high throughput screening

Decades of Discovery: How the NCI-60 Revolutionized Cancer Drug Screening

Posted on by Kendra Hanslik

The National Cancer Institute’s NCI-60 drug screening panel, comprised of 60 diverse human cancer cell lines, has been a cornerstone in advancing cancer research and drug discovery since its inception in the late 1980s. Developed in response to the need for more predictive and comprehensive preclinical models, the NCI-60 facilitates the screening of thousands of compounds annually, aiming to identify potential anti-cancer drugs across a broad spectrum of human cancers. This article traces the origins, development, and evolution of the NCI-60 panel, highlighting its significant role in advancing our understanding of cancer and therapeutic agents.  

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Deubiquitinases: A Backdoor into Undruggable Targets?

Posted on by Michele Arduengo

Molecular model of the yeast proteasome.
Molecular model of the yeast proteasome.

Ubiquitin modification of a protein directs events such as targeting for proteasomal degradation. Targeting a protein for degradation through ubiquitin modification is one way to regulate the amount of time a signaling protein, such as a kinase or other enzyme, is available to participate in cell signaling events. Deubiquitinases (DUBs) are enzymes that cleave the ubiquitin tags from proteins, and they have been implicated in several diseases, including cancer.

With their roles in the stabilization of proteins involved in cell cycle progression and other critical processes, DUBs are promising targets for small molecule inhibitors, particularly since they may provide a “back door” for targeting otherwise intractable, undruggable proteins by modulating their half lives. However, finding small molecule inhibitors of the ubiquitin proteases to date has not been trivial. Here we highlight two papers describing the identification and characterization of small molecule inhibitors against the DUB USP7. Continue reading “Deubiquitinases: A Backdoor into Undruggable Targets?”

Review: Validation of a high throughput cell-based assay to screen compounds for mitochondrial toxicity

Posted on by Radhika Ganeshan
mitotox_covertip

Drug-induced mitochondrial toxicity is a concern for pharmaceuticals that was, until recently, limited by the availability of a cell-based assay that is amenable to rapid high-throughput screening. Incorporating high-throughput assay chemistry that can detect mitochondrial dysfunction early in drug discovery programs provides the opportunity to identify potential mitotoxicants before they reach clinical trials or the market population.

In the paper reviewed here (1) Swiss and colleagues have validated the use of a commercially available Mitochondrial ToxGlo™ Asssay (2) to replace traditional methods that not only require expensive reagents and/or equipment but also do not lend themselves easily to high-throughput screening formats.

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Shining a Bright Light on Deep Questions in Biology with Bioluminescence

Posted on by Michele Arduengo
artists view inside a cell

Search the PubMed database for “dual-luciferase” and citations abound. The Dual-Luciferase® Reporter Assay is a powerful tool that allows researchers to ask a multitude of questions about gene control and expression in a system that itself could be normalized and internally controlled. For more than 15 years, firefly and Renilla luciferases  have formed the basis of a range of powerful assays and research tools for scientists who are asking questions about the deep and complex genetic and cellular story associated with cancer. Here we talk a bit of about bioluminescent chemistries, some of the newest bioluminescent tools available, and how some of these tools can be used to probe the deeper questions of cell biology, including cancer biology.

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Screening for Inhibitors of CD73 (5´-ectonucleotidase) Using a Metabolite Assay

Posted on by Michele Arduengo
CD73

CD73 also known as 5´-Ectonucleotidase (NT5E) is a membrane-anchored protein that acts at the outer surface of the cell to convert AMP to adenosine and free phosphate. CD73 activity is associated with immunosuppression and prometastatic effects, including angiogenesis. CD73 is highly expressed on the surfaces of many types of cancer cells and other immunosuppressive cells (1). A recent study by Quezada and colleagues showed that the high concentration of adenosine produced by the CD73-catalyzed reaction on glioblastoma multiforme cells, which are characterized by extreme chemoresistance, triggered adenosine signaling and in turn, the multi-drug resistance (MDR) phenotype of these cells (2).

Because of the roles of adenosine in immunosuppression, angiogenesis and MDR phenotypes, CD73 (NT5E) is an attractive therapeutic target. However, the current methods of assaying for the ectonucleotidase activity, HPLC and a malachite green assay, are cumbersome and not suited to high-throughput screening. The HPLC assay is expensive and difficult to automate and miniaturize (3). The malachite green assay is sensitive to phosphate found in media, buffers and other solutions used in the compound-screening environment.

To address the problem of developing a reliable high-throughput screening assay for CD73, Sachsenmeier and colleagues (3) looked to a luminescent ATP-detection reagent.

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Screening for Drug-Drug Interactions with PXR and CYP450 3A4 Activation

Posted on by Promega

The pregnane X receptor (PXR) is a nuclear receptor known to regulate expression of cytochrome P450 (CYP450) drug-metabolizing enzymes (1). PXR has even been designated the “master xenosensor” due to its ability to upregulate cellular levels of a variety of drug-metabolizing enzymes in response to drugs and foreign chemicals. Elevated levels of CYP450 enzymes can elicit alterations in the pharmacokinetics of co-administered drugs, which can result in adverse drug-drug interactions (DDI) or diminished bioavailability. By assessing PXR activation and CYP450 enzyme induction early in the drug development process, many companies hope to reduce late-stage clinical failures and minimize the high costs associated with bringing a new drug to market.

Proportion of drugs metabolized by different CYPs

A paper by Shukla et al. (2) examined over 2,800 clinically used drugs for their ability to activate human PXR (hPXR) and rat PXR (rPXR), induce human cytochrome P450 3A4 enzyme (CYP3A4) at the cellular level, and bind hPXR at the protein level. Several studies have identified PXR as playing a key role in regulating the expression of CYP3A4, an enzyme involved in the metabolism of more than 50% of all drugs prescribed in humans. Since PXR activation and CYP3A4 induction have an impact on drug metabolism and pharmacokinetics, the authors wanted to obtain data that would be valuable in understanding structure-activity relationships (SARs), the connection between chemical structure and biological activity, when prioritizing new molecular entities (NMEs) for further in vitro and in vivo studies.

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Describing Life and Death in the Cell

Posted on by Michele Arduengo

4621CALife is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading “Describing Life and Death in the Cell”