Model organisms are essential tools in the pursuit of understanding biological processes, elucidating the mechanisms of diseases, and developing potential treatments and therapies. Use of these organisms in scientific research has paved the way for groundbreaking discoveries across various fields of biology. In particular, non-mammalian models can be valuable due to characteristics such as rapid life cycles, low cost, and amenability to use with advanced genetic tools, including bioluminescent reporters such as NanoLuc® Luciferase.
NanoLuc® is a small (19.1 kDa) luciferase enzyme originating from deep sea shrimp that is 100x brighter than firefly or Renilla luciferase. It utilizes a furimazine substrate to produce its bright glow-type luminescence. In the decade following its development, the NanoLuc® toolbox has expanded to include NanoBiT® complementation, NanoBRET™ energy transfer methods, and new reagents such as the Nano-Glo® Fluorofurimazine In Vivo Substrate (FFz) which was designed for in vivo detection of NanoLuc® Luciferase, NanoLuc® fusion proteins or reconstituted NanoBiT® Luciferase. In addition to the aqueous-soluble reagents increased substrate bioavailability in vivo, with fluorofurimazine, NanoLuc® and firefly luciferase can be used together in dual-luciferase molecular imaging studies.
Here we spotlight some recent research that demonstrates how the expanded NanoLuc® toolbox can be adapted to use in non-mammalian models, shedding new light on fundamental biological processes and advancing our understanding of complex mechanisms in these diverse organisms.
A new study published in Nature Chemical Biology shows that the most commonly mutated protein in cancer might not be as “undruggable” as previously believed. Promega R&D scientists collaborated with the research group led by Kevan Shokat at the University of California – San Francisco to develop strategies for targeting mutants of KRAS that have evaded previous drug discovery efforts. Their paper opens new possibilities for developing small molecule inhibitors against KRAS(G12D) and other clinically significant mutants.
While PROTACs might not be the topic of conversation at high society cocktail parties, or merit cover stories in glamor magazines, they’re certainly shaking up the drug discovery industry. PROTAC® degraders, together with related compounds like molecular glues and LYTACs, are the basic tools for a targeted protein degradation strategy. Research in this field is advancing rapidly, enabling the development of therapies for disease targets disease targets previously thought to be “undruggable”. This blog post provides an overview of PROTACs based on frequently asked questions.
G protein-coupled receptors (GPCRs) comprise a large group of cell surface receptors, characterized by the unique structural property of crossing the cell membrane seven times. They respond to a diverse group of signaling molecules, such as peptides, neurotransmitters, cytokines, hormones and other small molecules (1). Upon activation, GPCRs interact with GTP-binding (G) proteins and arrestins to regulate a wide variety of signaling pathways. This broad range of functions makes GPCRs attractive targets for drug discovery. The importance of GPCR research was highlighted in 2012, with the Nobel Prize in chemistry being awarded to Robert Lefkowitz and Brian Kobilka “for studies of G-protein–coupled receptors”.
Based on structure and function, GPCRs are categorized into six classes, A–F. The class A GPCRs, or rhodopsin-like receptors, have been studied extensively due to their association with many types of diseases (2). Within the class A GPCRs is a group that share a highly conserved structural motif (3) and respond to chemokines—small “chemotactic cytokines” that stimulate cell migration, especially that of white blood cells (4). A subfamily of class A GPCRs respond to chemokines that have two cysteine residues near the N-terminus, known as CC chemokines. GPCRs activated by CC chemokines are called CC chemokine receptors or CCRs, and these interactions have been implicated in both pro- and anti-cancer pathways (5).
For decades now, peptides have been a molecule of interest for drug discovery research. Peptides offer a unique opportunity for therapeutic intervention that closely mimics natural pathways, as many physiological functions utilize peptides as intrinsic signaling molecules. Macrocyclic peptides, in particular, have recently proven to be promising candidates for targeting intracellular protein–protein interactions (PPIs), an attractive but hard-to-reach therapeutic target for conventional small molecule and biological drugs.
As with any opportunity, there are also challenges that accompany the peptide therapeutic development. Peptide ligands typically have poor membrane permeability, so thus far the majority of peptide therapeutics predominantly target extracellular proteins and receptors. There are also multiple mechanisms for cellular uptake of peptides, including both energy-dependent routes like endocytosis, and energy-independent, like passive diffusion or membrane translocation. Multiple mechanisms of cellular uptake paired with poor permeability makes engineering enough membrane permeability into peptides in order to advance them through drug discovery pipelines extremely difficult.
There are other factors to consider in developing peptide therapeutics, such as solubility, protein/lipid binding and stability, which can also have an affect on the overall cytosolic concentration and, ultimately impact the ability of the peptide to effectively engage its desired intracellular targets.
With so many challenging factors, the ability to have a predictive, high-throughput assay to assess cell permeability, independent of the mechanism(s) of entry, would be a critical and invaluable tool to support peptide drug discovery research.
The development of NanoLuc® luciferase technology has provided researchers with new and better tools to study endogenous biology: how proteins behave in their native environments within cells and tissues. This small (~19kDa) luciferase enzyme, derived from the deep-sea shrimp Oplophorus gracilirostris, offers several advantages over firefly or Renilla luciferase. For an overview of NanoLuc® luciferase applications, see: NanoLuc® Luciferase Powers More than Reporter Assays.
The small size of NanoLuc® luciferase, as well the lack of a requirement for ATP to generate a bioluminescent signal, make it particularly attractive as a reporter for in vivo bioluminescent imaging, both in cells and live animals. Expression of a small reporter molecule as a fusion protein is less likely to interfere with the biological function of the target protein. NanoLuc® Binary Technology (NanoBiT®) takes this approach a step further by creating a complementation reporter system where one subunit is just 11 amino acids in length. This video explains how the high-affinity version of NanoBiT® complementation (HiBiT) works:
Transcriptional activation of genes within the nucleus of eukaryotic cells occurs by a variety of mechanisms. Typically, these mechanisms rely on the interaction of regulatory proteins (transcriptional activators or repressors) with specific DNA sequences that control gene expression. Upon DNA binding, regulatory proteins also interact with other proteins that are part of the RNA polymerase II transcriptional complex.
One type of transcriptional activation relies on inducing a conformational change in chromatin, the DNA-protein complex that makes up each chromosome within a cell. In a broad sense, “extended” or loosely wound chromatin is more accessible to transcription factors and can signify an actively transcribed gene. In contrast, “condensed” chromatin hinders access to transcription factors and is characteristic of a transcriptionally inactive state. Acetylation of lysine residues in histones—the primary constituents of the chromatin backbone—results in opening up the chromatin and consequent gene activation. Disruption of histone acetylation pathways is implicated in many types of cancer (1).
What can you do with a small, super bright luciferase? Amazing things. We’ve highlighted many of the papers and new applications that NanoLuc® luciferase has enabled on this blog. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating bioluminescent tools used to study endogeneous protein dynamics, target engagement, protein degradation, immunodetection and more. So where did the NanoLuc luciferase come from and how does one enzyme power so many research capabilities? Read further for a primer on the various technologies and applications developed from this enzyme over the last 10 years.
Understanding the expression, function and dynamics of
proteins in their native environment is a fundamental goal that’s common to
diverse aspects of molecular and cell biology. To study a protein, it must
first be labeled—either directly or indirectly—with a “tag” that allows
specific and sensitive detection.
Using a labeled antibody to the protein of interest is a
common method to study native proteins. However, antibody-based assays, such as
ELISAs and Western blots, are not suitable for use in live cells. These
techniques are also limited by throughput and sensitivity. Further, suitable
antibodies may not be available for the target protein of interest.
Autism Spectrum Disorder, or ASD, is nothing if not unique.
The way ASD manifests itself in people is unique; although it most often presents as some form of variable impairment in social interaction and communication, each individual has behaviors and habits that are as unique to them as snowflakes are to one another.
ASD has also proven itself to be a uniquely challenging disorder to study. In the past decade, de novo (new) mutations have been identified as key contributors to causality of ASD. However, the majority of these identified de novo mutations are located in protein-coding genes, which comprise only 1–2% of the entire human genome.
Up to this point, a majority of previous research has focused on identifying mutations located in the 20,000 identified genes in the protein-coding region, which would seem like a promising approach. Genes are the genetic blueprints for creating proteins, which control and perform crucial tasks in our bodies, such as fighting off infections, communicating between your organs, tissues, and cells as chemical messengers, and regulating your blood sugar levels. It seems like basic math: Genes + Mutations = Mutated Proteins. Mutated Proteins = Disrupted Protein Function.
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