The perfect couple for GPCR
19/07/2017
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19/07/2017
Obtaining detailed structures of any membrane protein has been challenging. A detergent is required to extract the protein from the lipid environment which does not contribute to a favorable environment for crystallization using traditional vapor diffusion methods. To overcome this problem the lipid cubic phase (LCP) crystallization method was developed. LCP was originally limited only by the difficulties associated with handling and pipetting the extremely viscous cubic phase (monoolein and water) a hurdle that has now been overcome by the development of SPT Labtech’s robotic system, mosquito® LCP.
Crystallography has clearly made many contributions to science over the last century. The ongoing process of developing new therapeutic drugs has driven research towards a desire to understand drug-target interactions at a molecular level. One group of potential targets attracting a great deal of attention is the G-protein coupled receptors (GPCRs). GPCRs are a large and diverse group of eukaryotic membrane receptors that play a role in a plethora of biological functions from olfaction to mood regulation.
Many drugs exert their effects by binding to GPCRs and thus understanding the structure of this interaction is of great importance. The use of mosquito® LCP has alleviated many of the technical limitations around the crystallization of membrane proteins and allowed the crystallography research community to successfully determine many new GPCR structures that relate to drug therapy. Below are a few examples of the these which have all used mosquito LCP as an essential tool in determining the structures.
Research at Heptares Therapeutics has resulted in several publications in Nature describing the crystal structures of class B and class C GPCRs. Corticotropin-releasing factor receptor type 1 (CRF1R) is a class B GPCR involved in mediating the body’s response to stress and has thus been a target of drugs designed to treat both anxiety and depression. This was the first crystal structure of a class B GPCR to be determined1. More recent work from Heptares has determined the crystal structure of a class C GPCR, metabotropic glutamate receptor 5 transmembrane domain (mGlu5). mGlu5 responds to the neurotransmitter glutamate, which may open up research into the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders2.
Type 2 diabetes affects over 3 million people in the UK alone and is the result of an insufficient insulin generation leading to higher than normal levels of blood glucose. A specific GPCR known as the human GPR40 receptor (hGPR40) has been shown to enhance glucose-dependent insulin secretion. Recent work has led to the development of TAK-875, a selective agonist of hGPR40 from Takeda that reached Phase III trials for the treatment of type 2 diabtes3. Crystallographic methods, involving the use of automated robotic systems (including mosquito), successfully described novel hGPR40-TAK-875 interactions4.
Recent work in crystallography has looked at a particular type of GPCR known as the P2Y12 receptor (P2Y12R), which is part of a family making up one of the most common targets of drugs that work to inhibit platelet aggregation5. Platelets play a critical role in thrombus formation (blood clotting), and drugs targeting P2Y12R have been approved for the prevention of stroke and myocardial infarction. Studies by Zhang et al revealed exciting and surprising structural findings:
P2Y12R was found to be capable of undergoing striking extracellular conformational changes akin to an open or closed ‘binding pocket’ depending on the type of agonist of antagonist present.
Automated pipettors such as the mosquito® range (SPT Labtech), used in all of the research detailed here, have been described as ‘the iPads of robotics’ by a recent user. Technology has allowed many users to progress from manually setting up crystallography screens and conducting multiple experiments in 24-well plates to rapidly seeding a 96-well screen. In the process the added advantages are the use of very low volumes of reagents with varying viscosities and, with a high degree of both accuracy and precision. These features have proven essential in taking the challenge of crystallizing membrane proteins to a relatively routine procedure.
Image: LCP dispensing using mosquito® LCP
Using these new systems has meant that even proteins that have been notoriously difficult to crystallize due to their complex structure, poor availability or unique characteristics, can now be studied. Whether using hanging drop, microbatch, vapor diffusion, sitting drop or lipid cubic phase (LCP) set-ups, there are now systems available to help alleviate many of the experimental hurdles experienced in the past.