The Wide World of Bioprocessing: Science for the Greater Good

My former research career was spent in academic laboratories, and I don’t have first-hand experience in the world of bioprocessing. However in my current job as a science writer/copy editor, I create product information and literature about products that are useful to bioprocessing engineers and technicians, and thus wanted to learn more about this diverse area, where discovery and processing of biomaterials results in better therapeutic drugs, better biofuels and even healthier foods.

Bioprocessing is a combination of biological science and chemistry, and a burgeoning science field. Burgeoning is an understatement. Exploding is a much more apt description.

This 2011 Science magazine careers article defines bioprocessing thusly:

“Bioprocessing is an expanding field encompassing any process that uses living cells or their components (e.g., bacteria, enzymes, or chloroplasts) to obtain desired products, such as biofuels and therapeutics.”

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Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.

Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.

The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading

A New Way to Avoid False Hits During Compound Screening for Drug Discovery

One goal of drug discovery and research programs is to reduce false hits as early as possible in the process. Follow-up on false hits is costly in terms of time and resources, and the longer the false hits remain in the drug development pipeline, the more costly they are. So methods that can easily reduce the number of false hits during compound screening early in the discovery process are particularly sought after.

Reporter assays have proven to be invaluable tools for elucidating the mechanisms of action of small molecules or other agents on signaling pathways within cells, and the luciferase reporter assay has become a standard research tool in the biological research laboratory.

However, one caveat of using standard luciferase-based reporter assays for larger-scale compound screening efforts is the frequency of false hits that result from direct interaction of compounds with the luciferase reporter. This issue can be mitigated with a “coincidence reporter” system where two independent reporter proteins are produced from a single transcript. In this type of assay, a bicistronic transcript is stoichiometrically translated into two nonhomologous reporters by means of a 2A “ribosomal skipping” sequence. Since it is unlikely that compounds will interact with two distinct types of reporter, “coincident” responses will indicate on-target activity. Such a coincident reporter system provides an important control against costly false hits early in drug discovery research programs.

A paper published online in ACS Chem Biol in February describes the first successful application of the firefly/NanoLuc luciferase coincidence reporter system to identify new pathways that up-regulate PARK2 expression. Continue reading

Piecing the Puzzle Together: Using Multiple Assays to Better Understand What Is Happening with Your Cells

You often need several pieces of information to really understand what is happening within a cell or population of cells. If your cells are not proliferating, are they dying? Or, are you seeing cytostasis? If they are dying, what is the mechanism? Is it apoptosis or necrosis? If you are seeing apoptosis, what is the pathway: intrinsic or extrinsic?

If you are measuring expression of a reporter gene and you see a decrease in expression, is that decrease due to transfection inefficiencies, cytotoxicity, or true down regulation of your reporter gene?

To investigate these multiple parameters, you can run assays in parallel, but that requires more sample, and sample isn’t always abundant.

Multiplexing assays allows you to obtain information about multiple parameters or events (e.g., reporter gene expression and cell viability; caspase-3 activity and cell viability) from a single sample. Multiplexing saves sample, saves time and gives you a more complete picture of the biology that is happening with your experimental sample.

What information do you need about your cells to complete the picture?

What information do you need about your cells to complete the picture?

Multiplexing assay reagents to measure biomarkers in the same sample has often been considered an application only accomplished with antibodies or dyes and sophisticated detection instrumentation. However, Promega has developed microwell plate based assays for cells in culture that allow multiplexed detection of biomarkers in the same sample well using standard multimode multiwell plate readers. Continue reading

Measuring Changing Metabolism in Cancer Cells

Because of the central role of energy metabolism in health and disease, and its effect on other cellular processes, assays to monitor changes in cellular metabolic state have wide application in both basic research and drug discovery. In the Webinar “Tools for Cell Metabolism: Bioluminescent NAD(P)/NAD(P)H-Glo™ Assays” Jolanta Vidurigiene, a Senior Research Scientist at Promega, introduces three new metabolism assays for measuring oxidized and reduced forms of NAD and NADP.

In this webinar, Jolanta provides background information on why it is important to be able to accurately measure metabolites such as NAD/NADH and NADP/NADPH. She outlines the roles of each, and highlights some of the challenges involved in developing assays that can accurately measure these metabolites. She discusses key considerations for successful NAD(P)/NAD(P)H assays and provides examples of how to use these assays to measure either total (both oxidized and reduced) forms of NAD and NADP, or to measure oxidized and reduced forms individually in a single assay plate.

NAD(P)H-Glo™ Assay Mechanism

NAD(P)H-Glo™ Assay Mechanism

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Shining a Bright Light on Deep Questions in Biology with Bioluminescence

artists view inside a cellA quick search of the PubMed database for “dual luciferase” quickly returns over 1,000 papers. 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 been valuable tools for researchers asking many different kinds of questions in the life sciences. In a recent webinar, Biologically Relevant Assays for Oncology: Harnessing the Power of Bioluminescence, Neal Cosby discussed how bioluminescent chemistries 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. Continue reading

The 64 billion dollar question: Is my compound or treatment toxic?

CellTox™ Green Dye is excluded from viable cells, but it binds to DNA from cells with compromised membrane integrity.

CellTox™ Green Dye is excluded from viable cells, but it binds to DNA from cells with compromised membrane integrity.

Determining the exact cause/effect relationship between a treatment and a cellular outcome is not a simple matter, but is critical for really understanding how therapeutic treatments affect target cells or exercise any off-target effects.

Four key factors are critical for determining whether or not a particular treatment or compound is toxic.

  1. Dosage (usually addressed by a dilution series)
  2. Exposure time
  3. Mechanism of Action
  4. Cell Type

In a recent Promega Webinar, A Cytotoxicity Assay That Fits Your Timeline, Promega scientist Dr. Andrew Niles presented the CellTox™ Green Cytotoxicity Assay—a new tool that gives researchers more power to answer the question “Is my compound or treatment toxic?” Continue reading

The Ideal Kinase Assay

Kinome_FullIf you could design the ideal kinase assay system what would it look like?

  • Would it be able to match, point for point, the results of the tried-and-true isotopic assay methods but not have any of the associated safety and waste disposal issues?
  • Would it avoid the use of specific antibodies?
  • Would it minimize false hits associated with many of the fluorescence-based assays?
  • Would it be affordable technology, adaptable to any laboratory’s throughput from 96-well to 1,536-well automated screening?
  • Would it be universal—able to assess the function of any kind of kinase (protein, lipid or sugar) that uses any kind of substrate?
  • Would it be able to detect low conversion rates (low-activity enzymes) with a high signal-to-background ratio?
  • Would you be able to use it with substrates that are multiphosphorylated?

If you answered “yes” to any of the above questions, you might want to take a look at the Promega Webinar “Enabling Kinase Research with a Luminescent ADP Detection Platform and Complete Kinase Enzyme Systems”, presented by Hicham Zegzouti, PhD, research scientist at Promega. Here he describes the ADP-Glo™ Assay platform, which meets these and several other criteria.

The precise molecular lesion that occurs with the Philadelphia Chromosome translocation—a rearrangement that creates a bcr-abl fusion in which the abl tyrosine kinase is constitutively active leading to the development of chronic myeloid leukemia is the first description of dysregulation of a kinase leading to a particular disease state. However, the human genome contains 518 protein kinases and many other atypical kinases, and one-third of all human proteins are phosphorylated. It is now estimated that over 400 human diseases are caused by dysregulation or mutation of kinases, making kinases a major target for drug discovery efforts. Continue reading

Exploiting Bacterial Toxins for Good (Making Lemonade from Lemons?)

Bacterial exotoxins are scary things. The names of the big three: Tetanus, Anthrax and Botulinum, are sufficient to evoke fear and conjure up images of agony, paralysis, mass hysteria, and permanently frozen Hollywood faces. The worst toxin stories are hard to forget. I can still remember the gruesome textbook case studies that accompanied my bacteriology college lectures. There were the home-canning-gone-horribly-awry botulism stories, the historical examples of agonizing tetanus poisonings, and the less lethal but still nasty cases of fast-acting staph toxins delivered to unsuspecting airline passengers in re-heated meals (avoid the ham sandwiches!). It’s all coming flooding back to me.

So, a healthy respect for bacterial toxins is not a bad thing. The worst ones are highly potent and lethal, others may be less potent but are still capable of delivering effects from temporary misery to long-lasting debilitation. But it’s not all bad news. As any microbiology student knows, studies of bacterial toxins have led to some of the most significant advances in the history of medicine–the most well-known example being the development of vaccines based on denatured, inactive forms of toxins. Tetanus and diphtheria are the classic examples where knowledge of the properties of the toxin itself proved to be the key to developing treatment strategies. Continue reading

A New Method that Marks Proteins for Destruction

The ability to manipulate genes and proteins and observe the effects of specific changes is a foundational aspect of molecular biology. From the first site-directed mutagenesis systems to the development of knockout mice and RNA interference, technologies for making targeted changes to specific proteins to eliminate their expression or alter their function have made tremendous contributions to scientific discovery.

A recent paper highlights a novel application of HaloTag technology to enable the targeted destruction of specific HaloTag fusion proteins in vivo. The paper, published online in the July issue of Nature Chemical Biology, details a promising new method with application for validation of potential drug targets by specific in vivo inhibition, and for studying the function of specific genes in organogenesis or disease development. Continue reading