of the methylene analogue with the α‐phosphorus in the “gauche” conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of α,β‐methylene‐dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while α,β‐methylene‐dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein. Proteins 2008.

Improving thermostability and catalytic activity of pyranose 2‐oxidase from Trametes multicolor by rational and semi‐rational design

The fungal homotetrameric flavoprotein pyranose 2‐oxidase (P2Ox; EC 1.1.3.10) catalyses the oxidation of various sugars at position C2, while, concomitantly, electrons are transferred to oxygen as well as to alternative electron acceptors (e.g. oxidized ferrocenes). These properties make P2Ox an interesting enzyme for various biotechnological applications. Random mutagenesis has previously been used to identify variant E542K, which shows increased thermostability. In the present study, we selected position Leu537 for saturation mutagenesis, and identified variants L537G and L537W, which are characterized by a higher stability and improved catalytic properties. We report detailed studies on both thermodynamic and kinetic stability, as well as the kinetic properties of the mutational variants E542K, E542R, L537G and L537W, and the respective double mutants (L537G/E542K, L537G/E542R, L537W/E542K and L537W/E542R). The selected substitutions at positions Leu537 and Glu542 increase the melting temperature by approximately 10 and 14 °C, respectively, relative to the wild‐type enzyme. Although both wild‐type and single mutants showed first‐order inactivation kinetics, thermal unfolding and inactivation was more complex for the double mutants, showing two distinct phases, as revealed by microcalorimetry and CD spectroscopy. Structural information on the variants does not provide a definitive answer with respect to the stabilizing effects or the alteration of the unfolding process. Distinct differences, however, are observed for the P2Ox Leu537 variants at the interfaces between the subunits, which results in tighter association.