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No mean feat
September 2013
by Lloyd Dunlap  |  Email the author


MELBOURN, United Kingdom—For the first time, researchers have been able to successfully report the structure of a Class B G-Protein Coupled Receptor (GPCR) using lipidic cubic phase (LCP) protein crystallization. The work performed at Heptares Therapeutics Ltd., a drug discovery and development company, and published recently in Nature, details the structural identification of the stress receptor, corticotropin-releasing factor receptor 1 (CRF1), which opens up the technique to an entirely new group of molecules with important roles in the development of diseases as diverse as diabetes, osteoporosis, depression and anxiety. It is hoped that the Class B GPCRs will become valuable drug targets.  
The mosquito LCP, a solution for LCP crystallography designed by TTP Labtech, was a significant technical driving force enabling the research.
"TTP Labtech's mosquito LCP overcomes the common problems encountered with accurately dispensing the highly viscous LCP mixture used in membrane protein crystallization," notes Joby Jenkins, mosquito product manager and global director of automation at TTP Labtech. "It allows you to fully automate LCP set-ups accurately and repeatedly, dispensing LCP volumes as low as 25 nL, while automated calibration of syringe and pipette positioning ensures precise drop-on-drop placement for easy automated imaging."  
Basically, he says, the mosquito operates on the principle of positive displacement. The viscous LCP lipid is heated with a protein in the absence of air while in contact with a plunger, which then deposits the mixture on a glass membrane. An auto calibration feature ensures the accurate placement of each drop.  
GPCRs are a family of proteins that play an essential part in cell signaling and are thus important drug targets for modulating cell function and influencing disease outcome. The Class B subset of GPCRs includes many peptide hormone receptors relevant for treating disease, such as glucagon, glucagon-like peptide, calcitonin and parathyroid peptide hormone. However, until the recent work carried out by Heptares, it had proven almost impossible to provide structural data for this class, severely hampering drug development efforts across the industry.  
The finding that the structure of CRF1, a Class B GPCR, contains a novel binding pocket for the small-molecule antagonist, towards the intracellular side of the receptor and far from the position of other Class A GPCR ligands, underlines the importance of the discovery, particularly in terms of modeling other Class B receptors and potentially unlocking drug design across the subset.
Generating crystalline structures of key biological receptors is an important step in drug discovery, as it enables protein-protein interactions to be reliably predicted and fuels the rational design of molecules engineered to influence receptor activity. However, protein crystallography, often referred to as the "black art," is frequently a long and complex process requiring great technical skill, precision and patience. This is especially true when working with complex transmembrane structures such as GPCRs, which require the optimization of specific experimental and environmental conditions for each discrete structure.
Dr. Andrew S. Doré, head of crystallography at Heptares and co-author of the recent article in Nature, said, "In any crystallographic project, eliminating variables is key in producing a steady stream of crystallization-grade protein for structure determination. This, of course, also extends to crystallization itself. We had to stitch together data from 35 isomorphous crystals to produce a full dataset for the CRF1 structure solution. Mosquito LCP provided a reliable platform for our CRF1 LCP crystallization, deploying reproducible low volume boli at speed, and ultimately yielding a large sample set of CRF1 crystals for subsequent diffraction analysis."
TTP Labtech's mosquito LCP is used by many leading pharmaceutical and biotechnology companies for protein crystallization screening and production, playing an important role in the identification of many protein structures, including important GPCRs. However, this is perhaps the most significant, high-profile discovery published to date using the technology.  
"We've worked closely with Doré's team at Heptares since 2010—in fact, they were one of the earliest adopters of the mosquito LCP," noted Jenkins. "Heptares has now solved in excess of 35 GPCR structures to-date—no mean feat when working with such a complex experimental process."
Code: E091320



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