Charlotte Petit

A Rational Approach for the Identification of Non-Hydroxamate HDAC6-Selective Inhibitors

The human histone deacetylase isoform 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, being interesting for the treatment of multiple tumour types and neurodegenerative conditions. Currently, most HDAC inhibitors in preclinical or clinical evaluations are non-selective inhibitors, characterised by a hydroxamate zinc-binding group (ZBG) showing off-target effects and mutagenicity. The identification of selective HDAC6 inhibitors with novel chemical properties has not been successful yet, also because of the absence of crystallographic information that makes the rational design of HDAC6 selective inhibitors difficult. Using HDAC inhibitory data retrieved from the ChEMBL database and ligand-based computational strategies, we identified 8 original new non-hydroxamate HDAC6 inhibitors from the SPECS database, with activity in the low μM range. The most potent and selective compound, bearing a hydrazide ZBG, was shown to increase tubulin acetylation in human cells. No effects on histone H4 acetylation were observed. To the best of our knowledge, this is the first report of an HDAC6 selective inhibitor bearing a hydrazide ZBG. Its capability to passively cross the blood-brain barrier (BBB), as observed through PAMPA assays, and its low cytotoxicity in vitro, suggested its potential for drug development.

Prediction of the Passive Intestinal Absorption of Medicinal Plant Extract Constituents with the Parallel Artificial Membrane Permeability Assay (PAMPA)

At the early drug discovery stage, the high-throughput parallel artificial membrane permeability assay is one of the most frequently used in vitro models to predict transcellular passive absorption. While thousands of new chemical entities have been screened with the parallel artificial membrane permeability assay, in general, permeation properties of natural products have been scarcely evaluated. In this study, the parallel artificial membrane permeability assay through a hexadecane membrane was used to predict the passive intestinal absorption of a representative set of frequently occurring natural products. Since natural products are usually ingested for medicinal use as components of complex extracts in traditional herbal preparations or as phytopharmaceuticals, the applicability of such an assay to study the constituents directly in medicinal crude plant extracts was further investigated. Three representative crude plant extracts with different natural product compositions were chosen for this study. The first extract was composed of furanocoumarins (Angelica archangelica), the second extract included alkaloids (Waltheria indica), and the third extract contained flavonoid glycosides (Pueraria montana var. lobata). For each medicinal plant, the effective passive permeability values Pe (cm/s) of the main natural products of interest were rapidly calculated thanks to a generic ultrahigh-pressure liquid chromatography-UV detection method and because Pe calculations do not require knowing precisely the concentration of each natural product within the extracts. The original parallel artificial membrane permeability assay through a hexadecane membrane was found to keep its predictive power when applied to constituents directly in crude plant extracts provided that higher quantities of the extract were initially loaded in the assay in order to ensure suitable detection of the individual constituents of the extracts. Such an approach is thus valuable for the high-throughput, cost-effective, and early evaluation of passive intestinal absorption of active principles in medicinal plants. In phytochemical studies, obtaining effective passive permeability values of pharmacologically active natural products is important to predict if natural products showing interesting activities in vitro may have a chance to reach their target in vivo.

HPLC Profiling with At-line Microdilution Assay for the Early Identification of Anti-fungal Compounds in Plants from French Polynesia

INTRODUCTION The search for anti-fungal compounds has maintained a scientific interest notably due to existing difficulties in the treatment of mycoses and their increasing occurrence in hospitals. OBJECTIVE Development of a simple method to rapidly identify anti-fungal compounds in crude plant extracts based on a HPLC microfractionation approach combined with an at-line anti-Candida assay. METHODS The scale of the semi-preparative HPLC microfractionation was adapted to fit the sensitivity of the Candida albicans anti-fungal in a 96-well microdilution assay. This format is also compatible for MS and NMR dereplication of the active compounds. RESULTS Based on the screening of 12 crude extracts of plants from French Polynesia, three plants, which displayed various levels of anti-fungal activities, were selected to assess the efficiency of the HPLC anti-fungal profiling and the scale necessary for microfractionation. The same anti-Candida assay was performed on the HPLC microfractions collected using a generic profiling method. Analysis of active microfractions by MS and NMR issued from the most active extract enabled an efficient dereplication of the compounds responsible for the anti-fungal activity. CONCLUSION A generic HPLC anti-fungal profiling method was developed which revealed that only 50 mg of crude extract were sufficient for a rapid identification of compound(s) responsible for the anti-Candida activity. This approach was illustrated by the study of Alphitonia zizyphoides, a plant traditionally used to treat dermatomycoses.

5-Benzylidene-hydantoin is a new scaffold for SIRT inhibition: From virtual screening to activity assays.

Sirtuins (SIRTs) are a family of enzymes able to catalyze the deacetylation of the N-acetyl lysines of both histone and non-histone substrates. Inhibition of SIRTs catalytic activity was recently reported in the literature as being beneficial in human diseases, with very promising applications in cancer therapy and enzymatic neurodegeneration. By combining a structure-based virtual screening of the Specs database with cell-based assays, we identified the 5-benzylidene-hydantoin as new scaffold for the inhibition of SIRT2 catalytic activity. Compound 97 (Specs ID AH-487/41657829), active in the low μM range against SIRT2, showed the optimal physicochemical properties for passive absorption as well as relatively low cytotoxicity in vitro. Further studies revealed non-competitive and mixed-type kinetics toward acetyl-lysine substrates and NAD(+), respectively, and a non-selective profile for SIRT inhibition. A binding mode consistent with the experimental evidence was proposed by molecular modeling. Additionally, the levels of acetyl-p53 were shown to be increased in HeLa cells treated with 97. Taken together, these results encourage further investigation of 5-benzylidene-hydantoin derivatives for their SIRT-related therapeutic effects.

Phospholipidosis effect of drugs by adsorption into lipid monolayers

Drug-induced phospholipidosis indicates an accumulation of phospholipids within lysosomes, which can occur during therapeutic treatment. Whether or not phospholipidosis represents a toxicological phenomenon is still under investigation, and in the last decade the Food and Drug Administration has been raising concerns about the possible consequences of this adverse event. Cationic amphiphilic drugs represent the majority of phospholipidosis inducers, followed by aminoglycoside and macrolide antibiotics. Although the mechanism of phospholipidosis induction is still uncertain, the interaction of drugs with phospholipids in the lysosomal membrane represents a key step. Therefore, the study of the drug/lipid complex formation will provide valuable insight into the causation of phospholipidosis at the molecular level and to identify the potential phospholipidosis risk associated with drug. In this study, we investigated the insertion profile of eleven drugs with known phospholipidosis effect into preformed Langmuir monolayers of various lipid compositions, to evaluate for the first time the drug/lipid interaction for phospholipidosis inducers and non-inducers in a dynamic approach. We found that the addition of dipalmitoylphosphatidylserine (DPPS) to dipalmitoylphosphatidylcholine (DPPC) to form the lipid monolayer allowed a clear identification of the phospholipidosis effect of the selected drugs based on the variation of the surface pressure, not only for cationic amphiphilic drugs but also for the aminoglycoside and the macrolide antiobiotics tested. Compared to a standard PAMPA assay, the new method appears to be more effective for the study of poorly soluble drugs.

Passive Intestinal Absorption of Representative Plant Secondary Metabolites: A Physicochemical Study

Natural products are generally ingested as part of traditional herbal decoctions or in the current diet. However, in natural product research, the bioavailability of secondary metabolites is often poorly investigated. In this work, a systematic study was carried out in order to highlight the physicochemical parameters that mainly influence the passive intestinal absorption of natural products. For this, a representative set of natural products including alkaloids, coumarins, flavonoid aglycones and glycosides, and carboxylic acids was selected and their physicochemical properties were predicted using relevant Volsurf+ descriptors. The chemical space obtained with this unbiased method was then correlated with experimental passive intestinal permeability data, which highlighted the main influence of lipophilicity, global hydrophilicity, size, and the ionisation state on passive intestinal absorption of natural products. Since the pH range encountered in the intestine is wide, the influence of the ionisation was investigated deeper experimentally. The ionisation state of weakly ionisable natural products, such as flavonoid aglycones, alkaloids, and carboxylic acids, was found to prevent the passive intestinal absorption of such natural products completely. In addition, the impact of solubility issues on passive permeability results was evaluated in cases of poorly water-soluble natural products, such as flavonoid aglycones and coumarins. The biomimetic fasted state simulated fluid-version 2 was found to improve the apparent solubility of such poorly soluble natural products without influencing their permeability behaviours. The use of such a solubilising buffer was found to be well adapted to the hexadecane membrane-parallel artificial membrane permeability assay and can circumvent the solubility issues encountered with poorly soluble natural products in such an assay.

HDM-PAMPA to predict gastrointestinal absorption, binding percentage, equilibrium and kinetics constants with human serum albumin and using 2 end-point measurements

&NA; The parallel artificial membrane permeability assay (PAMPA) is a high‐throughput screening (HTS) technique developed to predict passive permeability through numerous different biological membranes, such as the gastrointestinal tract (GIT), the blood brain barrier (BBB), and the dermal layer. PAMPA is based on an artificial membrane, such as hexadecane (HDM), which separates two compartments (i.e., a donor and an acceptor compartment). In the present study, an HDM‐PAMPA method was developed with human serum albumin (HSA) under iso‐pH and gradient‐pH conditions to predict the percentage of binding, dissociation/association constants (Kd and Ka, respectively) and dissociation/association kinetic rates (koff and kon, respectively) between a given drug and HSA. Thanks to the kinetic properties of PAMPA, a two end‐point assay was implemented to obtain all three properties. The assay was used to measure basic, acidic, and amphoteric compounds. The protein was free in solution, allowing a direct comparison between this assay and equilibrium dialysis (ED). The developed PAMPA enabled screening of up to 96 compounds in a single run, generating valuable information on absorption and distribution in a high‐throughput and high‐repeatable manner. Graphical abstract Figure. No caption available.

Synthesis of a selective HDAC6 inhibitor active in neuroblasts

In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.