Detecting, quantifying, and discriminating the mechanism of mosaic chromosomal aneuploidies using MAD-seq.

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost, or the need to culture cells. We describe the mosaic aneuploidy detection by massively parallel sequencing (MAD-seq) capture assay and the MADSEQ analytical approach that allow low (<10%) levels of mosaicism for chromosomal aneuploidy or regional loss of heterozygosity to be detected, assigned to a meiotic or mitotic origin, and quantified as a proportion of the cells in the sample. We show results from a multi-ethnic MAD-seq (meMAD-seq) capture design that works equally well in populations of diverse racial and ethnic origins and how the MADSEQ analytical approach can be applied to exome or whole-genome sequencing data, revealing previously unrecognized aneuploidy or copy number neutral loss of heterozygosity in samples studied by the 1000 Genomes Project, cell lines from public repositories, and one of the Illumina Platinum Genomes samples. We have made the meMAD-seq capture design and MADSEQ analytical software open for unrestricted use, with the goal that they can be applied in clinical samples to allow new insights into the unrecognized prevalence of mosaic chromosomal aneuploidy in humans and its phenotypic associations.

UHPLC-MS-based HDAC Assay Applied to Bio-guided Microfractionation of Fungal Extracts.

INTRODUCTION: Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites. OBJECTIVE: To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains. METHODS: Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I isoform HDAC1 and the class II isoform HDAC6. RESULTS: Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in P. griseofulvum is described here for the first time. CONCLUSION: The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.

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.

Cross metathesis with hydroxamate and benzamide BOC-protected alkenes to access HDAC inhibitors and their biological evaluation highlighted intrinsic activity of BOC-protected dihydroxamates.

Conditions for the metathesis of alkenes in the convergent synthesis of HDAC inhibitors have been improved by continuous catalyst flow injection in the reaction media. Intermediate and target compounds obtained were tested for their ability to induce HDAC inhibition and tubulin acetylation, revealing the key role of the tert-butyloxycarbonyl (BOC) group for more HDAC6 selectivity. Molecular modelling added rationale for this BOC effect.

Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors.

Histone deacetylases (HDAC) are involved in several diseases including cancer, cardiovascular and neurodegenerative disorders, and the search for inhibitors is a current topic in drug discovery. Four HDAC inhibitors have already been approved by the FDA for cancer therapy and others are under clinical studies. However, the clinical utility of some of them is limited because of unfavorable toxicities associated with their broad range of HDAC inhibitory effects. Toxicity could be decreased by using HDAC inhibitors with improved specificity. To date, the most popular screening assays are based on fluorescence-labeled substrates incubated with an enzymatic source (cells extracts or recombinant isoforms). Here, we describe a high-throughput cell-based UHPLC-ESI-MS/MS assay able to rapidly predict activity against HDAC1 and HDAC6 in a cell environment. This method is predicted to be a useful tool to accelerate the search for class-selective HDAC inhibitors in drug discovery.

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.

Alkaloids from psychotria target sirtuins: in silico and in vitro interaction studies.

Epigenetic enzymes such as histone deacetylases play a crucial role in the development of ageing-related diseases. Among the 18 histone deacetylase isoforms found in humans, class III histone deacetylases, also known as sirtuins, seem to be promising targets for treating neurodegenerative conditions. Recently, Psychotria alkaloids, mainly monoterpene indoles, have been reported for their inhibitory properties against central nervous system cholinesterase and monoamine oxidase proteins. Given the multifunctional profile of these alkaloids in the central nervous system, and the fact that the indole scaffold has been previously associated with sirtuin inhibition, we hypothesized that these indole derivatives could also interact with sirtuins. In the present study, alkaloids previously isolated from Psychotria spp. were evaluated for their potential interaction with human sirtuin 1 and sirtuin 2 by molecular docking and molecular dynamics simulation approaches. The in silico results allowed for the selection of five potentially active compounds, namely, prunifoleine, 14-oxoprunifoleine, E-vallesiachotamine, Z-vallesiachotamine, and vallesiachotamine lactone. The sirtuin inhibition of these compounds was confirmed in vitro in a dose-response manner, with preliminary information on their pharmacokinetics properties.

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.

Study of leaf metabolome modifications induced by UV-C radiations in representative Vitis, Cissus and Cannabis species by LC-MS based metabolomics and antioxidant assays.

UV-C radiation is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. To assess these modifications from a global and untargeted perspective, the effects of the UV-C radiation of the leaves of three different model plant species, Cissus antarctica Vent. (Vitaceae), Vitis vinifera L. (Vitaceae) and Cannabis sativa L. (Cannabaceae), were evaluated by an LC-HRMS-based metabolomic approach. The approach enabled the detection of significant metabolite modifications in the three species studied. For all species, clear modifications of phenylpropanoid metabolism were detected that led to an increased level of stilbene derivatives. Interestingly, resveratrol and piceid levels were strongly induced by the UV-C treatment of C. antarctica leaves. In contrast, both flavonoids and stilbene polymers were upregulated in UV-C-treated Vitis leaves. In Cannabis, important changes in cinnamic acid amides and stilbene-related compounds were also detected. Overall, our results highlighted phytoalexin induction upon UV-C radiation. To evaluate whether UV-C stress radiation could enhance the biosynthesis of bioactive compounds, the antioxidant activity of extracts from control and UV-C-treated leaves was measured. The results showed increased antioxidant activity in UV-C-treated V. vinifera extracts.

Monoamine oxidase inhibition by monoterpene indole alkaloids and fractions obtained from Psychotria suterella and Psychotria laciniata.

Alkaloid fractions of Psychotria suterella (SAE) and Psychotria laciniata (LAE) as well as two monoterpene indole alkaloids (MIAs) isolated from these fractions were evaluated against monoamine oxidases (MAO-A and -B) obtained from rat brain mitochondria. SAE and LAE were analysed by HPLC-PDA and UHPLC/HR-TOF-MS leading to the identification of the compounds 1, 2, 3 and 4, whose identity was confirmed by NMR analyses. Furthermore, SAE and LAE were submitted to the enzymatic assays, showing a strong activity against MAO-A, characterized by IC(50) values of 1.37 ± 1.05 and 2.02 ± 1.08 µg/mL, respectively. Both extracts were also able to inhibit MAO-B, but in higher concentrations. In a next step, SAE and LAE were fractionated by RP-MPLC affording three and four major fractions, respectively. The RP-MPLC fractions were subsequently tested against MAO-A and -B. The RP-MPLC fractions SAE-F3 and LAE-F4 displayed a strong inhibition against MAO-A with IC(50) values of 0.57 ± 1.12 and 1.05 ± 1.15 µg/mL, respectively. The MIAs 1 and 2 also inhibited MAO-A (IC(50) of 50.04 ± 1.09 and 132.5 ± 1.33 µg/mL, respectively) and -B (IC(50) of 306.6 ± 1.40 and 162.8 ± 1.26 µg/mL, respectively), but in higher concentrations when compared with the fractions. This is the first work describing the effects of MIAs found in neotropical species of Psychotria on MAO activity. The results suggest that species belonging to this genus could consist of an interesting source in the search for new MAO inhibitors.

How to increase the safety and efficacy of compounds against neurodegeneration? A multifunctional approach.

Successful drug design requires not only the detailed knowledge of the pharmacokinetic and pharmacodynamic profiles of the drug candidate portfolio but also a thorough documentation of the possible toxic effects on humans and the environment. Thus, experimental and computational strategies able to measure or predict specific profiles of designed compounds related to their potential toxicity are highly desired. Moreover, a strategy to avoid toxic effects thus enhancing the potential efficacy of drug candidates is of great interest. To fulfil this aim, the pharmacochemistry research unit at the EPGL has recently developed and improved methodologies that detect the potential human health and environmental hazards of compounds active against neurodegeneration at an early stage. A three-step strategy is presented herein. In particular, i) an alternative index to model the bioconcentration of chemicals in the environment was determined; ii) the antioxidant activity of chemical species against free radicals was evaluated. Moreover, since antioxidants play a key role in both toxicity prevention and neuroprotection, iii) the potential interaction of such compounds with enzymatic targets involved in the neurodegenerative cascade was investigated in silico.

Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

A Cellular Stress Response Induced by the CRISPR-dCas9 Activation System Is Not Heritable Through Cell Divisions.

The CRISPR-Cas9 system can be modified to perform "epigenetic editing" by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions-an epigenetic regulatory property. Here, we show how distinct components of this CRISPR-dCas9 activation system, particularly dCas9 with untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR-dCas9 components in human cells, which is transient and not maintained through cell divisions.

A TLC bioautographic method for the detection of alpha- and beta-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.

High-efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells using a single-stranded donor oligonucleotide strategy.

While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for 'scarless' editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5-10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding.

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.

Novel histone deacetylase 6 (HDAC6) selective inhibitors: a patent evaluation (WO2014181137).

INTRODUCTION: Histone deacetylases (HDACs) are known to deacetylate histones and other proteins, which makes HDAC inhibitors able to affect cell survival, cell signaling, transport, and gene expression. Those effects have been associated to the therapeutic success of HDAC inhibitors. Class I-selective or pan-HDAC inhibitors have been approved for cancer therapy by the US Food and Drug Administration (FDA). Moreover, HDAC6 selective inhibitors entered phase I and II clinical trials for treating multiple myeloma. The development of potent and selective HDAC inhibitors is a hot topic in current drug discovery. Areas covered: The invention described in this patent (WO2014181137) is related to hydroxamic acid derivatives with inhibitory activity towards HDACs, their synthetic process and pharmaceutical formulations, as well as a method for treating patients suffering from a list of selected tumoral, inflammatory, cardiac and chronic disorders. Expert opinion: The compounds disclosed within this patent are selective against HDAC6 and their structure is related to tubastatin A, a known HDAC6 selective inhibitor. They are newly synthesized diarylamines showing an improved selectivity profile compared to other diarylamines under clinical investigation.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

Hydroxyl Ketone-Based Histone Deacetylase Inhibitors To Gain Insight into Class I HDAC Selectivity versus That of HDAC6.

Little is known about the biological and structural features that govern the isoform selectivity for class I histone deacetylases (HDACs) over HDAC6. In addition to that for known inhibitors, like benzamides, psammaplin A, and cyclodepsipeptide-derived thiols, selectivity was also observed for naturally occurring cyclopeptide HDAC inhibitors with an aliphatic flexible linker and ketonelike zinc-binding group (ZBG). The present study reports that this isoform selectivity is mainly due to the linker and ZBG, as replacement of the cyclopeptide cap region by a simple aniline retained class I HDAC isoform selectivity toward HDAC6 in enzymatic assays. The best cyclopeptide-free analogues preserved efficacy against Plasmodium falciparum and cancer cell lines. Molecular modeling provided hypotheses to explain this selectivity and suggests different behaviors of the flexible linker on HDAC1 and HDAC6 pockets, which may influence, on the basis of the strength of the ZBG, its coordination with the zinc ion.

Synthesis of an Uncharged Tetra-cyclopeptide Acting as a Transmembrane Carrier: Enhanced Cellular and Nuclear Uptake.

A small uncharged cyclopeptide scaffold inspired by a natural product and designed to undergo postfunctionalizations was used as a new transmembrane vector. A bioactive and fluorescent triazole aminocoumarin was bound to this carrier to facilitate its moving across cell and subcellular membranes, and this led to an increase in its cell toxicity.