Novel insights into biological roles of inducible cAMP early repressor ICER.

ICER corresponds to a group of alternatively spliced Inducible cAMP Early Repressors with high similarity, but multiple roles, including in circadian rhythm, and are involved in attenuation of cAMP-dependent gene expression. We present experimental and in silico data revealing biological differences between the isoforms with exon gamma (ICER) or without it (ICERγ). Both isoforms are expressed in the liver and the adrenal glands and can derive from differential splicing. In adrenals the expression is circadian, with maximum at ZT12 and higher amplitude of Icerγ. In the liver, the expression of Icerγ is lower than Icer in the 24 h time frame. Icer mRNA has a delayed early response to forskolin. The longer ICER protein binds to three DNA grooves of the Per1 promoter, while ICERγ only to two, as deduced by molecular modelling. This is in line with gel shift competition assays showing stronger binding of ICER to Per1 promotor. Only Icerγ siRNA provoked an increase of Per1 expression. In conclusion, we show that ICER and ICERγ have distinct biochemical properties in tissue expression, DNA binding, and response to forskolin. Data are in favour of ICERγ as the physiologically important form in hepatic cells where weaker binding of repressor might be preferred in guiding the cAMP-dependent response.

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes.

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

Rapid Mechanistic Evaluation and Parameter Estimation of Putative Inhibitors in a Single-Step Progress-Curve Analysis: The Case of Horse Butyrylcholinesterase.

Highly efficient and rapid lead compound evaluation for estimation of inhibition parameters and type of inhibition is proposed. This is based on a single progress-curve measurement in the presence of each candidate compound, followed by the simultaneous analysis of all of these curves using the ENZO enzyme kinetics suite, which can be implemented as a web application. In the first step, all of the candidate ligands are tested as competitive inhibitors. Where the theoretical curves do not correspond to the experimental data, minimal additional measurements are added, with subsequent processing according to modified reaction mechanisms.

Disentangling the formation, mechanism, and evolvement of the covalent methanesulfonyl fluoride acetylcholinesterase adduct: Insights into an aged-like inactive complex susceptible to reactivation by a combination of nucleophiles.

Chemical warfare nerve agents and pesticides, known as organophosphorus compounds inactivate cholinesterases (ChEs) by phosphorylating the serine hydroxyl group located at the active site of ChEs. Over the course of time, phosphorylation is followed by loss of an organophosphate-leaving group and the bond with ChEs becomes irreversible, a process known as aging. Differently, structurally related irreversible catalytic poisons bearing sulfur instead of phosphorus convert ChEs in its aged form only by covalently binding to the key catalytic serine. Kinetic and crystallographic studies of the interaction between Torpedo californica acetylcholinesterase (TcAChE) and a small organosulfonate, methanesulfonyl fluoride (MSF), indeed revealed irreversibly methylsulfonylated serine 200, to be isosteric with the bound aged sarin/soman analogues. The potent bulky reversible inhibitor 7-bis-tacrine (BTA) adopts, in the active site of the crystal structure of the MSF-enzyme adduct, a location and an orientation that closely resemble the one being found in the crystal structure of the BTA-enzyme complex. Remarkably, the presence of BTA accelerates the rate of methanesulfonylation by a factor of two. This unexpected result can be explained on the basis of two facts: i) the steric hindrance exerted by BTA to MSF in accessing the active site and ii) the acceleration of the MSF-enzyme adduct formation as a consequence of the lowering of the rotational and translational degrees of freedom in the proximity of the catalytic serine. It is well known that pralidoxime (2-Pyridine Aldoxime Methyl chloride, 2-PAM) alone or in the presence of the substrate acetylcholine cannot reactivate the active site serine of the TcAChE-MSF adduct. We show that the simultaneous presence of 2-PAM and the additional neutral oxime, 2-[(hydroxyimino)methyl]-l-methylimidazol (2-HAM), triggers the reactivation process of TcAChE within the hour timescale. Overall, our results pave the way toward the likely use of a cocktail of distinctive oximes as a promising recipe for an effective and fast reactivation of aged cholinesterases.

Kinetics of Torpedo californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group.

Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimer's disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser(200) with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc (Torpedo californica) AChE and, on the basis of the results, crystallized the complex between TcAChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser(200) and His(440), disrupting the existing interactions within the catalytic triad, and it stacks with Trp(84) at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.

Insights into subtle conformational differences in the substrate-binding loop of fungal 17ß-hydroxysteroid dehydrogenase: a combined structural and kinetic approach.

The 17ß-HSD (17ß-hydroxysteroid dehydrogenase) from the filamentous fungus Cochliobolus lunatus (17ß-HSDcl) is a NADP(H)-dependent enzyme that preferentially catalyses the interconversion of inactive 17-oxo-steroids and their active 17ß-hydroxy counterparts. 17ß-HSDcl belongs to the SDR (short-chain dehydrogenase/reductase) superfamily. It is currently the only fungal 17ß-HSD member that has been described and represents one of the model enzymes of the cP1 classical subfamily of NADPH-dependent SDR enzymes. A thorough crystallographic analysis has been performed to better understand the structural aspects of this subfamily and provide insights into the evolution of the HSD enzymes. The crystal structures of the 17ß-HSDcl apo, holo and coumestrol-inhibited ternary complex, and the active-site Y167F mutant reveal subtle conformational differences in the substrate-binding loop that probably modulate the catalytic activity of 17ß-HSDcl. Coumestrol, a plant-derived non-steroidal compound with oestrogenic activity, inhibits 17ß-HSDcl [IC50 2.8 µM; at 100 µM substrate (4-oestrene-3,17-dione)] by occupying the putative steroid-binding site. In addition to an extensive hydrogen-bonding network, coumestrol binding is stabilized further by π-π stacking interactions with Tyr212. A stopped-flow kinetic experiment clearly showed the coenzyme dissociation as the slowest step of the reaction and, in addition to the low steroid solubility, it prevents the accumulation of enzyme-coenzyme-steroid ternary complexes.

Substrate-dependent inactivation of recombinant paraoxonase 1 during catalytic dihydrocoumarin turnover and the protective properties of surfactants.

Human paraoxonase-1 (PON1) is the most studied member of the paraoxonases (PONs) family and catalyzes the hydrolysis of various substrates (lactones, aryl esters, and paraoxon). Numerous studies link PON1 to oxidative stress-related diseases such as cardiovascular disease, diabetes, HIV infection, autism, Parkinson's, and Alzheimer's, where the kinetic behavior of an enzyme is characterized by initial rates or by modern methods that obtain enzyme kinetic parameters by fitting the computed curves over the entire time-courses of product formation (progress curves). In the analysis of progress curves, the behavior of PON1 during hydrolytically catalyzed turnover cycles is unknown. Hence, progress curves for enzyme-catalyzed hydrolysis of the lactone substrate dihydrocoumarin (DHC) by recombinant PON1 (rePON1) were analyzed to investigate the effect of catalytic DHC turnover on the stability of rePON1. Although rePON1 was significantly inactivated during the catalytic DHC turnover, its activity was not lost due to the product inhibition or spontaneous inactivation of rePON1 in the sample buffers. Examination of the progress curves of DHC hydrolysis by rePON1 led to the conclusion that rePON1 inactivates itself during catalytic DHC turnover hydrolysis. Moreover, human serum albumin or surfactants protected rePON1 from inactivation during this catalytic process, which is significant because the activity of PON1 in clinical samples is measured in the presence of albumin.

8-Hydroxyquinolylnitrones as multifunctional ligands for the therapy of neurodegenerative diseases.

We describe the development of quinolylnitrones (QNs) as multifunctional ligands inhibiting cholinesterases (ChEs: acetylcholinesterase and butyrylcholinesterase-hBChE) and monoamine oxidases (hMAO-A/B) for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molecular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition, yet unexpectedly showed potent inhibition of hBChE (IC50 = 1.06 ± 0.31 nmol/L) and hMAO-B (IC50 = 4.46 ± 0.18 µmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood-brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson's disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Virtual screening yields inhibitors of novel antifungal drug target, benzoate 4-monooxygenase.

Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.

Automatic Assembly and Calibration of Models of Enzymatic Reactions Based on Ordinary Differential Equations.

Enzymatic reactions have been studied for more than a 100 years. Indeed, isolation of enzymes from biological materials is no longer the main source of enzymes today, as they are now largely produced using recombinant technology, or can even be synthesized from scratch. Studies of the three-dimensional structures of enzymes can provide answers to many questions, but the kinetics of enzymatic reactions is the only method that can lead to better understanding of their function. The complexity of high-throughput analysis of progress curves of data obtained can only be achieved through numerical solutions of a suitable system of ordinary differential equations. We have developed the freely available server ENZO: a web tool for derivation and evaluation of kinetic models of enzyme-catalyzed reactions ( http://enzo.cmm.ki.si/ ). ENZO can be used for simultaneous analysis of a series of progress curves obtained under many different conditions. In this chapter, we exemplify the principles and possibilities of this type of high-throughput analysis.

From tryptophan-based amides to tertiary amines: Optimization of a butyrylcholinesterase inhibitor series.

Lead optimization of a series of tryptophan-based nanomolar butyrylcholinesterase (BChE) inhibitors led to tertiary amines as highly potent, achiral, sp3-rich analogues with better synthetic accessibility and high selectivity over acetylcholinesterase (one to ten thousandfold). Taking it one step further, the introduction of a carbamate warhead on the well-explored reversible scaffold allowed conversion to pseudoirreversible inhibitors that bound covalently to BChE and prolonged the duration of inhibition (half-life of 14.8 h for compound 45a-carbamoylated enzyme). Additionally, N-hydroxyindole was discovered as a novel leaving group chemotype. The covalent mechanism of action was confirmed by time-dependency experiments, progress curve analysis, and indirectly by co-crystallization with the human recombinant enzyme. Two crystal structures of BChE-inhibitor complexes were solved and coupled with the supporting molecular dynamics simulations increased our understanding of the structure-activity relationship, while also providing the necessary structural information for future optimization of this series. Overall, this research demonstates the high versatility and potential of this series of BChE inhibitors.

Ruthenium complexes show potent inhibition of AKR1C1, AKR1C2, and AKR1C3 enzymes and anti-proliferative action against chemoresistant ovarian cancer cell line.

In this study, we present the synthesis, kinetic studies of inhibitory activity toward aldo-keto reductase 1C (AKR1C) enzymes, and anticancer potential toward chemoresistant ovarian cancer of 10 organoruthenium compounds bearing diketonate (1-6) and hydroxyquinolinate (7-10) chelating ligands with the general formula [(η6-p-cymene)Ru(chel)(X)]n+ where chel represents the chelating ligand and X the chlorido or pta ligand. Our studies show that these compounds are potent inhibitors of the AKR enzymes with an uncommon inhibitory mechanism, where two inhibitor molecules bind to the enzyme in a first fast and reversible step and a second slower and irreversible step. The binding potency of each step is dependent on the chemical structure of the monodentate ligands in the metalloinhibitors with the chlorido complexes generally acting as reversible inhibitors and pta complexes as irreversible inhibitors. Our study also shows that compounds 1-9 have a moderate yet better anti-proliferative and anti-migration action on the chemoresistant ovarian cancer cell line COV362 compared to carboplatin and similar effects to cisplatin.

Novel inhibitors of trihydroxynaphthalene reductase with antifungal activity identified by ligand-based and structure-based virtual screening.

Curvularia lunata is a dark pigmented fungus that is the causative agent of several diseases in plants and in both immunodeficient and immunocompetent patients. 1,8-Dihydroxynaphthalene-melanin is found in the cell wall of C. lunata and is believed to be the important virulence factor of dematiaceous fungi. Trihydroxynaphthalene reductase is an enzyme of the 1,8-dihydroxynaphthalene-melanin biosynthetic pathway, and it thus represents an emerging target for the development of novel fungicides and antimycotics. In the present study, we describe novel inhibitors of trihydroxynaphthalene reductase from C. lunata. These inhibitors were identified by ligand-based three-dimensional similarity searching and docking to a homology-built model and by subsequent biochemical and antifungal evaluation. Discovery of competitive inhibitors with K(i) values in low micromolar and even nanomolar concentration range proves the aplicability of homology-built model of 3HNR for hit finding by virtual screening methods.

Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system. RESULTS: By using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase. CONCLUSION: These findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.

Mutations that affect coenzyme binding and dimer formation of fungal 17beta-hydroxysteroid dehydrogenase.

The 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/reductase superfamily, and it functions as a dimer that is composed of two identical subunits. By constructing the appropriate mutants, we have examined the M204 residue that is situated in the coenzyme binding pocket, for its role in the binding of the coenzyme NADP(H). We have also studied the importance of hydrophobic interactions through F124, F132, F133 and F177 for 17beta-HSDcl dimer formation. The M204G substitution decreased the catalytic efficiency of 17beta-HSDcl, suggesting that M204 sterically coerces the nicotinamide moiety of the coenzyme into the appropriate position for further hydride transfer. Phenylalanine substitutions introduced at the dimer interface produced inactive aggregates and oligomers with high molecular masses, suggesting that these hydrophobic interactions have important roles in the formation of the active dimer.

The mechanism and benefit of human butyrylcholinesterase activation by what would otherwise be inhibitors.

Activation of human butyrylcholinesterase by small quaternary ammonium ions is known. Here, additional ligands in this series are presented: edrophonium and choline, and the reactivator pyridine-2-aldoxime methochloride. Kinetic analysis of the progress curves with these compounds indicates the mechanism of enhanced deacylation by the ligand bound to the catalytic anionic site (Trp82) at the base of the active site. The larger, bis-quaternary ligands examined, as propidium, hexamethonium, decamethonium, and bis-thiocholine, show only competitive inhibition of butyrylcholinesterase, by preventing substrate approach. This hypothesis of enhanced deacylation was tested for reactivation of methanesulfonylfluoride-inactivated butyrylcholinesterase, a complex analogous to organophosphate-aged cholinesterases. The combination of substrate/products and pyridine-2-aldoxime methochloride improved butyrylcholinesterase activity over 2 h of continuous measurements, before which time substrate depletion prevailed. Similar reactivation of Torpedo californica acetylcholinesterase was unsuccessful, but both of these cholinesterases regain some activity if they have been inhibited and aged for days by diisopropylfluorophosphate.

DEPTOR polymorphisms influence late complications in Type 2 diabetes patients.

Aim: We investigated if DEPTOR polymorphisms influence metabolic parameters and risk for vascular complications in Type 2 diabetes (T2D) patients. Methods: T2D patients were genotyped for DEPTOR rs7840156, rs2271900 and rs4871827. We built low homology model of DEPTOR to check the position of two investigated substitutions within the protein 3D structure. Results: Carriers of polymorphic DEPTOR rs4871827 A allele had higher HDL cholesterol than noncarriers (p = 0.008). Risk for macrovascular and microvascular complications was increased in rs4871827 GG normal genotype carriers (p = 0.006 and p = 0.021, respectively). Low homology model of DEPTOR has shown that p.Ser389Asn substitution resulting from rs4871827 polymorphism is located at the interaction surface with mTOR. Conclusion: Our data suggest role of DEPTOR polymorphism in T2D vascular complication. First draft submitted: xxx; Accepted for publication: xxx; Published online: TBC.

Kinetic mechanism of Enterococcus faeciumd-aspartate ligase.

Enterococcus faeciumd-aspartate ligase (Aslfm) is a peptide bond-forming enzyme that is involved in the peptidoglycan assembly pathway. It catalyzes the ATP-dependent ligation of the ß-carboxylate of D-Asp to the ε-amino group of L-Lys in the nucleotide precursor UDP- MurNAc-pentapeptide. The enzyme is of interest as a target of new, potential, narrow-spectrum antibiotics directed against multiresistant E. faecium. The kinetic mechanism of Aslfm has not been fully characterized. To determine it, a progress curve analysis of Aslfm catalytic process using pyruvate kinase/lactate dehydrogenase ATPase detection assay was performed. With an inspection of the shape of measured progress curves and the results of specific qualitative experiments, the Aslfm reaction mechanism was singled out. The proposed Aslfm kinetics reaction scheme was evaluated by fitting the parameters of the corresponding differential equations to progress curves using the computer program ENZO. The complete kinetic analysis result is consistent with the substrate binding order 1) ATP, 2) D-Asp, and 3) UDP-MurNAc-pentapeptide. The analysis suggests that slowly establishing non-productive equilibria between the free and ATP-bound enzyme with the participating pentapeptide are responsible for initial reaction burst followed by a steady-state period before the complete depletion of the reactant added in the lowest concentration.

Evidence for subdomain flexibility in Drosophila melanogaster acetylcholinesterase.

The catalytic domain of the acetylcholinesterases is composed of a single polypeptide chain, the folding of which determines two subdomains. We have linked these two subdomains by mutating two residues, I327 and D375, to cysteines, to form a disulfide bridge. As a consequence, the hydrodynamic radius of the protein was reduced, suggesting that there is some flexibility in the subdomain connection. In addition to the smaller size, the mutated protein is more stable than the wild-type protein. Therefore, the flexibility between the two domains is a weak point in terms of protein stability. As expected from the location of the disulfide bond at the rim of the active site, the kinetic studies show that it affects interactions with peripheral ligands and the entrance of some of the bulkier substrates, like o-nitrophenyl acetate. In addition, the mutations affect the catalytic step for o-nitrophenyl acetate and phosphorylation by organophosphates, suggesting that this movement between the two subdomains is connected with the cooperativity between the peripheral and catalytic sites.