Getting the whole picture: High content screening using three-dimensional cellular model systems and whole animal assays.

Phenotypic or High Content Screening (HCS) is becoming more widely used for primary screening campaigns in drug discovery. Currently the vast majority of HCS campaigns are using cell lines grown in well-established monolayer cultures (2D tissue culture). There is widespread recognition that the more biologically relevant 3D tissue culture technologies such as spheroids and organoids and even whole animal assays will eventually be run as primary HCS. Upgrading the IT infrastructure to cope with the increase in data volumes requires investments in hardware (and software) and this will be manageable. However, the main bottleneck for the effective adoption and use of 3D tissue culture and whole animal assays in HCS is anticipated to be the development of software for the analysis of 3D images. In this review we summarize the current state of the available software and how they may be applied to analyzing 3D images obtained from a HCS campaign. © 2016 International Society for Advancement of Cytometry.

An integrative approach identified genes associated with drug response in gastric cancer.

Gastric cancer (GC) is the second leading cause of global cancer mortality worldwide. However, the molecular mechanism underlying its carcinogenesis and drug resistance is not well understood. To identify novel functionally important genes that were differentially expressed due to combinations of genetic and epigenetic changes, we analyzed datasets containing genome-wide mRNA expression, DNA copy number alterations and DNA methylation status from 154 primary GC samples and 47 matched non-neoplastic mucosa tissues from Asian patients. We used concepts of 'within' and 'between' statistical analysis to compare the difference between tumors and controls within each platform, and assessed the correlations between platforms. This 'multi-regulated gene (MRG)' analysis identified 126 differentially expressed genes that underwent a combination of copy number and DNA methylation changes. Most genes were located at genomic loci associated with GC. Statistical enrichment analysis showed that MRGs were enriched for cancer, GC and drug response. We analysed several MRGs that previously had not been associated with GC. Knockdown of DDX27, TH1L or IDH3G sensitized cells to epirubicin or cisplatin, and knockdown of RAI14 reduced cell proliferation. Further studies showed that overexpression of DDX27 reduced epirubicin-induced DNA damage and apoptosis. Levels of DDX27 mRNA and protein were increased in early-stage gastric tumors, and may be a potential diagnostic and prognostic marker for GC. In summary, we used an integrative bioinformatics strategy to identify novel genes that are altered in GC and regulate resistance of GC cells to drugs in vitro.

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

Identification of molecular subtypes of gastric cancer with different responses to PI3-kinase inhibitors and 5-fluorouracil.

BACKGROUND & AIMS: Almost all gastric cancers are adenocarcinomas, which have considerable heterogeneity among patients. We sought to identify subtypes of gastric adenocarcinomas with particular biological properties and responses to chemotherapy and targeted agents. METHODS: We compared gene expression patterns among 248 gastric tumors; using a robust method of unsupervised clustering, consensus hierarchical clustering with iterative feature selection, we identified 3 major subtypes. We developed a classifier for these subtypes and validated it in 70 tumors from a different population. We identified distinct genomic and epigenomic properties of the subtypes. We determined drug sensitivities of the subtypes in primary tumors using clinical survival data, and in cell lines through high-throughput drug screening. RESULTS: We identified 3 subtypes of gastric adenocarcinoma: proliferative, metabolic, and mesenchymal. Tumors of the proliferative subtype had high levels of genomic instability, TP53 mutations, and DNA hypomethylation. Cancer cells of the metabolic subtype were more sensitive to 5-fluorouracil than the other subtypes. Furthermore, in 2 independent groups of patients, those with tumors of the metabolic subtype appeared to have greater benefits with 5-fluorouracil treatment. Tumors of the mesenchymal subtype contain cells with features of cancer stem cells, and cell lines of this subtype are particularly sensitive to phosphatidylinositol 3-kinase-AKT-mTOR inhibitors in vitro. CONCLUSIONS: Based on gene expression patterns, we classified gastric cancers into 3 subtypes, and validated these in an independent set of tumors. The subgroups have differences in molecular and genetic features and response to therapy; this information might be used to select specific treatment approaches for patients with gastric cancer.

Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria.

UNLABELLED: A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. IMPORTANCE: During the last decade, antibacterial drug discovery relied on biochemical assays, rather than whole-cell approaches, to identify molecules that interact with purified target proteins derived by genomics. This approach failed to deliver antibacterial compounds with whole-cell activity, either because of cell permeability issues that medicinal chemistry cannot easily fix or because genomic data of essentiality insufficiently predicted the vulnerability of the target identified. As a consequence, the field largely moved back to a whole-cell approach whose main limitation is its black-box nature, i.e., that it requires trial-and-error chemistry because the cellular target is unknown. We developed a novel type of antibacterial screening method, target mechanism-based whole-cell screening, to combine the advantages of both approaches. We engineered a mycobacterial reporter strain with a synthetic phenotype allowing us to identify inhibitors of the caseinolytic protease (ClpP1P2) inside the cell. This approach identified bortezomib, an anticancer drug, as a specific inhibitor of ClpP1P2. We further confirmed the specific "on-target" activity of bortezomib by independent approaches including, but not limited to, genetic manipulation of the target level (over- and underexpressing strains) and by establishing a dynamic structure-activity relationship between ClpP1P2 and growth inhibition. Identifying an "on-target" compound is critical to optimize the efficacy of the compound without compromising its specificity. This work demonstrates the feasibility of target mechanism-based whole-cell screening methods, validates ClpP1P2 as a druggable target, and delivers a lead compound for tuberculosis therapy.

Development of a plasmepsin II fluorescence polarization assay suitable for high throughput antimalarial drug discovery.

Despite decades of research, malaria remains the world's most deadly parasitic disease. New treatments with novel mechanisms of action are urgently needed. Plasmepsin II is an aspartyl protease that has been validated as an antimalarial therapeutic target enzyme. Although natural products form the basis of most modern antimalarial drugs, no systematic high-throughput screening has been reported against this target. We have designed an effective strategy for carrying out high-throughput screening of an extensive library of natural products that uses a fluorescence resonance energy transfer primary screening assay in tandem with a fluorescence polarization assay. This strategy allows rapid screening of the library coupled with effective discrimination and elimination of false-positive samples and selection of true hits for chemical isolation of inhibitors of plasmepsin II.

Spermine alkaloids from Albizia adinocephala with activity against Plasmodium falciparum plasmepsin II.

Crude MeOH extracts from the stem bark and leaves of a Panamanian specimen of Albizia adinocephala (Leguminosae) were found to inhibit the malarial enzyme plasmepsin II. Bioassay guided fractionation led to the isolation of two new bioactive spermine alkaloids, budmunchiamines L4 and L5.

The use of high-throughput screening in identifying chemotherapeutic agents for gastric cancer.

Gastric cancer claims many lives around the world, particularly in Asia. Although diagnosis and treatment has improved, long-term survival of patients is still poor and there is an urgent need to develop more effective treatments for this disease. This review outlines some of the more innovative high-throughput screening-based approaches and strategies that may be used to identify compounds that have new or novel mechanisms of action and could be developed further as possible gastric cancer treatments in the future.

Establishment of a novel whole animal HTS technology platform for melioidosis drug discovery.

Melioidosis is a serious emerging endemic infectious disease caused by Burkholderia pseudomallei, a gram-negative pathogen. Septicemic melioidosis has a mortality rate of 50% even with treatment. Like other gram-negative bacteria, B. pseudomallei is resistant to a number of antibiotics and multi-drug resistant B. pseudomallei is beginning to be encountered in hospitals. There is a clear medical need to develop new treatment options to manage this disease. We used Burkholderia thailandensis (a BSL-2 class organism) to infect Caenorhabditis elegans and set up a surrogate whole animal infection model of melioidosis that we could run in a 384 microtitre plate and establish a whole animal HTS assay. We have optimized and validated this assay in a fluorescence-based format that can be run on our automated screening platforms. This assay has now been used to screen over 300,000 compounds from our small molecule library and we are in the process of characterizing the hits obtained and select compounds for further studies. We have thus established a biologically relevant assay technology platform to screen for antibacterial compounds and used this platform to identify new compounds that may find application in treating melioidosis infections.

Isolation and synthesis of falcitidin, a novel myxobacterial-derived acyltetrapeptide with activity against the malaria target falcipain-2.

A 384-well microtitre plate fluorescence cleavage assay was developed to identify inhibitors of the cysteine protease falcipain-2, an important antimalarial drug target. Bioassay-guided isolation of a MeOH extract from a myxobacterium Chitinophaga sp. Y23 isolated from soil collected in Singapore, led to the identification of a new acyltetrapeptide, falcitidin (1), which displayed an IC50 value of 6 µM against falcipain-2. The planar structure of 1 was secured by NMR and MS/MS analysis. Attempts to isolate further material for biological testing were hampered by inconsistent production and by a low yield (<100 µg l(-1)). The absolute configuration of 1 was determined by Marfey's analysis and the structure was confirmed through total synthesis as isovaleric acid-D-His-L-Ile-L-Val-L-Pro-NH2. Falcitidin (1) is the first member of a new class of falcipain-2 inhibitors and, unlike other peptide-based inhibitors, does not contain reactive groups that irreversibly bind to active cysteine sites.

Analysis of p53 binding to DNA by fluorescence imaging microscopy.

Transcription factors play a central role in cell biology through binding to target DNA elements and regulating gene expression. In this study, we used the p53 tumour suppressor as a model transcription factor to develop an imaging based assay to measure DNA binding. The assay utilizes fluorescence imaging microscopy to detect labelled p53 bound to DNA coated on microbeads. We demonstrate the ability to multiplex the assay by interrogating simultaneous binding to variant DNA sequences present on tractable beads. Additionally, the assay measures activation of p53 for increased DNA binding by a known peptide in addition to reactivation of mutant p53 by a small molecule. It may therefore be adaptable to a high-content imaging screen for compounds capable of restoring the function of mutant p53 associated with cancer.

An automated high-content screening image analysis pipeline for the identification of selective autophagic inducers in human cancer cell lines.

Automated image processing is a critical and often rate-limiting step in high-content screening (HCS) workflows. The authors describe an open-source imaging-statistical framework with emphasis on segmentation to identify novel selective pharmacological inducers of autophagy. They screened a human alveolar cancer cell line and evaluated images by both local adaptive and global segmentation. At an individual cell level, region-growing segmentation was compared with histogram-derived segmentation. The histogram approach allowed segmentation of a sporadic-pattern foreground and hence the attainment of pixel-level precision. Single-cell phenotypic features were measured and reduced after assessing assay quality control. Hit compounds selected by machine learning corresponded well to the subjective threshold-based hits determined by expert analysis. Histogram-derived segmentation displayed robustness against image noise, a factor adversely affecting region growing segmentation.

Overview of high-throughput screening.

High-throughput screening (HTS) is a key process used in drug discovery to identify hits from compound libraries that may become leads for medicinal chemistry optimization. This updated overview discusses the utilization of compound libraries, compounds derived from combinatorial and parallel synthesis campaigns and natural product sources; creation of mother and daughter plates; and compound storage, handling, and bar coding in HTS. The unit also presents an overview of established and emerging assay technologies (i.e., time-resolved fluorescence, fluorescence polarization, fluorescence-correlation spectroscopy, functional whole cell assays, and high-content assays) and their integration in automation hardware and IT systems. This revised unit provides updated descriptions of state-of-the-art instrumentation and technologies in this rapidly changing environment. The section on assay methodologies now also covers enzyme complementation assays and methods for high-throughput screening of ion channel activities. Finally, a section on criteria for assay robustness is included discussing the Z'-factor, which is now a widely accepted criterion for evaluation and validation of high throughput screening assays.

Pyrrole carboxamidine tryptase inhibitors from Leptonychia pubescens.

The root and stem bark extracts of a Nigerian sample of Leptonychia pubescens Keay (Sterculiaceae) were found to inhibit the serine protease tryptase, a potential therapeutic target for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Bioassay-guided isolation led to the identification of 1-beta-ribofuranosylbrunfelsamidine as the active component with a tryptase IC (50) of 3 microM. Brunfelsamidine was also isolated, but was only weakly active.

Agonodepsides a and B: two new depsides from a filamentous fungus F7524.

Two new compounds, agonodepsides A (1) and B (2), were isolated from a nonsporulating filamentous fungus, F7524. The compounds were purified via reversed-phase chromatography and their structures determined by spectroscopic methods. Agonodepside A (1) was found to inhibit the mycobacterial InhA enzyme with an IC50 value of 75 microM, while 2 was inactive at 100 microM.

Identification of chelerythrine as an inhibitor of BclXL function.

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins.

New antiinfective and human 5-HT2 receptor binding natural and semisynthetic compounds from the Jamaican sponge Smenospongia aurea.

In addition to the sesquiterpene-phenol aureols (1), 6'-chloroaureol (2), and aureol acetate (3), eight indole alkaloids including the new N-3'-ethylaplysinopsin (9) have been isolated from the Jamaican sponge Smenospongia aurea. Makaluvamine O (10), a new member of the pyrroloiminoquinone class, was also isolated. The structures were characterized by spectroscopic methods, and two new derivatives of aureol were prepared to optimize the biological activity. Aureol N,N-dimethyl thiocarbamate (1a) and 6-bromoaplysinopsin (7) exhibit significant antimalarial and antimycobacterial activity in vitro. Compound 6 showed activity against the Plasmodium enzyme plasmepsin II. The 6-bromo-2'-de-N-methylaplysinopsin (6), 6-bromoaplysinopsin (7), and N-3'-ethylaplysinopsin (9) displaced high-affinity [(3)H]antagonist ligands from cloned human serotonin 5-HT(2) receptor subtypes, whereas the other compounds tested did not. Remarkably, the 6-bromo-2'-de-N-methylaplysinopsin (6) showed a > 40-fold selectivity for the 5-HT(2C) subtype over the 5-HT(2A) subtype.