Claudia Simões-Pires

HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs?

Histone deacetylase (HDAC) inhibitors have been demonstrated to be beneficial in animal models of neurodegenerative diseases. Such results were mainly associated with the epigenetic modulation caused by HDACs, especially those from class I, via chromatin deacetylation. However, other mechanisms may contribute to the neuroprotective effect of HDAC inhibitors, since each HDAC may present distinct specific functions within the neurodegenerative cascades. Such an example is HDAC6 for which the role in neurodegeneration has been partially elucidated so far. The strategy to be adopted in promising therapeutics targeting HDAC6 is still controversial. Specific inhibitors exert neuroprotection by increasing the acetylation levels of α-tubulin with subsequent improvement of the axonal transport, which is usually impaired in neurodegenerative disorders. On the other hand, an induction of HDAC6 would theoretically contribute to the degradation of protein aggregates which characterize various neurodegenerative disorders, including Alzheimer’s, Parkinson’s and Hutington’s diseases. This review describes the specific role of HDAC6 compared to the other HDACs in the context of neurodegeneration, by collecting in silico, in vitro and in vivo results regarding the inhibition and/or knockdown of HDAC6 and other HDACs. Moreover, structure, function, subcellular localization, as well as the level of HDAC6 expression within brain regions are reviewed and compared to the other HDAC isoforms. In various neurodegenerative diseases, the mechanisms underlying HDAC6 interaction with other proteins seem to be a promising approach in understanding the modulation of HDAC6 activity.

Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors

Thirteen Psychotria alkaloids were evaluated regarding their interactions with acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A and MAO-B), which are enzymatic targets related with neurodegenerative diseases. Two quaternary β-carboline alkaloids, prunifoleine and 14-oxoprunifoleine, inhibited AChE, BChE and MAO-A with IC(50) values corresponding to 10 and 3.39 μM for AChE, 100 and 11 μM for BChE, and 7.41 and 6.92 μM for MAO-A, respectively. Both compounds seem to behave as noncompetitive AChE inhibitors and time-dependent MAO-A inhibitors. In addition, the monoterpene indole alkaloids (MIAs) angustine, vallesiachotamine lactone, E-vallesiachotamine and Z-vallesiachotamine inhibited BChE and MAO-A with IC(50) values ranging from 3.47 to 14 μM for BChE inhibition and from 0.85 to 2.14 μM for MAO-A inhibition. Among the tested MIAs, angustine is able to inhibit MAO-A in a reversible and competitive way while the three vallesiachotamine-like alkaloids display a time-dependent inhibition on this target. Docking calculations were performed in order to understand the binding mode between the most active ligands and the selected targets. Taken together, our findings established molecular details of AChE, BChE and MAO-A inhibition by quaternary β-carboline alkaloids and MIAs from Psychotria, suggesting these secondary metabolites are scaffolds for the development of multifunctional compounds against neurodegeneration.

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

INTRODUCTION Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of beta-glucosidase inhibitors but not for alpha-glucosidase. Nonetheless, alpha-glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti-viral infections. OBJECTIVE To develop a TLC bioautographic method to detect alpha- and beta-glucosidase inhibitors in plant extracts. METHODOLOGY The enzymes alpha- and beta-d-glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of alpha-d-glucosidase, and 37 degrees C for 20 min in the case of beta-d-glucosidase. For detection of the active enzyme, solutions of 2-naphthyl-alpha-D-glucopyranoside or 2-naphthyl-beta-D-glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for alpha-d-glucosidase) or 1 : 4 (for beta-d-glucosidase) and sprayed onto the plate to give a purple background colouration after 2-5 min. RESULTS Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited alpha-d-glucosidase and beta-d-glucosidase down to 0.1 microg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 microg for alpha-glucosidase and 50 microg for beta-glucosidase. CONCLUSION The screening test was able to detect inhibition of alpha- and beta-glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts.

How the flexibility of human histone deacetylases influences ligand binding: an overview

Over the past decade, human histone deacetylases (HDACs) have become interesting as therapeutic targets because of the benefits that their modulation might provide in aging-related disorders. Recently, studies using crystallography and computational chemistry have provided information on the structure and conformational changes related to HDAC-mediated recognition events. Through the description of the key mass and one-off movements observed in metal-dependent HDACs, here we highlight the impact of flexibility on drug-binding affinity and specificity. The collected information will be useful for not only a better understanding of the biological functions of HDACs, but also the conception of new selective binders.

Aurones as histone deacetylase inhibitors: identification of key features.

In this study, a total of 22 flavonoids were tested for their HDAC inhibitory activity using fluorimetric and BRET-based assays. Four aurones were found to be active in both assays and showed IC50 values below 20 μM in the enzymatic assay. Molecular modelling revealed that the presence of hydroxyl groups was responsible for good compound orientation within the isoenzyme catalytic site and zinc chelation.

Anti-inflammatory, antimicrobial and antioxidant activities of Diospyros bipindensis (Gurke) extracts and its main constituents

ETHNOPHARMACOLOGICAL RELEVANCE Diospyros bipindensis (Gürke) stem bark is used in Cameroon by Baka Pygmies for the treatment of respiratory disorders. AIM OF THE STUDY To assess the anti-inflammatory, antibacterial and antioxidant properties of constituents from the bark extracts through bioassay-guided fractionation. MATERIALS AND METHODS The anti-inflammatory activity of extracts, fractions and pure compounds was assessed through the inhibition of the pro-inflammatory mediator nuclear factor-kappa B (NF-κB) transcriptional activity and nitric oxide (NO) production. DPPH, ABTS and ORAC assays were used for determining the antioxidant properties. The activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli and Klebsiella pneumoniae, was evaluated on the basis of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by the macrodilution method. RESULTS The water extract showed antimicrobial activity against S. pneumoniae (MIC: 300 μg/ml) and S. pyogenes (MIC: 300 μg/ml). The dichloromethane extract efficiently inhibited NF-κB transcriptional activity and NO production and exhibited significant antioxidant activity in the ORAC assay. An interesting activity was also found against S. pneumoniae (MIC: 200 μg/ml), S. aureus (MIC: 400 μg/ml) and S. pyogenes (MIC: 200 μg/ml). The phytochemical investigation of the dichloromethane extract afforded plumbagin, canaliculatin, ismailin, betulinic acid and 4-hydroxy-5-methyl-coumarin as the main constituents. Plumbagin and ismailin were found to be responsible for the main biological activities observed. CONCLUSIONS These results may provide a rational support for the traditional use of Diospyros bipindensis stem bark in the treatment of respiratory disorders, since the anti-inflammatory, antimicrobial and antioxidant compounds isolated from the dichloromethane extract were also present in the traditional water extract.

Cancer chemopreventive diterpenes from Salvia corrugata

NMR and NP-HPLC-UV profiling of the exudate of Salvia corrugata revealed that its secondary metabolite composition was largely dominated by α-hydroxy-β-isopropyl-benzoquinone diterpenoids. Among them, four diterpenes not described previously were isolated and identified as fruticulin C (3), 7α-methoxy-19-acetoxy-royleanone (4), 7α,19-diacetoxy-royleanone (5), and 7-dehydroxy-conacytone (7). In addition, the known diterpenes fruticulin A (1), demethyl-fruticulin A (2) and 7α-O-methyl-conacytone (6) were also obtained. The isolated compounds were evaluated for their cancer chemopreventive activity by measuring quinone reductase induction activity and histone deacetylase inhibition. Three compounds (1, 2 and 5) showed promising activity.

Non-specific SIRT inhibition as a mechanism for the cytotoxicity of ginkgolic acids and urushiols

Ginkgolic acids and urushiols are natural alkylphenols known for their mutagenic, carcinogenic and genotoxic potential. However, the mechanism of toxicity of these compounds has not been thoroughly elucidated so far. Considering that the SIRT inhibitory potential of anacardic acids has been hypothesized by in silico techniques, we herein demonstrated through both in vitro and computational methods that structurally related compounds such as ginkgolic acids and urushiols are able to modulate SIRT activity. Moreover, their SIRT inhibitory profile and cytotoxicity were comparable to sirtinol, a non-specific SIRT inhibitor (SIRT1 and SIRT2), and different from EX-527, a SIRT1 specific inhibitor. This is the first report on the SIRT inhibition of ginkgolic acids and urushiols. The results reported here are in line with previously observed effects on the induction of apoptosis by this class of compounds, and the non-specific SIRT inhibition is suggested as a new mechanism for their in vitro cytotoxicity.

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.

Prevention of False-Positive Results: Development of an HPTLC Autographic Assay for the Detection of Natural Tyrosinase Inhibitors.

A simple and rapid high-performance thin-layer chromatography-based autographic assay was established to screen plant extracts for the presence of tyrosinase-inhibiting substances. Three mobile phases were selected for the chromatographic separation of different types of extracts. After development, the plate was sprayed with the substrate solution Levodopa followed by a solution of the enzyme tyrosinase. Several known tyrosinase inhibitors were tested simultaneously as positive controls. They were detected as white spots with white light in remission from the plate as well as with white light transmitted through the plate. Some of the investigated extracts included spots showing a different behaviour; some lipophilic substances appeared as white spots in white light remission but were black in white light transmission. This behaviour, which could lead to false-positive results, was due to poor wettability of the corresponding spots. False-positive results were eliminated by adding Triton X-100 to the Levodopa solution and drying the plate after 10 minutes incubation with a molecular sieve. Tyrosinase inhibitors can be clearly identified as white spots against a dark background in white light remission as well as in white light transmitted through the plate. The established high-performance thin-layer chromatography autographic assay was validated and can be used as a standard method for the detection of tyrosinase inhibitors in plant extracts without causing false-positive results.