Unravelling the mechanisms of a protein refolding process based on the association of detergents and co-solvents.

A new technique associating the detergent Sodium Dodecyl Sulphate (SDS) and an alcohol-type co-solvent has been set up, showing an unexpected efficiency to refold several types of soluble or membrane proteins. The present contribution deepens the fundamental knowledge on the phenomena underlying this process, considering the refolding of two model peptides featuring the main protein secondary structures: α-helix and ß-sheet. Their refolding was monitored by fluorescence and circular dichroism, and it turns out that: (i) 100% recovery of the folded structure is observed for both peptides, (ii) the highest the SDS concentration, the more co-solvent to be added to recover the peptides' native structures, (iii) a high alcohol concentration is required to alter the SDS denaturing properties, (iv) the co-solvent performance relies on its specific lipophilic-hydrophilic balanced character, (v) the size of the micelle formed by the detergent does not enter the process critical parameters, and (vi) increasing the salt concentration up to 1 M NaCl has a beneficial impact on the process efficiency. These mechanistic aspects will help us to improve the method and extend its application. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Low potency and limited efficacy of antiepileptic drugs in the mouse 6 Hz corneal kindling model.

Corneal kindling is a useful alternative to electrically induced amygdala or hippocampal kindling, which requires advanced surgical and EEG techniques that may not be easily available in many laboratories. Therefore the first aim of this study was to evaluate whether repeated 6 Hz corneal stimulation in mice would lead to an increased and persistent seizure response as described for higher frequency (50/60 Hz) corneal kindling. Male NMRI mice stimulated twice daily (except weekends) for 3 s with 6 Hz electrical current at 44 mA displayed robust kindling development, i.e., a progressive increase in seizure severity. The majority of the animals (about 90%) developed a fully kindled state, defined as at least 10 consecutive stage 3-5 seizures within 5 weeks of corneal stimulation. Afterwards, the fully kindled state was maintained for at least 8 weeks with only two days of stimulations per week. Next, the protective efficacy of four mechanistically different antiepileptic drugs (AEDs; clonazepam, valproate, carbamazepine and levetiracetam) was assessed and compared between 6 Hz and 50 Hz fully kindled mice. All tested AEDs showed a relatively lower potency in the 6 Hz kindling model and a limited efficacy against partial seizures was observed with carbamazepine and levetiracetam. We can conclude that 6 Hz kindling may be more advantageous than the previously described 50/60 Hz corneal kindling models due to its robustness and persistence of the fully kindled state. Furthermore, the observed low potency and limited efficacy of AEDs in 6 Hz fully kindled mice suggest that this model could be a useful tool in the discovery of novel AEDs targeting treatment resistant epilepsy.

Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry.

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC-MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.

Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella ß-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-ß-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.

Brivaracetam does not alter spatial learning and memory in both normal and amygdala-kindled rats.

Several antiepileptic drugs (AEDs) may induce memory deficits when tested in preclinical models at doses that exert significant protection against seizures. Brivaracetam (BRV) is a novel high-affinity SV2A ligand also displaying inhibitory activity at neuronal voltage-gated sodium channels. In the present study we have investigated the effects of BRV, at doses that exerted marked anticonvulsant effects in kindled rats, upon cognitive functioning and memory in both normal and amygdala-kindled rats using place learning version of Morris water maze. In addition the effect of BRV on long-term potentiation (LTP) in rat hippocampal slices has been investigated. BRV (2.1, 6.8 or 21.0mg/kg i.p.) was injected daily, 60min before each session. Results indicated that in both normal and amygdala-kindled rats BRV did not alter the latency to find the hidden platform or swimming speed during the four consecutive days of learning. Similarly, the time spent in the target quadrant, used as a further independent index of spatial memory, was not modified by BRV treatment. Likewise, BRV did not affect the LTP induction in CA1 hippocampal region when tested at 3-30microM concentration range, which had been demonstrated to significantly reduce epileptiform activity in slice models. Based on the results of the present study it can be expected that BRV will not have detrimental effects on hippocampal-dependent cognitive functions in patients with epilepsy.

Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A.

BACKGROUND AND PURPOSE: Screening of 12,000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. EXPERIMENTAL APPROACH: The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure-susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. KEY RESULTS: Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure-susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. CONCLUSIONS AND IMPLICATIONS: Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index.

The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents.

1. In this study in vitro and in vivo approaches were combined in order to investigate if the anti-epileptic mechanism(s) of action of levetiracetam (LEV; Keppra) may involve modulation of inhibitory neurotransmission. 2. GABA- and glycine-gated currents were studied in vitro using whole-cell patch-clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound-susceptible mice. The effect of LEV was compared with reference anti-epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. 3. LEV contrasted the reference AEDs by an absence of any direct effect on glycine-gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA-gated currents. A similar action on GABA-elicited currents was observed with the reference AEDs, except ethosuximide. 4. These minor direct effects contrasted with a potent ability of LEV (EC(50)=1 - 10 microM) to reverse the inhibitory effects of the negative allosteric modulators zinc and beta-carbolines on both GABA(A) and glycine receptor-mediated responses. 5. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of beta-carbolines on GABA-gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine-gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. 6. LEV (17 mg kg(-1)) produced a potent suppression of sound-induced clonic convulsions in mice. This protective effect was significantly abolished by co-administration of the beta-carboline FG 7142, from a dose of 5 mg kg(-1). In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg(-1)) was without any effect on the protection afforded by LEV. 7. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti-epileptic mechanism(s) of action of LEV.

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Kinetic study of two novel enantiomeric tricyclic beta-lactams which efficiently inactivate class C beta-lactamases.

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative beta-lactamases and D-alanyl-D-alanine peptidases (DD-peptidases) is presented. Both compounds are very efficient inactivators of the Enterobacter cloacae 908R beta-lactamase, which is usually resistant to inhibition. Preliminary experiments indicate that various extended-spectrum class C beta-lactamases (ACT-1, CMY-1, and MIR-1) are also inactivated. With the E. cloacae 908R enzyme, complete inactivation occurs with a second-order rate constant, k(2)/K', of 2 x 10(4) to 4 x 10(4) M(-1) s(-1), and reactivation is very slow, with a half-life of >1 h. Accordingly, Lek 157 significantly decreases the MIC of ampicillin for E. cloacae P99, a constitutive class C beta-lactamase overproducer. With the other serine beta-lactamases tested, the covalent adducts exhibit a wide range of stabilities, with half-lives ranging from long (>4 h with the TEM-1 class A enzyme), to medium (10 to 20 min with the OXA-10 class D enzyme), to short (0.2 to 0.4 s with the NmcA class A beta-lactamase). By contrast, both carbapenems behave as good substrates of the Bacillus cereus metallo-beta-lactamase (class B). The Streptomyces sp. strain R61 and K15 extracellular DD-peptidases exhibit low levels of sensitivity to both compounds.

Pharmacological evaluation of a diarylmethylene-piperidine derivative: a new potent atypical antipsychotic?

A new diaryl-methylene piperidine derivative, 2, displayed an atypical antipsychotic profile both in vitro and in vivo. The main pharmacological characteristics of this compound appears to reside in a more potent antagonism of the 5-HT2 serotonergic receptor than of the D2 dopaminergic receptor. This confirms that molecules displaying a D2/5-HT2 binding ratio < 1 possess clozapine-like antipsychotic activity.

Thermal unfolding of an intermediate is associated with non-Arrhenius kinetics in the folding of hen lysozyme.

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T(m) approximately 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ( approximately 25 %) and slow ( approximately 75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the alpha-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the alpha-domain intermediate (the dominant factor at high temperatures). Destabilization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.

Technique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases.

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.

Evidence for a unique profile of levetiracetam in rodent models of seizures and epilepsy.

The protective and adverse effect potentials of levetiracetam ((S)-alpha-ethyl-2-oxo-pyrrolidine acetamide) in rodent models of seizures and epilepsy were compared with the profile of several currently prescribed and newly developed antiepileptic drugs. Levetiracetam was devoid of anticonvulsant activity in the acute maximal electroshock seizure test and in the maximal pentylenetetrazol seizure test in mice (up to 540 mg/kg, i.p.) but exhibited potent protection against generalised epileptic seizures in electrically and pentylenetetrazol-kindled mice (ED50 values = 7 and 36 mg/kg, respectively, i.p.). This differs markedly from established and most new antiepileptic drugs which induce significant protection in both the acute seizure tests and the kindling models. Furthermore, levetiracetam was devoid of anticonvulsant activity in several maximal chemoconvulsive seizure tests although an interesting exception was the potent protection observed against secondarily generalised activity from focal seizures induced by pilocarpine in mice (ED50 value = 7 mg/kg, i.p.), pilocarpine and kainic acid in rats (minimum active dose = 17 and 54 mg/kg, respectively, i.p.). The protection afforded by levetiracetam on the threshold for methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)-induced seizures persisted after chronic administration (17-170 mg/kg, i.p., twice daily/14 days) and levetiracetam did not lower the seizure threshold for the proconvulsant action of the inverse benzodiazepine receptor agonist, N-methyl-beta-carboline-3-carboxamide (FG 7142). The main metabolite of levetiracetam (ucb L057; (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid) was found to be inactive in sound-sensitive mice after acute administration of doses up to 548 mg/kg, i.p. Levetiracetam induced only minor behavioural alterations in both normal and amygdala-kindled rats (54-1700 mg/kg, i.p.) resulting in an unusually high safety margin between rotarod impairment and seizure suppression of 148 in corneally kindled mice and 235 in Genetic Absence Epilepsy Rats from Strasbourg. In comparison, existing antiepileptic drugs have ratios between 2 and 17 in the corneally kindled mouse model. These studies reveal a unique profile of levetiracetam in rodent models. Characteristics are a general lack of anticonvulsant activity against maximal, acute seizures and selective protection with a very high safety margin in genetic and kindled animals and against chemoconvulsants producing partial epileptic seizures. This activity differs markedly from that of the established and newly introduced antiepileptic drugs and appears to derive from the parent compound since its major metabolite was inactive in all models studied. Together these results therefore suggest that levetiracetam may offer an effective, broad-spectrum treatment of epileptic seizures in patients, with a minimum of adverse effects.

Validation of corneally kindled mice: a sensitive screening model for partial epilepsy in man.

Epileptogenesis induced by electrical kindling of rats appears to be superior to the acute maximal electroshock seizure (MES) test in normal animals in predicting the efficacy and adverse effect potential of drugs in patients with partial epilepsy. Unfortunately, inclusion of such kindling models in primary screening is hampered by the laborious and expensive procedure of stimulation and recording with implanted brain electrodes. Furthermore the size of the rats excludes their use in initial testing where compound availability is often limited for the 'first batch synthesis'. The present study demonstrates that chronic electrical stimulation with corneal electrodes in mice can rapidly yield large numbers of kindled animals with a seizure phenomenology reflecting partial seizures in man. A pharmacological characterisation showed that corneally kindled mice can be used repeatedly for several drug experiments with reproducible results. The seizure protection and adverse effect potential obtained with proven antiepileptic drugs were similar to the effects observed in amygdala kindled rats and their corresponding clinical profile in partial epilepsy. Protection was obtained with vigabatrin and levetiracetam in this new model despite their lack of anticonvulsant activity in the acute MES test. Furthermore, in agreement with clinical findings with NMDA antagonists, MK-801 revealed more severe adverse effects in corneally kindled mice than in normal animals. These results suggest that corneal kindling of mice represents a sensitive and valid screening model for the identification of new therapies for partial epilepsy in man.

The folding process of hen lysozyme: a perspective from the 'new view'.

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the 'new view' of protein folding based on energy surfaces and landscapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together.

The origin of the alpha-domain intermediate in the folding of hen lysozyme.

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. In addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not seen at 50 degrees C. Addition of 2 M NaCl to the refolding buffer at 50 degrees C, however, regenerates both the hydrogen exchange and optical properties associated with the alpha-domain intermediate but has no significant effect on the overall refolding kinetics. Together with previous findings, these results indicate that non-native interactions within the alpha-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the beta-domain constitutes the rate-limiting step for the majority of molecules.

Catalytic properties of class A beta-lactamases: efficiency and diversity.

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.