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.
This blog was written by guest author, Amy Landreman, PhD.
Drug repurposing, identifying new uses for approved or investigational drugs, is an attractive strategy when looking for new disease treatments. Because the compounds have already gone through some level of pre-clinical optimization and safety testing, this approach can reduce risk, reduce cost, and speed up the timeline for further drug development. An additional benefit of this approach is that it can result in new biological insights or a better understanding of disease mechanisms since these compounds usually already have some level of mechanistic characterization. Indeed, there are now a number of compound collections openly available specifically for the purpose of facilitating drug repurposing efforts. For example, the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library is a collection of 12,000 compounds developed by Scripps Research Center and has been screened to identify novel candidate therapeutics for Cryptosporidium infection (1). The Broad Institute also offers a drug repurposing hub that contains an annotated collection of over 7,000 compounds.
Drug repurposing libraries, although often smaller than novel compound small molecule libraries, are designed for implementation into high-throughput screening workflows in order to efficiently triage compounds for the desired result. Effective compound screens require assays that can be scaled to 384 or 1536-well microplate formats and implemented in batch or continuous processing workflows. The firefly luciferase reaction has been leveraged to create many assays that are well-suited to these types of high-throughput screening approaches. In particular, the generation of “Glow” assays that have stable luminescent signals and homogenous assay design is a good fit. The signal stability allows for multi-plate processing and because the reagent is added directly to cells in culture, pre-processing steps are eliminated allowing for automated workflows. Assay reagents such as the CellTiter-Glo® Cell Viability Assay and the ADP-Glo™ Kinase Assay are commonly used in screening efforts including those done with repurposing libraries. In addition, there are several firefly luciferase reporter assay reagents such as Steady-Glo® and Bright-Glo™ Luciferase Assays that have been optimized for high-throughput detection of firefly luciferase activity making them well-suited to repurposing screens.
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.
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.
The ability to target protein interactions with low solubility or weak binding affinities can present a significant challenge when it comes to drug screening. The beauty of these types of challenges we often face in the lab is that finding solutions to these problems doesn’t necessarily require brand new tools. Sometimes we already have the right tools in our arsenal and, with just a little creativity and collaboration, they can be adapted to address the challenge at hand.
In the following video, Dr. Mohamed (Soly) Ismail, a Postdoctoral Fellow at the Downward Lab of the Francis Crick Institute, presents the perfect example of this with his novel approach to the NanoBiT® Protein:Protein Interaction Assay. Through a collaboration with Promega R&D Scientists, Dr. Ismail has translated the assay into a cell-free, biochemical format, termed the NanoBiT Biochemical Assay (NBBA).
Malaria affects nearly half of the world’s population, with almost 80% of cases in sub-Saharan Africa and India. While there have been many strides in education and prevention campaigns over the last 30 years, there were over 200 million cases documented in 2017 with over 400,000 deaths, and the majority were young children. Despite being preventable and treatable, malaria continues to thrive in areas that are high risk for transmission. Recently, clinicians started rolling out use of the first approved vaccine, though clinical trials showed it is only about 30% effective. Meanwhile, researchers must continue to focus on innovative efforts to improve diagnostics, treatment and prevention to reduce the burden in these areas.
Tissue culture using primary or cultured cell lines has long been a mainstay of testing compounds for inhibiting cell growth or promoting apoptosis during screening for cancer drugs. However, the standard culture conditions result in monolayers of cells, dividing and growing across the bottom of a well, plate or flask in a single layer. The drawback of this technique is that organisms do not come in monolayers; a three-dimensional (3D) spheroid is closer to the in vivo state, especially if the spheroids are made up of more than one cell type like tumors in multicellular organisms. Even more beneficial would be using 3D cultured cells in high-throughput screening to facilitate compound profiling for target effectiveness and cytotoxicity. In a recent PLOS ONE article, researchers used normal and breast cancer cells both in monoculture and coculture to test a set of compounds and found results differed between 2D and 3D cultured cells. Continue reading “Improving Cancer Drug Screening with 3D Cell Culture”
Working with bacteria and viruses that cause life-threatening diseases with no currently available treatment options takes guts. Most scientists are familiar with the routine requirements of good aseptic technique, are highly aware of laboratory safety requirements, and are more than familiar with autoclaves and sterilization issues, but if we make a mistake the consequences are usually only lost time or a spoiled experiment—not a lost life.
Scientists working with highly virulent organisms deal with a whole other level of risk that requires adherence to the strictest of safety regulations, and these containment regulations can sometimes place constraints on the type of experiment that can be performed with dangerous pathogens. A paper published in the April issue of Assay and Drug Development Technologies brought this to my attention and reminded me of the serious issues some scientists face on a daily basis as they research ways to combat infectious diseases. Continue reading “Screening for Antiviral Compounds under Level 4 Containment Conditions”
When you hold a position as a scientific communication specialist at a biotech company, you never know what you are going to need to write. Most of the time I really like the fact that I have to master new subject matter on a daily basis. I’m using my degree and my brain, and articulating science in a way that connects with the reader is incredibly rewarding. It’s why I do what I do.
Dysfunction of histone deacetylases (HDACs) is associated with many diseases including cancers, asthma and allergies, inflammatory diseases and disorders affecting the central nervous system. Because of their involvement in such a wide range of pathologies, HDACs have become a target for drug discovery. Traditional HDAC activity assays are either isotopic or fluorescent assays using artificial substrates that are prone to artifacts or fluorescence interference. There is a need for a functional assay that is sensitive, accurate and amenable to drug-screening activities.
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