Capillary electrophoretic mobility shift displacement assay for the assessment of weak drug-protein interactions.

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic mobility shift displacement assay that can be used in FBDD for primary screening of weak biomolecular interactions between fragments and target protein. The accuracy and reliability of the present method was demonstrated by measuring the IC50 value of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat. Furthermore, we built a small chemical library to evaluate the performance and the advantage of our newly developed screening-bioassay compared to the direct affinity capillary electrophoresis-binding assay. The results demonstrate the high discriminatory power of the method and above all its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, greatly expanding the scope of the assay. Finally, we proved that this approach is able to discriminate between reversible and irreversible binders. Altogether, this work demonstrates that capillary electrophoresis could constitute an important added value in the arsenal used to screen fragments in drug discovery.

Transverse diffusion of laminar flow profiles as a generic capillary electrophoresis method for in-line nanoreactor mixing: Application to the investigation of antithrombotic activity.

Capillary electrophoresis (CE) instrument was used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened molecules was monitored in a quick and fully automated fashion using only few nanoliters of reactants. To achieve this goal, the targeted enzyme (thrombin) and the chromogenic substrate with or without the screened inhibitor were injected as separate plugs. The mixing of the reactants was then realized using electrophoretically mediated microanalysis (EMMA) or fast transverse diffusion of laminar flow profiles (TDLFP) procedure. The latest provided better mixing performance and was chosen to investigate the inhibitory potency of a fragment library. This very straightforward and fast CE activity assay showed results in good accordance with a previously developed CE affinity assay that confirms the potential of CE at the early stages of drug discovery, providing not only an efficient nanoscale bioreactor but also a selective and integrated separation device.

Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the µM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD.

Optimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases.

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25°C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1-0.3%) and the concentration of DEA (0.01-0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed.

Use of low-dose pregabalin in patients undergoing laparoscopic cholecystectomy.

BACKGROUND: The objective of this study was to examine the effects of low-dose pregabalin on the analgesic efficacy, side-effects, and recovery profile in patients undergoing laparoscopic cholecystectomy. METHODS: One hundred and sixty-two patients aged 18-65 yr, of ASA physical status I-III, undergoing elective outpatient laparoscopic cholecystectomy were recruited and randomized in this prospective, placebo-controlled, double-blind study to receive one of the following study medications orally: pregabalin 50 mg, pregabalin 75 mg, or placebo, 1 h before surgery and then every 12 h after operation for a total of three doses. Postoperative numeric pain scores, analgesic consumption, recovery score (QoR-40), and side-effects (opioid-related symptom distress scale) were assessed in the early postoperative period (every 15 min during the first hour, at 90, 120 min, 6, and 12 h) and at days 1, 2, and 7. Data were analysed using an intention-to-treat method. RESULTS: Compared with the placebo group, the pain scores were lower in the pregabalin 75 mg group in the first 90 min after surgery (P<0.05). Pregabalin 50 mg resulted in pain reduction at 30 and 45 min (P<0.05) relative to placebo. The analgesic consumption, side-effects, and recovery scores were similar among the three groups. CONCLUSIONS: Perioperative administration of pregabalin 75 mg provided limited analgesic benefit in the postoperative period. An updated meta-analysis confirms this finding (see Supplementary material).