Capillary electrokinetic chromatography for chiral separation of potential SARS-CoV-2 3CL protease inhibitors.

In this work, a capillary electrophoresis method was developed as a quality control tool to determine the enantiomeric purity of a series of five chiral compounds evaluated as potential severe acute respiratory syndrome coronavirus 2 3CL protease inhibitors. The first cyclodextrin tested, that is, highly sulfated-ß-cyclodextrin, at 6% (m/v) in a 25 mM phosphate buffer, using a capillary dynamically coated with polyethylene oxide, at an applied voltage of 15 kV and a temperature of 25°C, was found to successfully separate the five derivatives. The limits of detection and quantification were calculated together with the greenness score of the method in order to evaluate the method in terms of analytical and environmental performance. In addition, it is noteworthy that simultaneously high-performance liquid chromatography separation of the enantiomers of the same compounds with two different columns, the amylose tris(3,5-dimethylphenylcarbamate)-coated and the cellulose tris(3,5-dichlorophenylcarbamate)-immobilized on silica stationary phases, was studied. Neither the former stationary phase nor the latter was able to separate all derivatives in a mobile phase consisting of n-heptane/propan-2-ol 80/20 (v/v).

Electrophoretic separation of multiple chiral center analyte with a three cyclodextrins mixture.

A capillary electrokinetic chromatography method (CEKC) was developed for complete stereoisomeric separation of a neutral, hydrophobic, multiple chiral center dihydropyridone analogue, a drug candidate proposed in type 2 diabetes treatment. A background electrolyte comprising three cyclodextrins was found to successfully separate the eight isomers. First an anionic cyclodextrin, the SBE-ß-CD, was selected to allow the chiral separation of our neutral compound and partial resolutions of the eight isomers were obtained. Then, the effects of different parameters such as the nature and concentration of the other cyclodextrins added and pH of the buffer were examined. Finally, a triple CD-system consisted of 15 mM SBE-ß-CD plus 15 mM Î³-CD and 40 mM HP-γ-CD in a 50 mM borate background electrolyte at pH 10, was found to successfully separate the eight isomers. Last, the selectivity and limits of detection and quantification were evaluated for this optimized method.

Molecular Design in Practice: A Review of Selected Projects in a French Research Institute That Illustrates the Link between Chemical Biology and Medicinal Chemistry.

Chemical biology and drug discovery are two scientific activities that pursue different goals but complement each other. The former is an interventional science that aims at understanding living systems through the modulation of its molecular components with compounds designed for this purpose. The latter is the art of designing drug candidates, i.e., molecules that act on selected molecular components of human beings and display, as a candidate treatment, the best reachable risk benefit ratio. In chemical biology, the compound is the means to understand biology, whereas in drug discovery, the compound is the goal. The toolbox they share includes biological and chemical analytic technologies, cell and whole-body imaging, and exploring the chemical space through state-of-the-art design and synthesis tools. In this article, we examine several tools shared by drug discovery and chemical biology through selected examples taken from research projects conducted in our institute in the last decade. These examples illustrate the design of chemical probes and tools to identify and validate new targets, to quantify target engagement in vitro and in vivo, to discover hits and to optimize pharmacokinetic properties with the control of compound concentration both spatially and temporally in the various biophases of a biological system.

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

Single and dual cyclodextrins systems for the enantiomeric and diastereoisomeric separations of structurally related dihydropyridone analogues.

CD capillary electrophoresis methods were developed for complete enantiomeric and diastereoisomeric separations of a series of ten dihydropyridone analogues, of which eight were neutral, one was anionic, and one was cationic. Ten different systems comprising one or two CDs were found to successfully separate the isomers thanks to a screening approach. Among the tested CDs, highly sulfated-γ-CD (HS-γ-CD), either in a single or in a dual system, in a phosphate buffer using capillaries dynamically coated with polyethylene oxide, and SBE-ß-CD, either in a single or in a dual system, in a borate buffer using uncoated capillaries, were the most selective selectors. The effects of different parameters such as the nature and concentration of the CDs, nature and concentration of the buffer, and voltage were examined. The precision and LODs and limits of quantification were evaluated for the optimized methods.

Characterization of monoclonal antibodies by a fast and easy liquid chromatography-mass spectrometry time-of-flight analysis on culture supernatant.

Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain mass spectrometry profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates three fragments of 25 kDa that can be analyzed by liquid chromatography-mass spectrometry time-of-flight (LC-MS TOF) in one run: LC, Fd, and Fc/2. This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process was successfully compared with high-performance capillary electrophoresis with laser-induced fluorescence detection (HPCE-LIF), currently considered as the "gold standard" method.

Development of HPLC/fluorescence detection method for chiral resolution of dansylated benzimidazoles derivatives.

The development of high performance liquid chromatography method on amylose-based stationary phase (Chiralpak AD) with n-hexane-2-propanol (90:10, v/v) as mobile phase allowed the enantioseparation of non-dansylated and dansylated benzimidazole derivatives, prepared from potent-AMPK activators, to be achieved. Using both fluorescence and ultraviolet detection, limits of detection and quantification were determined. Fluorescence detection seems to be the most appropriate technique since the first enantiomer of dansylated benzimidazole 8 eluted with a limit of quantification of 2.25 nm. The quantification limit was improved 10-fold by fluorescence detection compared with a previous report describing mass spectrometry detection. Linearity and repeatability parameters were validated. These lower limits obtained by fluorescence detection, associated with good resolution observed for the dansylated derivatives, make this chiral methodology convenient for the use of these fluorescent potent-AMPK activators as probes to elucidate their cellular localization and their mechanism of action.

Identification of new correctors for traffic-defective ABCB4 variants by a high-content screening approach.

ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.

N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease.

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 µM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.

Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses.

Since end of 2019, the global and unprecedented outbreak caused by the coronavirus SARS-CoV-2 led to dramatic numbers of infections and deaths worldwide. SARS-CoV-2 produces two large viral polyproteins which are cleaved by two cysteine proteases encoded by the virus, the 3CL protease (3CLpro) and the papain-like protease, to generate non-structural proteins essential for the virus life cycle. Both proteases are recognized as promising drug targets for the development of anti-coronavirus chemotherapy. Aiming at identifying broad spectrum agents for the treatment of COVID-19 but also to fight emergent coronaviruses, we focused on 3CLpro that is well conserved within this viral family. Here we present a high-throughput screening of more than 89,000 small molecules that led to the identification of a new chemotype, potent inhibitor of the SARS-CoV-2 3CLpro. The mechanism of inhibition, the interaction with the protease using NMR and X-Ray, the specificity against host cysteine proteases and promising antiviral properties in cells are reported.

Discovery, Structure-Activity Relationships, and In Vivo Activity of Dihydropyridone Agonists of the Bile Acid Receptor TGR5.

A novel series of potent agonists of the bile acid receptor TGR5 bearing a dihydropyridone scaffold was developed from a high-throughput screen. Starting from a micromolar hit compound, we implemented an extensive structure-activity-relationship (SAR) study with the synthesis and biological evaluation of 83 analogues. The project culminated with the identification of the potent nanomolar TGR5 agonist 77A. We report the GLP-1 secretagogue effect of our lead compound ex vivo in mouse colonoids and in vivo. In addition, to identify specific features favorable for TGR5 activation, we generated and optimized a three-dimensional quantitative SAR model that contributed to our understanding of our activity profile and could guide further development of this dihydropyridone series.

Identification of indole-based activators of insulin degrading enzyme.

Insulin degrading enzyme (IDE) is a zinc metalloprotease that cleaves numerous substrates among which amyloid-ß and insulin. It has been linked through genetic studies to the risk of type-2 diabetes (T2D) or Alzheimer's disease (AD). Pharmacological activation of IDE is an attractive therapeutic strategy in AD. While IDE inhibition gave paradoxal activity in glucose homeostasis, recent studies, in particular in the liver suggest that IDE activators could be also of interest in diabetes. Here we describe the discovery of an original series of IDE activators by screening and structure-activity relationships. Early cellular studies show that hit 1 decreases glucose-stimulating insulin secretion. Docking studies revealed it has an unprecedented extended binding to the polyanion-binding site of IDE. These indole-based pharmacological tools are activators of both Aß and insulin hydrolysis by IDE and could be helpful to explore the multiple roles of IDE.

Supercritical fluid chromatography and liquid chromatography for isomeric separation of a multiple chiral centers analyte.

The development of a chiral separation strategy has always been a challenge of crucial importance, particularly in the pharmaceutical field. Chromatographic methods have become popular, particularly High Performance Liquid Chromatography and Supercritical Fluid Chromatography from a preparative scale point of view. A bioactive compound bearing three stereogenic centers was entrusted in our laboratory and the aim of this work was to obtain the complete resolution of the eight stereoisomers. Nine different polysaccharide-based columns were tested in SFC under various carbon dioxide-based mobile phases. The use of a single chiral column Lux Cellulose-2 under 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C, 120 bar outlet pressure allowed the obtention of eight peaks. To further improve the resolution of the two last isomers, two columns were serially coupled . The results obtained with the six different combinations are discussed. The tandem column supercritical fluid chromatography has demonstrated to be a useful technique to resolve the eight stereoisomers on Lux Cellulose-2//Cellulose-2 tandem of coupled columns with 30% 2-PrOH in carbon dioxide, at a flow-rate of 1 mL/min, column temperature of 40°C and 120 bar outlet pressure, despite a long analysis time. In order to compare the two methods (i.e supercritical and liquid), chiral liquid chromatography under polar aqueous-organic mode, polar organic mode and normal-phase mode, was implemented. The last mode allowed the full baseline resolution of the eight isomers on Cellulose-5 CSP, with 20% 2-PrOH in n-heptane at a flow-rate of 0.8 mL/min, at 25°C, λ = 220 nm. The limits of detection and of quantification were determined for this method and the best values obtained for isomer 8 were equal to 2.84 and 9.37 nM respectively. Finally, a small-scale preparative separation of the multiple chiral centers compound was implemented on Cellulose-5 CSP within 10% 2-PrOH in n-heptane in order to study the stereoisomer elution order on Cellulose-2, Cellulose-5 and Chiralpak AD-H, under EtOH or 2-PrOH in n-heptane mobile phases, and partial reversal elution orders were observed.

Farnesoid X Receptor Activation in Brain Alters Brown Adipose Tissue Function via the Sympathetic System.

The nuclear bile acid (BA) receptor farnesoid X receptor (FXR) is a major regulator of metabolic/energy homeostasis in peripheral organs. Indeed, enterohepatic-expressed FXR controls metabolic processes (BA, glucose and lipid metabolism, fat mass, body weight). The central nervous system (CNS) regulates energy homeostasis in close interaction with peripheral organs. While FXR has been reported to be expressed in the brain, its function has not been studied so far. We studied the role of FXR in brain control of energy homeostasis by treating wild-type and FXR-deficient mice by intracerebroventricular (ICV) injection with the reference FXR agonist GW4064. Here we show that pharmacological activation of brain FXR modifies energy homeostasis by affecting brown adipose tissue (BAT) function. Brain FXR activation decreases the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase (TH), and consequently the sympathetic tone. FXR activation acts by inhibiting hypothalamic PKA-CREB induction of TH expression. These findings identify a function of brain FXR in the control of energy homeostasis and shed new light on the complex control of energy homeostasis by BA through FXR.

Beyond the Rule of 5: Impact of PEGylation with Various Polymer Sizes on Pharmacokinetic Properties, Structure-Properties Relationships of mPEGylated Small Agonists of TGR5 Receptor.

PEGylation of therapeutic agents is known to improve the pharmacokinetic behavior of macromolecular drugs and nanoparticles. In this work, we performed the conjugation of polyethylene glycols (220-5000 Da) to a series of non-steroidal small agonists of the bile acids receptor TGR5. A suitable anchoring position on the agonist was identified to retain full agonistic potency with the conjugates. We describe herein an extensive structure-properties relationships study allowing us to finely describe the non-linear effects of the PEG length on the physicochemical as well as the in vitro and in vivo pharmacokinetic properties of these compounds. When appending a PEG of suitable length to the TGR5 pharmacophore, we were able to identify either systemic or gut lumen-restricted TGR5 agonists.

Hypothalamic bile acid-TGR5 signaling protects from obesity.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

Novel non-carboxylic acid retinoids: 1,2,4-oxadiazol-5-one derivatives.

We have successfully obtained 1,2,4-oxadiazol-5-one bioisoteres of Am580 or Tazarotene-like retinoids. In particular compound 4 displays an EC(50) of 26nM on RAR-beta.

Enantioseparation of chiral benzimidazole derivatives by electrokinetic chromatography using sulfated cyclodextrins.

Baseline separation of 18 new substituted benzimidazole derivatives, potent AMP-activated protein kinase (AMPK) activators, with one chiral center, was achieved by CD-EKC using sulfated and highly sulfated CDs (SCDs and HS-CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The SCDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times around 6 min using 25 mM phosphate buffer at pH 2.5 containing either beta-S-CD, HS-beta-CD, HS-gamma-CD (3 or 4% w/v) at 25 degrees C, with a voltage of 20 kV. The apparent association constants of the inclusion complexes were calculated. The study of the solute structure-enantioseparation relationships seems to show the high contribution of the interactions between the solutes phenyl ring and the CDs to the enantiorecognition process. The optimized method was briefly validated (LOD less than 1%) and the purity of enantiomers of compound 3 was determined. The enantiomer migration shows reversal order depending on the kind of CD.

Application of tandem coupling of columns in supercritical fluid chromatography for stereoisomeric separation: Optimization and simulation.

Chromatographic separation of compounds with more than one chiral center is challenging, requiring high resolution methods. Owing to the low viscosity of the mobile phase, Supercritical Fluid Chromatography (SFC) enables the tandem coupling of columns which increases resolution compared over a single column and can be effective in resolving stereoisomers. Enantioseparation of a dihydropyridone derivative with two chiral centers, synthetic API, was here studied using SFC. Six polysaccharide-based, chiral stationary phases with a mobile phase consisting of a carbon dioxide/methanol mixture (80:20 v:v) were investigated at 40 °C and a flow-rate of 3 mL/min, but only incomplete separation of the four expected stereoisomers was observed. We then examined different combinations of columns in tandem. It was found that, among the thirteen successful tandems, the OJ-H//AD-H system gave complete baseline resolution of the four stereoisomers with 4.98, 5.63, 6.06 and 6.89 as retention times and 2.97, 1.83 and 3.54 as resolution values. The conditions were further optimized to obtain the best resolution in the shortest elution time. The best conditions were transposed to semi-preparative scale to obtain the pure isomers, with yield increased by using stacked injections. The four fractions allowed the attribution of elution order on all tandem performed previously. The column order itself had no impact on the stereoisomeric elution order but the type of stationary phase and column order strongly influenced the resolution. In parallel, a previously reported mathematical model was used to predict the retention times of the four stereoisomers on each of the six polysaccharide-based, chiral stationary phase column tandems. This mathematical model was successfully applied to predict separation the dihydropyridone derivative's isomers on two columns with chlorinated stationary phases.

Identification of ebselen as a potent inhibitor of insulin degrading enzyme by a drug repurposing screening.

Insulin-degrading enzyme, IDE, is a metalloprotease implicated in the metabolism of key peptides such as insulin, glucagon, ß-amyloid peptide. Recent studies have pointed out its broader role in the cell physiology. In order to identify new drug-like inhibitors of IDE with optimal pharmacokinetic properties to probe its multiple roles, we ran a high-throughput drug repurposing screening. Ebselen, cefmetazole and rabeprazole were identified as reversible inhibitors of IDE. Ebselen is the most potent inhibitor (IC50(insulin) = 14 nM). The molecular mode of action of ebselen was investigated by biophysical methods. We show that ebselen induces the disorder of the IDE catalytic cleft, which significantly differs from the previously reported IDE inhibitors. IDE inhibition by ebselen can explain some of its reported activities in metabolism as well as in neuroprotection.