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J Biomol Screen [journal]
- Labware Additives Identified to Be Selective Monoamine Oxidase-B Inhibitors. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Oct 8.
Plastic labware is used in all processes of modern pharmaceutical research, including compound storage and biological assays. The use of these plastics has created vast increases in productivity and cost savings as experiments moved from glass test tubes and capillary pipettes to plastic microplates and multichannel liquid handlers. One consequence of the use of plastic labware, however, is the potential release of contaminants and their resultant effects on biological assays. We report herein the identification of biologically active substances released from a commonly used plastic microplate. The active contaminants were identified by gas chromatography-mass spectroscopy as dodecan-1-ol, dodecyl 3-(3-dodecoxy-3-oxopropyl)sulfanylpropanoate, and dodecanoic acid, and they were found to be selective monoamine oxidase-B inhibitors.
- A New Experimental Model for Assessing Drug Efficacy against Trypanosoma cruzi Infection Based on Highly Sensitive In Vivo Imaging. [REVIEW]
- J Biomol Screen 2014 Oct 8.
The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world's major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major niche of long-term infection, and has demonstrated that chagasic heart disease can develop in the absence of locally persistent parasites. Here, we review the parameters of the imaging system and describe how this experimental model can be incorporated into drug development programs as a valuable tool for assessing efficacy against both acute and chronic T. cruzi infections.
- Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Oct 3.
Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus, morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably, Z' factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal. Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.
- Metabolomic-Based Strategies for Anti-Parasite Drug Discovery. [REVIEW]
- J Biomol Screen 2014 Oct 3.
Metabolomics-based studies are proving of great utility in the analysis of modes of action (MOAs) and resistance mechanisms of drugs in parasitic protozoa. They have helped to determine the MOA of eflornithine, half of the gold standard combination therapy in use against human African trypanosomiasis (HAT), as well as the mechanism of resistance to this drug. In Leishmania, metabolomics has also given insight into the MOA of miltefosine, an alkylphospholipid. Several studies on antimony resistance in Leishmania have been conducted, analyzing the metabolic content of resistant lines, offering clues as to the MOA of this class of drugs. A study of chloroquine resistance in Plasmodium falciparum combined metabolomics techniques with other genetic and proteomic techniques to offer new insight into the role of the PfCRT protein. The MOA and mechanism of resistance to a group of halogenated pyrimidines in Trypanosoma brucei have also recently been elucidated. Effective as metabolomics techniques are, care must be taken in the design and implementation of these experiments, to ensure the resulting data are meaningful. This review outlines the steps required to conduct a metabolomics experiment as well as provide an overview of metabolomics-based drug research in protozoa to date.
- Lead Identification to Clinical Candidate Selection: Drugs for Chagas Disease. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Oct 3.
Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing β-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.
- Validation and Optimization of Novel High-Throughput Assays for Human Epithelial Sodium Channels. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Oct 2.
The epithelial sodium channel (ENaC) plays a crucial role in salt and water homeostasis and is primarily involved in sodium reabsorption in the kidney and lung. Modulators of ENaC function, particularly within lung epithelia, could offer potential treatments for a number of diseases. As a constitutively active sodium channel, ENaC expression at the cell membrane is highly regulated through rapid turnover. This short half-life of the channel at the membrane and cytotoxicity from overexpression pose a problem for reagent generation and assay development in drug discovery. We have generated an HEK293 stable cell line expressing ENaC β and γ subunits containing the PY motif trafficking mutations found in Liddle's syndrome to overcome rapid channel turnover at the membrane. A BacMam virus was used to transiently express the ENaC α subunit to reconstitute channel function to reduce the toxicity associated with long-term overexpression. We have configured a 384-well FLIPR membrane potential antagonist assay for high-throughput screening and an IonWorks Quattro electrophysiology antagonist assay that is predictive of potency values derived from primary lung epithelial cell short-circuit measurements. The triage strategy for compound screening and profiling against this target using these assays has resulted in the discovery of novel chemotypes.
- Development and Validation of a High-Throughput Anti-Wolbachia Whole-Cell Screen: A Route to Macrofilaricidal Drugs against Onchocerciasis and Lymphatic Filariasis. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Oct 2.
There is an urgent need to develop new, safe, and affordable macrofilaricidal drugs for onchocerciasis and lymphatic filariasis treatment and control. The Anti-Wolbachia Consortium (A·WOL) aims to provide a novel treatment with macrofilaricidal activity by targeting the essential bacterial symbiont Wolbachia. The consortium is currently screening a diverse range of compounds to find new chemical space to drive this drug discovery initiative and address this unmet demand. To increase the throughput and capacity of the A·WOL cell-based screen, we have developed a 384-well format assay using a high-content imaging system (Operetta) in conjunction with optimized Wolbachia growth dynamics in the C6/36 Aedes albopictus mosquito cell line. This assay uses texture analysis of cells stained with SYTO 11 as a direct measure of bacterial load. This validated assay has dramatically increased the capacity and throughput of the A·WOL compound library screening program 25-fold, enriching the number of new anti-Wolbachia hits identified for further development as potential macrofilaricides for onchocerciasis and lymphatic filariasis.
- Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Sep 29.
Bromodomain protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) protein family, acts as a central element in transcriptional elongation and plays essential roles in cell proliferation. Inhibition of BRD4 binding to acetylated histone tails via its two bromodomains, BD1 and BD2, with small-molecule inhibitors has been shown to be a valid strategy to prevent cancer growth. We have evaluated and established two novel assays that quantify the interaction of transfected BRD4 BD1 with chemical inhibitors inside cultured cells. Both methods are based on the principle of ligand-induced protein stabilization by which the binding of a small-molecule inhibitor stabilizes intracellular BRD4 BD1 and protects it from proteolytic degradation. We demonstrate the universal character of this principle by using two orthogonal, highly sensitive detection technologies for the quantification of BRD4 BD1 levels in cellular lysates: enzyme fragment complementation and time-resolved fluorescence resonance energy transfer (TR-FRET). Upon optimization of both assays to a miniaturized high-throughput format, the methods were validated by testing a set of small-molecule BET inhibitors and comparing the results with those from a cell-free binding assay and a biophysical thermal shift assay. In addition, point mutations were introduced into BRD4 BD1, and the corresponding mutants were characterized in the TR-FRET stabilization assay.
- Cell Lines Expressing Recombinant Transmembrane Domain-Activated Receptor Kinases as Tools for Drug Discovery. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Sep 26.
Many receptor tyrosine kinases (RTKs) represent bona fide drug targets in oncology. Effective compounds are available, but treatment invariably leads to resistance, often due to RTK mutations. The discovery of second-generation inhibitors requires cellular models of resistant RTKs. An approach using artificial transmembrane domains (TMDs) to activate RTKs was explored for the rapid generation of simple, ligand-independent cellular RTK assays, including resistance mutants. The RTKs epidermal growth factor receptor (EGFR), MET, and KIT were chosen in a proof-of-concept study. Their intracellular domains were inserted into a series of expression vectors encoding artificial TMDs, and they were tested for autophosphorylation activity in transient transfection assays. Active constructs could be identified for MET and EGFR, but not for KIT. Rat1 cell pools were generated expressing the MET or EGFR constructs, and their sensitivity to reference tool compounds was compared to that of MKN-45 or A431 cells. A good correlation between natural and recombinant cells led us to build a panel of clinically relevant MET mutant cell pools, based on the wild-type construct, which were then profiled via MET autophosphorylation and soft agar assays. In summary, a platform was established that allows for the rapid generation of cellular models for RTKs and their resistance mutants.
- Streptomyces: A Screening Tool for Bacterial Cell Division Inhibitors. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Sep 25.
Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the lack of essentiality, the streptomycetes divisome is similar to that of other prokaryotes. Therefore, the chemical inhibitors of sporulation in model streptomycetes may interfere with the cell division in rod-shaped bacteria as well. To test this, we investigated 196 compounds that inhibit sporulation in S. coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA-damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, Fil-2, and Fil-3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors.