Illuminating the Kinome: NanoBRET Target Engagement Technology in the Spotlight

In the life of a cell, phosphorylation of proteins is an everyday occurrence. The transfer of a phosphate group, from a molecule such as adenosine triphosphate (ATP) to a specific functional group on a protein, is catalyzed by a protein kinase. The vast majority of protein kinases are classified as either serine/threonine kinases or tyrosine kinases; over 500 kinase genes have been identified in the human genome (1).

nanobret-Target-Engagement-1024x512-1

Protein phosphorylation is a key step in most cell signaling pathways, in response to external or internal stimuli, and it is not surprising that dysregulation of these pathways contributes to a variety of cancers. The first oncogene to be characterized was SRC, a gene that encodes a tyrosine kinase (reviewed in 2). With more kinases being implicated in oncogenic pathways, significant drug discovery efforts have been devoted to developing and characterizing inhibitors of protein kinases. These efforts have accelerated ever since the first targeted small-molecule kinase inhibitor, imatinib, received US FDA approval in 2001 for the treatment of chronic myeloid leukemia (3). Since that time, many more protein kinase inhibitors have received FDA approval, with 67 small-molecule inhibitors listed as of September 2021.

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The 32nd International Symposium on Human Identification: Remembering Past Challenges and Developing a Path Forward

The global COVID-19 pandemic has changed the entire conference and tradeshow industry. Although plans for many in-person conferences were paused, this month the 32nd International Symposium on Human Identification offered the best of both worlds: an in-person symposium in Orlando, Florida (September 12–16), and a virtual conference where registrants could view the session recordings online. At the symposium, exhibits and poster presentations offered attendees the opportunity to reconnect in person after long absences, while various networking events gave attendees a chance to catch up and socialize.

Promega booth at ISHI 32
The Promega booth at ISHI 32 offered a welcoming environment for attendees to reconnect, with a backyard pool party theme.

As usual, workshops were held before and after the main symposium. In a sign of the changing times, Rachel Oefelein and Tarah Nieroda (DNA Labs International) presented a talk on the unique challenges and opportunities associated with virtual courtroom testimony.

The weekend before the symposium was marked by an event of great significance across the world: the 20th anniversary of the September 11, 2001, terrorist attacks on the World Trade Center, the Pentagon, and the attempt on the U.S. Capitol that was thwarted by the brave sacrifice of the passengers and crew on board United Airlines Flight 93. In particular, the DNA forensics community was reminded of how much technology has evolved over the years, in the efforts—still ongoing—to identify the victims of the attacks.

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LncRNA: The Long and Short of “Junk RNA”

The Central Dogma and Junk DNA

lncRNA, long noncoding RNA

On September 19, 1957, Francis Crick delivered a lecture during a symposium at University College London, titled “Protein Synthesis”. The lecture was published a year later (1); in it, Crick quotes his colleague James Watson as saying, “The most significant thing about the nucleic acids is that we don’t know what they can do.” In contrast, Crick argued that proteins play a central, indispensable role as enzymes within the cell that catalyze a variety of chemical reactions. He believed that the main role of genetic material was to control the synthesis of proteins, although the mechanism of that process was not known.

Crick’s hypothesis came to be known as the central dogma of molecular biology, and it was immortalized in his hand-written notes that described the flow of information from DNA to RNA to proteins. This achievement was all the more remarkable, considering that messenger RNAs were completely unknown at that time, and very little was known about how the cellular translational machinery functioned within the cytoplasm to synthesize proteins. Although the later discovery of retroviruses appeared to challenge Crick’s central dogma, he explained quite succinctly that his original statement had simply been misunderstood, and that information could flow in both directions between DNA and RNA (2).

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The NLRP3 Inflammasome: Flipping the Switch

It’s been just over 10 years since the world lost a pioneering immunologist and biochemist, Dr. Jürg Tschopp. He died tragically during a hiking trip in the Swiss Alps on March 22, 2011. A host of academic journals, including Science, Nature and Cell, paid tribute to Dr. Tschopp with obituaries that highlighted his many accomplishments in the fields of apoptosis and immunology.

In 2002, a team led by Dr. Tschopp at the University of Lausanne, Switzerland, was studying the role of the proinflammatory cytokine interleukin 1 beta (IL-1β). This cytokine is produced in the cytoplasm as an inactive precursor (pro-IL-1β). It is cleaved by caspase-1 to the active form, but the exact process by which caspase-1 itself is activated was unknown at the time. Several members of the caspase family contain a conserved region known as the caspase recruitment domain or CARD, and it was proposed that this domain was essential to caspase activation.

Based on similarity to another protein containing an N-terminal CARD motif (Apaf-1) that is involved in activation of caspase-9, the researchers examined the roles of a family of proteins known as NALP1, NALP2 and NALP3 (1). In particular, they were interested in NALP1, which is involved in the immune response. Unlike Apaf-1, NALP1 contains a CARD motif at the C terminus, while the N terminus contains a related motif known as a pyrin-like domain (PYD). The research team had previously showed that the PYD region of NALP1 interacted with an adapter protein known as PYCARD or ASC, which also contains an N-terminal PYD and C-terminal CARD.

The results of the team’s in vitro binding, activation and immunodetection studies showed that a multi-unit protein complex is responsible for caspase activation, and they called this complex the “inflammasome” (1). It is composed of caspase-1, caspase-5, PYCARD/ASC and NALP1.

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RAS-Targeted Drug Discovery: From Challenge to Opportunity

cancer cell, ras-targeted drug discovery

In 1963, Jennifer Harvey was studying Moloney murine leukemia virus (MMLV) at the cancer research department of the London Hospital Research Laboratories. After routine transfers of plasma from MMLV-infected rats to mice, she made an unusual discovery. In addition to the expected leukemia, the mice that received the plasma developed solid tumors (soft-tissue sarcomas), primarily in the spleen (1). A few years later, Werner Kirsten at the University of Chicago observed similar results working with mouse erythroblastosis virus (MEV) (2).

Subsequent research, with the advent of genome sequencing, showed that a cellular rat gene had been incorporated into the viral genome in both cases (3). These genomic sequences contained a mutation later shown to be responsible for the development of sarcomas, and the word “oncogene” became a common part of the vocabulary in cancer publications during the early 1980s (4). Harvey’s discovery led to the naming of the corresponding rat sarcoma oncogene as HRAS, while Kirsten’s related oncogene was named KRAS. Several laboratories, working independently, cloned the human homolog of the viral HRAS gene in 1982 (3). The human KRAS gene was cloned shortly thereafter, as well as a third RAS gene, named NRAS (3). Additional studies showed that a single point mutation in each of these genes led to oncogenic activation, and they have been popular targets for anticancer drug discovery efforts ever since.

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Endpoint PCR in 15 Minutes with New Master Mix and Thermal Cycler Combination

Since its invention in 1983, the polymerase chain reaction (PCR) has become a fundamental technology in life science laboratories across the world. Much of the technological innovation is driven by quantitative PCR and digital PCR (1); however, endpoint PCR remains a workhorse technology for applications such as gene cloning, mutagenesis and detection of microbial pathogens. Variations on the basic endpoint PCR method—for example, the use of multiplexed, fluorescently labeled primers followed by capillary electrophoresis to analyze the amplified DNA fragments—are popular in forensic DNA analysis and cell line authentication.

The COVID-19 pandemic has created an urgent need for PCR-based diagnostic testing for SARS-CoV-2. Most of these diagnostic tests use real-time, reverse-transcription quantitative PCR (RT-qPCR). However, RT-qPCR can be challenging for routine use in developing countries and in laboratories with limited access to real-time PCR thermal cyclers. A recent study described an endpoint PCR method for SARS-CoV-2 detection to address these limitations (2).

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COVID-19 Therapies: Are We There Yet?

A year after COVID-19 was declared a pandemic, collaborative efforts among pharma/biotech and academic researchers have led to remarkable progress in vaccine development. These efforts include novel mRNA vaccine technology, as well as more conventional approaches using adenoviral vectors. While vaccine deployment understandably has captured the spotlight in the fight against COVID-19, there remains an urgent need to develop therapeutic agents directed against SARS-CoV-2.

COVID-19 therapeutic drugs

In the March 12 issue of Science, an editorial by Dr. Francis Collins, director of the U.S. National Institutes of Health (NIH), examines lessons learned over the past 12 months (1). Collins points out that many clinical trials of potential therapeutics were not designed to suit a public health emergency. Some were poorly designed or underpowered, yet they received considerable publicity—as was the case with hydroxychloroquine. Collins advises developing antiviral agents targeted at all major known classes of pathogens, to head off the next potential pandemic before it becomes one. A news feature in the same issue discusses the current state of coronavirus drug development (2).

The present crop of drug candidates is remarkably diverse, including repurposed drugs that were originally developed to treat diseases quite different from COVID-19. Typically, however, the mainstream candidates belong to two broad classes: small-molecule antiviral agents and large-molecule monoclonal antibodies (mAbs).

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Fighting Plant Pathogens Worldwide with the Maxwell® RSC PureFood GMO and Authentication Kit

Among the one trillion or so species that share space on our planet, complex relationships have emerged over time. Such relationships, in which two or more species closely interact, are collectively termed symbiosis. Although it’s commonly assumed that symbiotic relationships are mutually beneficial, this example constitutes only one type of symbiosis (known as mutualism). The traditional predator-prey relationship, clearly a one-sided arrangement, is also an example of symbiosis.

Olive trees in Italy are being affected by the plant pathogen Xylella fastidiosa

The sheer diversity of microbial species has led to the development of many well-characterized relationships with plants and animals. Perhaps the best-known example of mutualism in this context is the process of nitrogen fixation. In this process, various types of bacteria that live in water, soil or root nodules convert atmospheric nitrogen into forms that are readily used by plants. On the other hand, some types of bacteria-plant relationships are parasitic: the bacteria rely on the plant for survival but end up damaging their host. Parasitic relationships can have devastating ecological and economic consequences when they affect food crops.

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Intranasal COVID-19 Vaccines: What the Nose Knows

COVID-19 vaccine distribution efforts are underway in several countries. Recently, the Serum Institute of India celebrated the nationwide rollout of its Covishield vaccine, kicking off the country’s largest ever vaccination program. Meanwhile, many other vaccines against the coronavirus that causes COVID-19 are in either preclinical studies or clinical trials. At present, 19 vaccine candidates are in Phase 3 clinical trials, while 8 vaccines have been granted emergency use authorization (EUA) in at least one country.

intranasal covid-19 vaccine coronavirus

In the US, mRNA vaccines from Pfizer/BioNTech and Moderna are in distribution. Adenoviral vector vaccines authorized for distribution include Oxford/AstraZeneca AZD1222 in the UK (Covishield in India) and Gamaleya Sputnik V in Russia. A third type of vaccine consists of inactivated coronavirus particles, such as those developed by Sinopharm and Sinovac in China.

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Adenoviral Vector Vaccines for COVID-19: A New Hope?

The global war against the coronavirus that causes COVID-19 rages on, spearheaded by efforts to develop effective and safe vaccines. At the time of writing, over 100 COVID-19 vaccine clinical trials were listed in the clinicaltrials.gov database. Recent attention has focused on mRNA vaccines developed by Pfizer/BioNTech and Moderna. If licensed, they would become the first mRNA vaccines for human use.

Other vaccine development efforts are relying on more conventional techniques—using an adenoviral vector to deliver a DNA molecule that encodes the SARS-CoV-2 spike (S) protein. Examples of these adenoviral vector vaccines include the vaccines from Oxford University/AstraZeneca (the UK), Cansino Biologics (China), Sputnik V (Russia) and Janssen Pharmaceuticals/Johnson & Johnson (the Netherlands and USA).

sars-cov-2 coronavirus covid-19 infection with antibodies from a vaccine attacking the virus; several vaccines are in development including adenoviral vector vaccines
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