Author: Ken Doyle

RAF Inhibitors: Quantifying Drug-Target Occupancy at Active RAS-RAF Complexes in Live Cells

Posted on by Ken Doyle

Mitogen-activated protein kinases (MAPKs) are a large family of proteins that regulate diverse cellular functions in eukaryotes, including gene expression, proliferation, differentiation and apoptosis (1). MAPK signaling pathways typically include three sequentially activated kinases, and these pathways are triggered in response to extracellular stimuli, such as cytokines, mitogens, growth factors and oxidative stress (1). Ultimately, the signal is transmitted to the nucleus, with the activation of a specific transcription factor that modulates the expression of one or more genes.

Among MAPK pathways, the RAS-RAF-MEK-ERK signaling pathway has been studied extensively. Mutations in RAS family proteins and resultant dysregulation of the signaling pathway are implicated in a variety of cancers. Therefore, this pathway is a popular target for anticancer drug development.

An overview of the RAS-RAF-MEK-ERK signaling pathway.
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Synthetic Biology: Minimal Cell, Maximal Opportunity

Posted on by Ken Doyle

According to the National Human Genome Research Institute, synthetic biology is “a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities”. Synthetic biology has a broad range of applications, from manufacturing pharmaceuticals and other biologically active chemicals and biofuels, to accelerating the adoption of plant-based burgers (1).

At the heart of the synthetic biology revolution is the rapid technological advancement—and accompanying drop in costs—of DNA oligonucleotide synthesis. Typically, synthetic biology researchers use oligonucleotides as building blocks to assemble genes of interest that are then introduced into, and expressed by, a different organism. For example, to create the plant-based Impossible Burger, the soy leghemoglobin gene (normally found in the root nodules of leguminous plants) was synthesized and expressed in yeast cells (1). This component gives the burger its meaty flavor and appearance of “bleeding” when cooked.

An Impossible Burger served with fries on the side

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Phage Therapy: Meeting the Challenge of Drug-Resistant Bacterial Infections

Posted on by Ken Doyle

Global pandemics, such as COVID-19, have taught us to abhor viruses. The emergence of new, highly infectious viruses is—rightfully so—a cause for concern. However, despite the average human body harboring 380 trillion viruses, most of them simply coexist with us and are harmless. When it comes to an ancient lineage of viruses within the realm Duplodnaviria, researchers are even using them as weapons in the battle against infectious diseases.

In 1915, Frederick William Twort, an English bacteriologist at the University of London, reported the discovery of an unusual “ultramicroscopic virus” (1). Twort was culturing vaccinia virus as part of an experiment to determine if he could prepare smallpox vaccines in vitro. These vaccines, made in calves, were typically contaminated with Staphylococcus bacteria. When Twort plated the vaccines, he found small, clear areas on the agar plates where the bacteria would not grow, and these clear areas were the source of his ultramicroscopic virus. Two years later, a French-Canadian microbiologist, Félix d’Hérelle, independently discovered a similar phenomenon when culturing Shigella bacteria from fecal samples of patients with bacillary dysentery. He called the new virus “un bactériophage obligatoire” (2). Shortly after his discovery, he found that bacteriophages (phages) could be used as powerful agents to treat a variety of bacterial infections, and the field of phage therapy was born (3).

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Genome-Wide CRISPR Screening: Putting Death on Hold

Posted on by Ken Doyle

We share this planet with approximately 8.7 million species of plants and animals. Within such a diverse environment, it’s only natural that many complex relationships have developed among different species. Some relationships are mutually beneficial, some are parasitic—and some are lethal.

Genome wide - crisper screening to help with toxic compounds to humans

Natural toxins and venoms are biologically active compounds produced by normal metabolic processes in an organism but are harmful to other organisms. Typically, toxins are encountered passively or ingested by the affected organisms, and have a specific mode of action and binding site within a cell. In contrast, venoms are introduced directly into the victim through a specialized delivery mechanism, and they may consist of a mixture of compounds that affect a range of cell types and tissues (1). Both types of poisons are produced for predation, defense, or to offer a competitive advantage (1).

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PARP and DDR Pathways: Targeting the DNA Damage Response for Cancer Treatment

Posted on by Ken Doyle

Our cells, and the DNA they contain, are under constant attack from external factors such as ionizing radiation, ultraviolet light and environmental toxins. Internal cellular processes can also generate metabolites, such as reactive oxygen species, that damage DNA. In most cases, DNA damage results in permanent changes to DNA molecules, including DNA mismatches, single-strand breaks (SSBs), double-strand breaks (DSBs), crosslinking, or chemical alteration of bases or sugars. If left unchecked, DNA damage can cause genome instability, mutations and aberrant transcription, and oncogenic transformation.

PARP DDR pathway for drug discovery

Fortunately, our cells have also evolved multiple pathways to repair damaged DNA, collectively known as the DNA damage response (DDR). The type of repair mechanism depends on the nature of the damage, and whether the damage occurs in mitochondrial or nuclear DNA. These mechanisms have been reviewed extensively (1,2). Recently, considerable attention has focused on pathways for repairing SSBs and DSBs, mediated by the ADP-ribosylating enzyme known as poly (ADP-ribose) polymerase 1, or PARP-1.

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Monoclonal Antibody (mAb) Therapy to Delay the Onset of Type 1 Diabetes

Posted on by Ken Doyle

On November 18, 2022, the US Food and Drug Administration (FDA) announced the approval of the first drug to delay the onset of stage 3 type 1 diabetes (T1D). The monoclonal antibody (mAb) drug, teplizumab, was approved for use in adults and pediatric patients 8 years and older.

3D illustration of a monoclonal antibody

The road to approval has been a bumpy one for the manufacturer, Provention Bio. In 2020, the FDA rejected the application for teplizumab due to several concerns, including the small size of the clinical trial. With the current approval, based on new clinical trial results, Provention Bio confirmed a co-promotion agreement with Sanofi US. The agreement included a $35 million Sanofi equity investment in the company.

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Left-Handed DNA: Is That Right?

Posted on by Ken Doyle

There’s a certain group of people (including this blog post author) who derive no small amount of amusement from seeing stock photos of DNA and pointing out flaws in the structure. It’s even more amusing when these photos are used in marketing by life science companies. The most common flaw: the DNA molecule is a left-handed double helix.

What does that even mean? DNA, like many organic chemicals in biology, is a chiral molecule. That is, it can exist in two structural forms that are mirror images of each other but are not superimposable (enantiomers). Just like your left and right hands are mirror images of each other, the two DNA structures are left-handed and right-handed double helices. The DNA double helix is chiral, because its building blocks (nucleotides) are chiral.

Two DNA helices that are mirror images

It can be challenging, at first glance, to tell whether an image of DNA is left-handed or right-handed. Various helpful hints are available; however, the one that I’ve found easiest to remember is described in a blog post by Professor Emeritus Larry Moran at the University of Toronto:

Imagine that the double helix is a spiral staircase, and you’re walking down the staircase. If you’re turning to the right as you descend, the DNA structure is right-handed; if turning to the left, it’s left-handed. In the image shown earlier, the DNA molecule on the right is a right-handed double helix, while its mirror image is left-handed.

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Cellular Senescence and Cancer Therapy: Overcoming Immortality?

Posted on by Ken Doyle

At the time of writing this post, no scientist had yet discovered the secret to immortality. In our world, we’ve come to accept that living things are born, grow old and die—the circle of life.

And yet, for many years, life scientists believed that the circle of life did not apply to our constituent cells when cultured in a laboratory. That is, cultured normal human cells were immortal, and they would continue to grow and proliferate forever, as long as they were provided with the necessary nutrients.

The animal cell cycle. Image by Kelvinsong; made available under the Creative Commons CC0 1.0 Universal Public Domain Dedication

Pioneering work published in 1961 by Leonard Hayflick and Paul Moorhead challenged that theory (reviewed in 1). Their research showed that normal cells in culture have a finite capacity to replicate. After they reach a certain number of replicative cycles, cells stop dividing. Hayflick and Moorhead made the important distinction between normal human cells and cultured cancer cells, which are truly immortal. In later years, the limit to the number of replicative cycles normal human cells can undergo became known as the Hayflick limit. Although some scientists still express skepticism about these findings, the Hayflick limit is widely recognized as a fundamental principle of cell biology.

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Neanderthal DNA and Modern Humans: Svante Pääbo Receives the 2022 Nobel Prize in Physiology or Medicine

Posted on by Ken Doyle

What makes humans “human”?

Neanderthal DNA sequencing from ancient bone samples

On October 3, 2022, the Nobel Assembly at Karolinska Institutet announced the 2022 Nobel Prize in Physiology or Medicine had been awarded to Svante Pääbo, director of the Department of Genetics at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany. The Assembly cited his “discoveries concerning the genomes of extinct hominins and human evolution”. They mentioned the highlight of his research: the seemingly impossible task, at the time, of sequencing the Neanderthal genome. The discoveries that followed from this sequencing project continue to redefine our understanding of modern human origins.

The award showcases the technological advancements made in the analysis of ancient DNA. However, Pääbo’s research had an inauspicious beginning. In 1985, he published the results of his early work, cloning and sequencing DNA fragments from a 2,400-year-old Egyptian mummy (1). Unfortunately, later analysis revealed that the samples could have been contaminated by the researchers’ own DNA (2).

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mRNA Vaccine Manufacturing: Responding Effectively to a Global Pandemic

Posted on by Ken Doyle

We’ve learned a few important lessons from the COVID-19 pandemic.

Perhaps the most significant one is the importance of an early and rapid global response to the initial outbreak. A coordinated response—including widespread use of masks and other personal protective equipment (PPE), travel restrictions, lockdowns and social distancing—could save lives and reduce long-term health effects (1). Widespread availability of effective vaccines goes hand in hand with these measures.

New Boosters to Fight Omicron

Last month, Pfizer/BioNTech announced the US Food and Drug Administration (FDA) had granted emergency use authorization (EUA) for a new adapted-bivalent COVID-19 booster vaccine for individuals 12 years and older. This vaccine combines mRNA encoding the wild-type Spike protein from the original vaccine with another mRNA encoding the Spike protein of the Omicron BA.4/BA.5 subvariants. Moderna also announced FDA EUA for its new Omicron-targeting COVID-19 booster vaccine. The Omicron variant of SARS-CoV-2 shows multiple mutations across its subvariants, and it is currently the dominant SARS-CoV-2 variant of concern across the world.

Genomic epidemiology of SARS-CoV-2 with subsampling focused globally over the past 6 months. This phylogenetic tree shows evolutionary relationships of SARS-CoV-2 viruses from the ongoing COVID-19 pandemic. Image from Nextstrain.org; generated September 20, 2022

Booster doses of vaccines have become a way of life, both due to declining effectiveness of the original vaccines especially in older adults (2), and the rapid mutation rate of SARS-CoV-2 (3). Clinical data for the new Pfizer/BioNTech booster vaccine showed superior effectiveness in eliciting an immune response against Omicron BA.1 compared to the original vaccine. Previously, Moderna published interim results from an ongoing phase 2-3 clinical trial, showing that the new bivalent booster vaccine elicited a superior neutralizing antibody response against Omicron, compared to its original COVID-19 vaccine (4).

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