- hERG Trafficking Assay in Development
We're getting ready to release a new trafficking assay for hERG, for cardiotox testing, and as an important selectivity screen for compounds aimed at correcting trafficking defects (especially for those studying correctors of CFTR mis-folding). Just like CFTR, we'll have wild-type and mutant hERG assays available.
The assays will initially be available as a service for profiling compounds, and as we get assay protocols fully optimized, we'll make cell-lines and assay kits available.
See http://www.sharpedgelabs.com/on-the-bench/herg-trafficking/ for details.?
- Cystic Fibrosis Assays Now Available
We've just moved our CFTR assay from "on the bench" to "off the shelf!"
The assay tracks cell-surface expression of both wild-type and ?F508 CFTR. By using cell-excluded, and cell-permeant dyes we can directly measure both surface and total protein. This allows us to distinguish different CFTR rescue mechanisms (for example, compounds that shift the Surface/ER equilibrium, versus compounds that increase total protein, and thus surface protein, without specifically increasing the fraction at the cell-surface).
We've also included an Application Note, where we test several known CFTR correctors, alone and in synergistic combinations.
The assay is available as a service, or as reagents for more advanced users (we can train in the use of the assay). The assay is running using high-speed flow cytometry.
We're excited to be able to bring this important new assay on-line for our customers. Have a look and contact us if you'd like to test some compounds in the assay.?
- For Immediate Release
Sharp Edge Labs and SpectraGenetics Sign Agreement to Provide Better Assays for Studying G-Protein Coupled Receptors.
Sharp Edge Labs and SpectraGenetics, two Pittsburgh-based biotechnology companies, have entered into an Assay Development Agreement that will allow the companies to offer their industry leading assays for studying the detailed function of an important class of pharmaceutical targets, the G-Protein Coupled Receptors, or GPCRs. GPCRs are the most common target of today?s blockbuster drugs, including drugs for diseases ranging from hypertension and allergy to migraines, pain and cancer. ?While GPCRs are among the most-studied drug targets, we are just now learning important new aspects of how they work. These assays provide unprecedented detail in understanding these new aspects of activity for the industry's most valuable drug targets? said Dr. Scott Sneddon, Ph.D., J.D., President and CEO of Sharp Edge Labs.
?Working together, we can now offer a comprehensive set of tools for pharmaceutical and academic researchers including reagents and read-to-run assay kits, as well as assay services for profiling and screening compounds? added Dr. Reid Asbury, Ph.D., CEO of SpectraGenetics. ?Several large-pharma customers are already using the technology to study so-called ?ligand bias? or ?functional selectivity? of GPCRs. By combining SpectraGenetics? catalog of over 150 GPCRs with Sharp Edge Labs? assay development expertise, we believe we can help researchers address any question in this important new field of drug research? he added.
SpectraGenetics provides research reagents to the pharma/biotech industry and academic labs. SpectraGenetics has a large catalog of tagged genes, including a large selection of tagged GPCRs. Sharp Edge Labs is the exclusive licensee of the Fluorogen Activating Peptide technology from CMU and provides assay products and services for pharma/biotech researchers in the areas of Receptors, Ion Channels and Transporters. The company?s web-sites are www.spectragenetics.com and www.sharpedgelabs.com. Detailed images of the technology in use suitable for print or web publication can be found at www.sharpedgelabs.com/media-images ?
- Our lead biologist, +Qi Yan describes the work that won this year's Nobel prize in Medicine and Physiology. And it involves trafficking!
Protein trafficking is a fundamental process for proper cell function. To move from one subcellular compartment to another or to be secreted into the extracellular space, protein cargo is packaged in tiny ?bubbles?, vesicles and delivered to their destinations. The 2013 Nobel Prize in medicine is awarded to James Rothman, Randy Schekman, and Thomas ?Suedhof for their discovery of the cellular machinery that is responsible for regulating the transport and delivery of molecular cargo in the cell.
Randy Schekman employed genetic approaches to study genes that are important for vesicle transport in yeast. From genetic screens, Schekman identified yeast temperature-sensitive mutants that showed defects in transporting proteins from the ER or the Golgi to the plasma membrane. These genes fall into three categories and are key elements in controlling vesicle transport.
James Rothman took advantage of biochemical tools and discovered a protein complex: SNARE that mediates the fusion between the vesicle and the target membrane. During membrane fusion, vesicle SNAREs and target SNAREs interact with each other specifically to initiate a series of events, including tethering, activation, fusion and proof-reading.
Thomas? Suedhof elucidated the mechanisms underlying the precise temporal control of neurotransmitter release from vesicles in neurons. Suedhof identified synaptotagmin as a calcium sensitive protein where upon binding to calcium ions, synaptotagmin triggers rapid synaptic fusion and thus release of signaling molecules.
The findings by these three scientists have revealed a basic system in cell biology and physiology. In addition, the increased knowledge of vesicle fusion has also provided a better understanding of a number of diseases, such as autism and diabetes.
Image is from:
- +Scott Sneddon just posted a mini-review on GPCRs and new modes of GPCR activation, inspired by the recent paper by Garland in JBS. Check out the video on GPCR internalization, which was created using our pH Sensors.Still lots of great unexploited molecular pharmacology in these targets. If you're interested in our GPCR internalization/desensitization and recycling assays contact us through the web-site or via G+.http://www.sharpedgelabs.com
GPCRs: Key Regulators, Still Great Targets
GPCRs, or G-Protein Coupled Receptors, are cell surface proteins that allow cells to sense their environment and respond accordingly. Because of their location at the cell surface, they act as key control points for the flow of information into and out of the cell, and have become the largest single source of drug targets for the Pharma industry. GPCRs are the quintessential "gold-plated druggable targets" and they have been for decades.
A recently published paper by Stephen Garland in the Journal of Biomolecular Screening poses the question "Are GPCRs Still a Source of New Targets?" (Open Access)
The short answer is definitely "YES," and the paper offers several reasons why.
? GPCRs are part of a large family of include that include Aminergic receptors (which bind dopamine, adrenaline, epinephine, histamine, etc), as well as GPCRs that bind lipids, peptides, as well as orphan receptors (receptors where the ligand is unknown).
? The individual classes can be broken down further in FamilyA/Peptide, Family B, C, and F
? Even within a single ligand binding receptor, there are often subtypes that are expressed in different tissues, and that lead to different pharmacology. For example, there are at least 4 different Dopamine Receptor (D1-D4), and 3 different opioid Receptors (mu, kappa and delta)
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013641/ "GPCRDB: Information System for G Protein-coupled Receptors"
? With all of this diversity in GPCRs, most drugs are directed at a single class of GPCRs, the Aminergic receptors, which are only 10% of the known GPCR universe. (Garland 2013)
But, it gets even more interesting than that, because GPCRs have more than just one mode of action, and the different modes provide opportunities for developing different drugs to treat different disorders.
? "Direct Inhibition" is the gold-standard, and involves a small molecule binding in place of the natural ligand and either acting like the natural ligand (an "agonist"), or preventing the natural ligand from inducing a response (an "antagonist"). Direct inhibition is the most common mechanism by which current drugs work, partly because the tools for measuring GPCR activity often involved displacing the known ligand.
? Allosteric inhibitors bind to the receptor, but not at the same site that the natural ligand. These binders alter the structure of the receptor, either changing the affinity for the natural ligand, or affecting the conformational change that takes place upon binding of the agonist, this altering down-stream signaling. These drugs open a whole new kind of inhibition, with its own dynamics, including the ability to respond to the underlying rhythms of the natural ligand, while altering the intensity or duration of the response.
? Alternative signaling pathways. It has recently been discovered that even a single GPCR does not have just one kind of signaling, and this leads to different kinds of pharmacology under different situations and in different cells. These alternative signaling pathways, provide further new points of intervening with drugs that hope to alter pharmacology and avoid side-effects. (The Garland paper has a nice overview of this)
? Finally, GPCR internalization/desensitization and recycling are becoming more well understood as key parts of how GPCRs behave in-vivo. Here, when a GPCR binds its ligand, the GPCR adopts a conformation that results in the GPCR being taken off the surface of the cell and stored within the cell in what's called an endosome. Since the GPCR is no longer at the surface, it can no longer respond to the signals from outside the cell, and the cell is "desensitized." While inside the endosome, GPCRs have been observed to send different kinds of signals when inside the cells.
A video of the agonist-driven internalization of ADBR2 receptor http://youtu.be/Ho7VgzmNI30
see http://www.ncbi.nlm.nih.gov/pubmed/20303186 "Signaling by internalized G-protein-coupled receptors." for related references.
? Industry has discovered, mostly "by accident", that some drugs appear to operate through several of the mechanisms listed above, and this makes sense, since the GPCR itself is common among all of them. This newly recognized property of GPCR ligands has been termed "ligand bias" and Big pharma and newly form startups alike are looking at it as a source of new gold from the industry's gold-plated targets.
There's lots more to be said about these important drug targets, but I wanted to share this recent paper that makes the point that these are not just nostalgic "golden oldies" in the drug industry, but that we're just scratching the surface of the opportunities for treating important diseases where good drugs just don't exist yet.
Image courtesy of ChEMBL blog at http://chembl.blogspot.com/2012/12/gpcr-structure-human-par1-receptor.html?