MELBOURN, United Kingdom—For the first time, researchershave been able to successfully report the structure of a Class B G-ProteinCoupled Receptor (GPCR) using lipidic cubic phase (LCP) proteincrystallization. The work performed at Heptares Therapeutics Ltd., a drugdiscovery and development company, and published recently in Nature, details the structural identification of the stressreceptor, corticotropin-releasing factor receptor 1 (CRF1), which opens up thetechnique to an entirely new group of molecules with important roles in thedevelopment of diseases as diverse as diabetes, osteoporosis, depression andanxiety. It is hoped that the Class B GPCRs will become valuable drug targets.
The mosquito LCP, a solution for LCP crystallographydesigned by TTP Labtech, was a significant technical driving force enabling theresearch.
"TTP Labtech's mosquito LCP overcomes the common problemsencountered with accurately dispensing the highly viscous LCP mixture used inmembrane protein crystallization," notes Joby Jenkins, mosquito product managerand global director of automation at TTP Labtech. "It allows you to fullyautomate LCP set-ups accurately and repeatedly, dispensing LCP volumes as lowas 25 nL, while automated calibration of syringe and pipette positioningensures precise drop-on-drop placement for easy automated imaging."
Basically, he says, the mosquito operates on the principleof positive displacement. The viscous LCP lipid is heated with a protein in theabsence of air while in contact with a plunger, which then deposits the mixtureon a glass membrane. An auto calibration feature ensures the accurate placementof each drop.
GPCRs are a family of proteins that play an essential partin cell signaling and are thus important drug targets for modulating cellfunction and influencing disease outcome. The Class B subset of GPCRs includesmany 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 impossibleto provide structural data for this class, severely hampering drug developmentefforts 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 theintracellular side of the receptor and far from the position of other Class AGPCR ligands, underlines the importance of the discovery, particularly in termsof modeling other Class B receptors and potentially unlocking drug designacross the subset.
Generating crystalline structures of key biologicalreceptors is an important step in drug discovery, as it enables protein-proteininteractions to be reliably predicted and fuels the rational design ofmolecules engineered to influence receptor activity. However, proteincrystallography, often referred to as the "black art," is frequently a long andcomplex process requiring great technical skill, precision and patience. Thisis especially true when working with complex transmembrane structures such asGPCRs, which require the optimization of specific experimental andenvironmental conditions for each discrete structure.
Dr. Andrew S. Doré, head of crystallography at Heptares andco-author of the recent article in Nature,said, "In any crystallographic project, eliminating variables is key inproducing a steady stream of crystallization-grade protein for structuredetermination. This, of course, also extends to crystallization itself. We hadto stitch together data from 35 isomorphous crystals to produce a full datasetfor the CRF1 structure solution. Mosquito LCP provided a reliable platform forour CRF1 LCP crystallization, deploying reproducible low volume boli at speed,and ultimately yielding a large sample set of CRF1 crystals for subsequentdiffraction analysis."
TTP Labtech's mosquito LCP is used by many leadingpharmaceutical and biotechnology companies for protein crystallizationscreening and production, playing an important role in the identification ofmany protein structures, including important GPCRs. However, this is perhapsthe most significant, high-profile discovery published to date using thetechnology.
"We've worked closely with Doré's team at Heptares since2010—in fact, they were one of the earliest adopters of the mosquito LCP,"noted Jenkins. "Heptares has now solved in excess of 35 GPCR structuresto-date—no mean feat when working with such a complex experimental process."