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J Biomol Screen [journal]
- Effects of a Ferulate-Derived Dihydrobenzofuran Neolignan on Angiogenesis, Steroidogenesis, and Redox Status in a Swine Cell Model. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 10.
In the ongoing search for new therapeutic compounds, lignans and neolignans, which are widely distributed in plants, deserve special attention because of their interactions with several biological targets. Searching for potential antiangiogenic agents related to natural lignans/neolignans, we were attracted by a previously studied synthetic dihydrobenzofuran neolignan. We synthesized the compound by means of an eco-friendly, enzyme-mediated biomimetic coupling of the methyl ester of ferulic acid, and the present study was aimed to deeply investigate its effect in angiogenesis bioassays validated in our laboratory. In addition, a previously well-defined granulosa cell model was employed to evaluate the effect of dihydrobenzofuran neolignan on cell viability, steroidogenesis, and redox status. Present data support the antiangiogenic effect of this neolignan. Moreover, we demonstrate that, at least at the highest concentrations tested, dihydrobenzofuran neolignan affects granulosa cell viability and steroidogenesis. In addition, the compound inhibits generation of free radicals and stimulates scavenger enzyme activities. The present data, which are a further deepening of the evaluation of the biological activities of the dihydrobenzofuran lignan in well-defined cell models, are of interest and worthy of special attention.
- Insights into PARP Inhibitors' Selectivity Using Fluorescence Polarization and Surface Plasmon Resonance Binding Assays. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 10.
PARP inhibitors are an exciting new class of antineoplastic drugs that have been proven to be efficacious as single agents in cancer settings with inherent DNA repair defects, as well as in combination with DNA-damaging chemotherapeutics. Currently, they are designed to target the catalytic domain of PARP-1, the most studied member of the family, with a key role in the DNA-damage repair process. Because PARP inhibitors are substrate (NAD(+)) competitors, there is a need for a deeper understanding of their cross-reactivity. This is particularly relevant for PARP-2, the PARP-1 closest homologue, for which an embryonic lethal phenotype has been observed in double knockout mice. In this study, we describe the development and validation of binding assays based on fluorescence polarization (FP) and surface plasmon resonance (SPR) techniques. PARP-1, PARP-2, PARP-3, and TNKS-1 FP displacement assays are set up by employing ad hoc synthesized probes. These assays are suitable for high-throughput screening (HTS) and selectivity profiling, thus allowing the identification of NAD(+) binding site selective inhibitors. The PARP-1 and PARP-2 complementary SPR binding assays confirm displacement data and the in-depth inhibitor characterization. Moreover, these formats have the potential to be broadly applicable to other members of the PARP family.
- Niemann-Pick Disease Type C: Induced Pluripotent Stem Cell-Derived Neuronal Cells for Modeling Neural Disease and Evaluating Drug Efficacy. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 6.
Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, and δ-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and δ-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-β-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.
- Development of Basal-Like HaCaT Keratinocytes Containing the Genome of Human Papillomavirus (HPV) Type 11 for Screening of Anti-HPV Effects. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 29.
Condylomata acuminata (CA), induced by low-risk human papillomaviruses (HPVs), is one of the most common sexually transmitted diseases. The increasing incidence and the high recurrence rate of CA have significantly contributed to public health problems around the world. Because HPVs cannot be cultured in vitro for a long time, there has been little progress in the development of HPV-speciﬁc antiviral agents. In this study, we established an HPV11.HaCaT system by introducing the recircularized genome of HPV-11 into HaCaT keratinocytes with transfection techniques and cultured them in a special medium. The existence and replication of HPV-11 DNA were positively detected in established HPV11.HaCaT cells. The HPV-11 DNA in HPV11.HaCaT cells has been stably replicated in definite passages of cells. We preliminarily studied the anti-HPV-11 effects of recombinant human interferon α1b (rhIFN-α) and 13-hexyl-palmatine hydrochloride (HP-13) in HPV11.HaCaT cells. The results suggest that HP-13 significantly inhibited the proliferation of HPV11.HaCaT cells in a dose-dependent manner, whereas rhIFN-α did not. HP-13 and rhIFN-α inhibited the replication of HPV-11 DNA and the expression of E1(∧)E4 mRNA in HPV11.HaCaT cells. In conclusion, the established HPV11.HaCaT cells can provide us with a convenient and relatively stable tool for screening anti-HPV-11 agents.
- High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 27.
Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the clinical IDO1 inhibitor INCB024360 shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384- or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.
- Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 27.
Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.
- Software, Database, and Information Services. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 19; 19(5):822-824.
- Knowledge from Small-Molecule Screening and Profiling Data. [EDITORIAL]
- J Biomol Screen 2014 May 19; 19(5):611-613.
- Rank Ordering Plate Data Facilitates Data Visualization and Normalization in High-Throughput Screening. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 14.
High-throughput screening (HTS) of chemical and microbial strain collections is an indispensable tool for modern chemical and systems biology; however, HTS data sets have inherent systematic and random error, which may lead to false-positive or false-negative results. Several methods of normalization of data exist; nevertheless, due to the limitations of each, no single method has been universally adopted. Here, we present a method of data visualization and normalization that is effective, intuitive, and easy to implement in a spreadsheet program. For each plate, the data are ordered by ascending values and a plot thereof yields a curve that is a signature of the plate data. Curve shape characteristics provide intuitive visualization of the frequency and strength of inhibitors, activators, and noise on the plate, allowing potentially problematic plates to be flagged. To reduce plate-to-plate variation, the data can be normalized by the mean of the middle 50% of ordered values, also called the interquartile mean (IQM) or the 50% trimmed mean of the plate. Positional effects due to bias in columns, rows, or wells can be corrected using the interquartile mean of each well position across all plates (IQMW) as a second level of normalization. We illustrate the utility of this method using data sets from biochemical and phenotypic screens.
- Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening. [JOURNAL ARTICLE]
- J Biomol Screen 2014 May 12.
A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.