Author: Darcia Schweitzer

Cytochrome P450 Inhibition: Old Drug, New Tricks

Posted on by Darcia Schweitzer
multiwell screening plate and various pills on a table

Cytochrome P450 (CYP) inhibitors are often used as boosting agents in combination with other drugs. This drug development strategy is front and center for Paxlovid, the new anti-SARS-CoV-2 treatment from Pfizer. Paxlovid is a combination therapy, comprised of two protease inhibitors, nirmatrelvir and ritonavir. It significantly reduces the risk of COVID-19 hospitalization in high-risk adults and is ingested orally rather than injected, which is an advantage over other SARS-CoV-2 treatments, such as Remdesivir.

Nirmatrelvir was originally developed by Pfizer almost 20 years ago to treat HIV and works by blocking enzymes that help viruses replicate. Pfizer created another version of this drug to combat SARS in 2003, but, once that outbreak ended, further development was put on pause until the advent of the COVID-19 pandemic. After developing an intravenous form of nirmatrelvir early in the pandemic, Pfizer created another version that can be taken orally and combined it with ritonavir.

When ritonavir was originally developed, it wasn’t considered particularly useful because it metabolized so quickly in the body. Now it is recognized as a pharmacokinetic enhancer in combination with other drugs. Ritonivir inhibits CYP3A4, an enzyme which plays a key role in the metabolism of drugs and xenobiotics. By inhibiting CYP3A4, ritonivir slows the metabolism of other drugs. In the case of Paxlovid, this allows nirmatrelvir to stay in the body longer at a high enough concentration to be effective against the virus. This ultimately means that patients can be given lower doses of the drug with reducing efficacy.

Diagram of Nirmaltrelvir mechanism of action.
Nirmatrelvir inhibits the viral 3CL protease, so that functional, smaller viral proteins cannot be produced.
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Firefly Luciferase Sheds Light on Development of New Malaria Treatments

Posted on by Darcia Schweitzer
field of fireflies at night; researchers are using firefly luciferase as a tool to power screening assays for new malaria treatments

Despite significant advancements in antimalarial drugs and widespread efforts to prevent transmission over the past decade, deaths from malaria remain high, particularly in younger children. New drugs with novel modes of action are urgently needed to continue reducing mortality and address drug resistance in the malaria parasite, Plasmodium falciparum. While tens of thousands of compounds have been identified as potential candidates through massive screening efforts, scalable methods for identifying the most effective compounds are needed.

The goal is to find a drug that is potent during all stages in the life cycle of P. falciparum and kills the parasite quickly. Focusing on assessing whether a compound can rapidly eliminate initial parasite burden, Paul Horrocks, PhD, and his colleagues developed a validated bioluminescence-based assay that rapidly determines the initial rate of kill for discovery antimalarials. One key to developing their assay was figuring out how to monitor when the parasite dies after introducing the drug. While measuring DNA content can be used to monitor parasite burden, it is too stable to use for a relevant time course assay.

See how Dr. Paul Horrocks uses a firefly luciferase-based system to understand the dynamics of drug action in the development of new malaria treatments.

Enter firefly luciferase, a dynamic reporter tool to investigate drug action. By creating transgenic P. falciparum that express the luc reporter gene, the researchers could monitor drug action over time. When the parasite is killed, it stops making the luciferase reporter. Since there is no new production of luciferase, levels fall quickly after the parasite dies, and a luciferase assay can determine how fast each drug killed the parasite.

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How to Train Your Instrument Service Team in a Pandemic

Posted on by Darcia Schweitzer
Service engineers engaged in remote instrument training.

When the Spectrum Compact CE System launched in June 2020, all the instrument service engineers that are part of the Promega Global Service & Support (GSS) Team needed to be trained on using and fixing the instrument. This is a challenging endeavor in the best of times, but the COVID-19 pandemic made it even more difficult. Thanks to the work of some dedicated teams and individuals, Promega service engineers around the world were able to receive remote instrument training. But how do you teach someone to repair an instrument when you can’t be in the same room?

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7 Tips for Creating an Individual Development Plan

Posted on by Darcia Schweitzer

Today’s guest blog is written by Jayme Miller, a Human Resources Generalist at Promega, who has some tips for creating an IDP that will help you achieve your goals. Individual Development Plans (IDPs) are common career development tools used in industry, and there has been a push for PhD programs to incorporate career development tools such as IDPs. By creating an IDP, employees and students both have a formal way to communicate their career goals and help them stay on track.

Employee development, the different paths you can take, arrows pointing

There is one question I am frequently asked by candidates during the interview process—“Is employee development a focus at this organization?” Employees frequently tell me they are looking for employers and opportunities where they will have the ability to learn, grow and develop. While that all sounds great, it is important to have an upfront and transparent discussion about roles, responsibilities and expectations when it comes to employee development.

Many organizations indicate that they have an employee development “program” at their organization, but when they begin talking about their program, they describe their performance management process. Often, they will describe how employees are evaluated and provided feedback from their manager. Feedback is a key component for employee development, but it is up to the employee to use that feedback to create action items that will give them the opportunity to learn and grow.  

Often employees believe that employee development is something provided by companies to employees, that it is something that employers make happen for employees. Good organizations will offer continuous learning opportunities and a feedback culture that allows employees to learn and grow. However, no employee development program will work for an employee who is not fully engaged in their own development and does not take ownership over the process. It is ultimately the employee’s responsibility to ensure they are actively taking the steps to develop within their role and within their organization.  

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CRISPR/Cas9 HiBiT Knock-In: A Scalable Approach for Studying Endogenous Protein Dynamics

Posted on by Darcia Schweitzer

Studying protein function in live cells is limited by the tools available to analyze the expression and interactions of those proteins. Although mass spectrometry and antibody-based protein detection are valuable technologies for protein analysis, both methods have drawbacks that limit the range of targets and contexts in which proteins can be investigated.

Mass spectrometry is often poor at detecting low-abundance proteins. Antibody-based techniques require high quality, specific antibodies, which can be difficult to impossible to acquire. Both methods require cell lysis, preventing real-time analysis and limiting the physiological relevance, and both methods can be limiting for higher-throughput analysis. While plasmid-based overexpression of tagged target proteins simplifies detection and can allow for real time analysis, protein levels don’t typically resemble endogenous levels. Overexpression also has the potential to create experimental artifacts or limit the dynamic range of an observed response.

In 2018, Promega R&D scientists published a paper in ACS Chemical Biology demonstrating the use of CRISPR/Cas9 to integrate the 11 amino acid, bioluminescent HiBiT tag directly into the genome to serve as an easily measured reporter for endogenous proteins. This provides a highly quantitative method for investigating cellular protein dynamics that sidesteps the need for cloning and other drawbacks to conventional methods, including the ability to measure changing protein dynamics in real-time. (For more details about CRISPR/Cas9 knock-in tagging and other applications, read this blog.)

While their findings showed that this method provides efficient and specific tagging of endogenous proteins, the research was limited to just five different proteins within a single signaling pathway in two cell lines. This left unanswered questions about whether this approach was scalable, had broader applications and how accurately the natural biology of the cells was represented.

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How Does Our Garden Grow?

Posted on by Darcia Schweitzer

It’s the time of year in the northern US when you start to the miss green grass, ample daylight and warm breezes that are still months away. The promise of spring’s renewal and seedlings sprouting from the snow-covered ground seems too far out to even indulge in a daydream of better weather.

But then again, I’m not a farmer.                                                    

The Promega Culinary Garden at Bluebird Farms.

Now is the time of year when farmers are reflecting on last year’s harvest, making decisions about changes that need to be made and planning for the upcoming growing season. This work includes choosing what plants and varieties will be planted, estimating how many of each are needed and ordering the seeds. Crop rotation and cover crops are also part of the considerations.

If you’re a regular reader of this blog, you may know that Promega has a culinary garden that supplies some of the produce for our cafeterias on the Madison campus. During the growing season our Culinary Gardener, Logan Morrow, oversees the operations of Bluebird Farms with the help of his colleage Mike Daugherty.

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Green Chemistry is Better Chemistry

Posted on by Darcia Schweitzer

When you think of sustainability, what comes to mind? Immediately, my brain imagines vast collections of plastic in the ocean and carbon emissions from millions of cars. I’m guessing that, like me, you didn’t think about optimizing the synthesis of chemical reactions to reduce toxicity or energy usage. Although we’re often focused on the more visible forms of waste, sustainability applies to an enormous range of human activities.

Promega is committed to integrating the principles of sustainability across all aspects of our business. One recent area of focus for our PBI branch is a shift toward Green Chemistry. PBI synthesizes reagents and small molecules used in Promega products. After deciding “it was the right thing to do for our customers and for the environment,” the leader of Promega’s Corporate Responsibility Program, Corey Meek, assembled a few individuals to start a conversation about implementing Green Chemistry principles.

“It was the right thing to do for our customers and for the environment.”

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NanoLuc: Tiny Tag with a Big Impact

Posted on by Darcia Schweitzer

Synthetic biology—genetically engineering an organism to do or make something useful—is the central goal of the iGEM competition each year. After teams conquer the challenge of cloning their gene, the next hurdle is demonstrating that the engineered gene is expressing the desired protein (and possibly quantifying the level of expression), which they may do using a reporter gene.

Reporters can also play a more significant role in iGEM projects when teams design their organism with reporter genes to detect and signal the presence of specific molecules, like environmental toxins or biomarkers. Three of the iGEM teams Promega sponsored this year opted to incorporate some version of NanoLuc® Luciferase into their projects.

NanoLuc® luciferase is a small monomeric enzyme (19.1kDa, 171 amino acids) based on the luciferase from the deep sea shrimp Oplophorus gracilirostris. This engineered enzyme uses a novel substrate, furimazine, to produce high-intensity, glow-type luminescence in an ATP-independent reaction. Unlike other molecules for tagging and detecting proteins, NanoLuc® luciferase is less likely to interfere with enzyme activity and affect protein production due to its small size.

NanoLuc® Luciferase has also been engineered into a structural complementation reporter system, NanoBiT® Luciferase, that contains a Large subunit (LgBiT) and two small subunit options: low affinity SmBiT and high affinity HiBiT. Together, these NanoLuc® technologies provide a bioluminescent toolbox that was used by the iGEM teams to address a diverse set of biological challenges.

Here is an overview of each team’s project and how they incorporated NanoLuc® technology.

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The Future of Synthetic Biology: A Recap of iGEM 2019

Posted on by Darcia Schweitzer

After attending the iGEM Giant Jamboree last year and being completely blown away by the projects presented (check out this article or this one), I didn’t think I’d be as astonished this year. I attributed part of the awe I felt over the caliber and quality of the projects to my wide-eyed naiveté, having never attended the event before. The second time around, the “first-time” novelty long worn off, I didn’t expect to feel that same level of amazement.

I couldn’t have been more wrong.

After three days of impressive presentations, I once again felt that same astonishment as I prepared to watch the presentations of the 6 finalists. With good reason—the projects presented by the six finalists completely blew my mind!

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Tales from the Trenches: Career Growth in Biotechnology

Posted on by Darcia Schweitzer

Building a successful career in the biotechnology industry is really just a series of transitions from one role to another. But the devil is in the details—when to make a change, how to create opportunities and who can be your champion as you pivot. So how do you navigate these factors to keep your career goals on course?

Bob Weiland answers a question posed by Michele Smith at the MS Biotech Alumni Symposium.

I recently attended a symposium (presented by the University of Wisconsin Master of Science in Biotechnology Program, of which I’m an alum) that addressed this topic through the lens of one individual with a storied career in the industry. Bob Weiland currently serves on the Board of Directors for CymaBay Therapeutics. He has held various roles, from sales and marketing to operations and strategy, within large, established companies (Abbot, Baxter, Takeda) and smaller ones (Pacira Pharmacueticals). He drew on this wide-ranging experience to provide advice to professionals at all career stages.

Bob began the talk by declaring that there will be points in your career when you reach a “hard spot” and will need to transition, whether to a new role, company or even industry, to meet your career goals. He suggested a good starting point is simply to be thinking about making a change. But in the same breath he emphasized, “What are you doing about it?” He identified four distinct actions that you can take to ensure role changes and career transitions support your professional growth and development.

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