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.
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).
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.
Recently, Gordon et al. published an atlas of protein:protein interactions of all proposed SARS-CoV-2 proteins expressed individually in HEK 293 cells (Table 1). The study tagged each of the viral proteins with an epitope tag and performed a pull-down of the expressed protein followed by trypsin digestion and mass spec analysis, a process referred to as affinity purification–mass spec analysis. The group identified 332 human proteins interacting with 27 SARS-CoV-2 proteins.
The interactions identified in the HEK 293 cells helped Appelberg et al. analyze interactions over time in SARS-CoV-2-infected Huh7 cells. Gordon et al. used the PPI data to identify FDA-approved drugs, drugs in clinical trials, and pre-clinical compounds that bound to the identified human proteins and labs in New York and Paris tested some of these drugs for antiviral effects.
G protein-coupled receptors (GPCRs) are a large family of receptors that traverse the cell membrane seven times. Functionally, GPCRs are extremely diverse, yet they contain highly conserved structural regions. GPCRs respond to a variety of signals, from small molecules to peptides and large proteins. Many GPCRs are involved in disease pathways and, not surprisingly, they present attractive targets for both small-molecule and biologic drugs.
In response to a signal, GPCRs undergo a conformational change, triggering an interaction with a G protein—a specialized protein that binds GDP in its inactive state or GTP when activated. Typically, the GPCR exchanges the G protein-bound GDP molecule for a GTP molecule, causing the activated G protein to dissociate into two subunits that remain anchored to the cell membrane. These subunits relay the signal to various other proteins that interact with or produce second-messenger molecules. Activation of a single G protein can result, ultimately, in the generation of thousands of second messengers.
Given the complexity of GPCR signaling pathways and their importance to human health, a considerable amount of research has been devoted to GPCR interactions, both with specific ligands and G proteins.
This is a guest post from Katarzyna Dubiel, marketing intern in Cellular Analysis and Proteomics.
“The objective of my experiment was to test the NanoBRET™ assay as if I was a customer, independent of the research and development team which develops the assay.”
Designing and implementing a new assay can be a challenging process with many unexpected troubleshooting steps. We wanted to know what major snags a scientist new to the NanoBRET™ Assay would encounter. To determine this, we reached out to Laurence Delauriere, a senior applications scientist at Promega-France, who had never previously performed a NanoBRET™ assay. Laurence went step-by-step through the experimental process looking at the CRAF-BRAF interaction in multiple cell lines. In an interview, Laurence provided us with some tips and insights from her work implementing the new NanoBRET™ assay.
In a few words, can you explain NanoBRET? “NanoBRET is used to monitor protein: protein interactions in live cells. It is a bioluminescence resonance energy transfer (BRET) based assay that uses NanoLuc® luciferase as the BRET energy donor and HaloTag® protein labeled with the HaloTag® NanoBRET™ 618 fluorescent ligand as the energy acceptor to measure the interaction of two binding partners.” Continue reading “Executing a NanoBRET™ Experiment: From Start to Data”
The review “Kinase Inhibitors: the road ahead” was recently published in Nature Reviews Drug Discovery. In it, authors Fleur Ferguson and Nathanael Gray provide an up-to-date look at the “biological processes and disease areas that kinase-targeting small molecules are being developed against”. They note the related challenges and the strategies and technologies being used to efficiently generate highly-optimized kinase inhibitors.
This review describes the state of the art for kinase inhibitor therapeutics. To understand why kinase inhibitors are so important in the development of cancer (and other) therapeutics research, let’s start with the role of kinases in cellular physiology.
I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).
My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.
Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.
When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.
They continue:
Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.
XWe use cookies and similar technologies to make our website work, run analytics, improve our website, and show you personalized content and advertising. Some of these cookies are essential for our website to work. For others, we won’t set them unless you accept them. To learn more about our approach to Privacy we invite you to Read More
By clicking “Accept All”, you consent to the use of ALL the cookies. However you may visit Cookie Settings to provide a controlled consent.
We use cookies and similar technologies to make our website work, run analytics, improve our website, and show you personalized content and advertising. Some of these cookies are essential for our website to work. For others, we won’t set them unless you accept them. To find out more about cookies and how to manage cookies, read our Cookie Policy.
If you are located in the EEA, the United Kingdom, or Switzerland, you can change your settings at any time by clicking Manage Cookie Consent in the footer of our website.
Necessary cookies are absolutely essential for the website to function properly. These cookies ensure basic functionalities and security features of the website, anonymously.
Cookie
Duration
Description
cookielawinfo-checbox-analytics
11 months
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Analytics".
cookielawinfo-checbox-functional
11 months
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Functional".
cookielawinfo-checbox-others
11 months
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Other.
cookielawinfo-checkbox-advertisement
1 year
The cookie is set by GDPR cookie consent to record the user consent for the cookies in the category "Advertisement".
cookielawinfo-checkbox-necessary
11 months
This cookie is set by GDPR Cookie Consent plugin. The cookies is used to store the user consent for the cookies in the category "Necessary".
cookielawinfo-checkbox-performance
11 months
This cookie is set by GDPR Cookie Consent plugin. The cookie is used to store the user consent for the cookies in the category "Performance".
gdpr_status
6 months 2 days
This cookie is set by the provider Media.net. This cookie is used to check the status whether the user has accepted the cookie consent box. It also helps in not showing the cookie consent box upon re-entry to the website.
lang
This cookie is used to store the language preferences of a user to serve up content in that stored language the next time user visit the website.
viewed_cookie_policy
11 months
The cookie is set by the GDPR Cookie Consent plugin and is used to store whether or not user has consented to the use of cookies. It does not store any personal data.
Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics the number of visitors, bounce rate, traffic source, etc.
Cookie
Duration
Description
SC_ANALYTICS_GLOBAL_COOKIE
10 years
This cookie is associated with Sitecore content and personalization. This cookie is used to identify the repeat visit from a single user. Sitecore will send a persistent session cookie to the web client.
vuid
2 years
This domain of this cookie is owned by Vimeo. This cookie is used by vimeo to collect tracking information. It sets a unique ID to embed videos to the website.
WMF-Last-Access
1 month 18 hours 24 minutes
This cookie is used to calculate unique devices accessing the website.
_ga
2 years
This cookie is installed by Google Analytics. The cookie is used to calculate visitor, session, campaign data and keep track of site usage for the site's analytics report. The cookies store information anonymously and assign a randomly generated number to identify unique visitors.
_gid
1 day
This cookie is installed by Google Analytics. The cookie is used to store information of how visitors use a website and helps in creating an analytics report of how the website is doing. The data collected including the number visitors, the source where they have come from, and the pages visted in an anonymous form.
Advertisement cookies are used to provide visitors with relevant ads and marketing campaigns. These cookies track visitors across websites and collect information to provide customized ads.
Cookie
Duration
Description
IDE
1 year 24 days
Used by Google DoubleClick and stores information about how the user uses the website and any other advertisement before visiting the website. This is used to present users with ads that are relevant to them according to the user profile.
test_cookie
15 minutes
This cookie is set by doubleclick.net. The purpose of the cookie is to determine if the user's browser supports cookies.
VISITOR_INFO1_LIVE
5 months 27 days
This cookie is set by Youtube. Used to track the information of the embedded YouTube videos on a website.
Performance cookies are used to understand and analyze the key performance indexes of the website which helps in delivering a better user experience for the visitors.
Cookie
Duration
Description
YSC
session
This cookies is set by Youtube and is used to track the views of embedded videos.
_gat_UA-62336821-1
1 minute
This is a pattern type cookie set by Google Analytics, where the pattern element on the name contains the unique identity number of the account or website it relates to. It appears to be a variation of the _gat cookie which is used to limit the amount of data recorded by Google on high traffic volume websites.