The perfect couple

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). Here we find out why overcoming a rather tricky liquid handling problem has been key to this work

There are currently estimated to be more than 250 G-protein coupled receptors (with almost 1,000 different isoforms in humans) each one specific to its signal. 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 they continue to be of growing interest to the scientific community.

However, obtaining detailed structures of GPCRs has been challenging due to the difficulties in removing protein from their natural lipid environment. A detergent is required which does not contribute to a favourable environment for crystallisation using traditional vapour diffusion methods. To overcome this problem the lipid cubic phase (LCP) crystallisation method was developed. LCP is a membrane mimetic-matrix that is suitable for the stabilisation and crystallisation of membrane proteins but in a lipid-based environment¹. 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.

With the technical limitations around the crystallisation of GPCRs alleviated, the crystallography research community has been investigating GPCRs in new detail, and making great strides in the discovery of novel small molecule drugs for use in a range of therapy areas.

As crystallographers continue to discover and describe new structures, we see just how powerful GPCR crystallography can be at detailing novel targets for structure based drug design. 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)² 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. Crystallographic data revealed by Heptares presented an unusual “V” shaped cavity in CRF₁R facing out into the extracellular space, which constitutes a novel binding site for corticotropin-releasing factor (CRF). This was the first crystal structure of a class B GPCR to be determined. Additional investigations in CRF may aid in the design of new small-molecule drugs for diseases of brain and metabolism. More recent work 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 disorders³. Both of these ground-breaking studies provide what will hopefully be a robust framework to be used in the modelling of related receptors as well as leading into work on structure-based small-molecule drug discovery.

Type 2 diabetes affects over 3 million people in the UK alone⁴ and is the result of an insufficient insulin generation leading to higher than normal levels of blood glucose, or the pancreatic ?-cells no longer reacting to insulin (insulin resistance). A specific GPCR known as the human GPR40 receptor (hGPR40) has been shown to bind long chain free fatty acids which enhance glucose-dependent insulin secretion⁵. This finding paves the way for research into novel treatments for type 2 diabetes by targeting hGPR40. 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 diabtes⁶. Crystallographic methods, involving the use of automated robotic systems (including mosquito), successfully described novel hGPR40-TAK-875 interactions⁷. These new properties of TAK-875 have led to the generation of a model which describes the binding of multiple ligands to the hGPR40 receptor to amplify insulin secretion. This represents a very interesting step forward in the development of drugs used to treat type 2 diabetes.

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 aggregation⁸. 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 (both typically resulting in reduced blow flow on account of thrombus formation). The manner in which the receptor interacts with agonists and antagonists at a molecular level is still relatively poorly understood. 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 present⁸. The level of plasticity demonstrated by P2Y₁₂R was completely unexpected and it is hoped that with further research, more might be learned about other closely related P2Y receptors and their possible benefits in clinical research.

Crystallography has previously been referred to as the limiting factor in gene-to-structure discovery on account of the inherently complex optimisation processes involved. Today the increased throughput of the crystallography screening and optimisation process – achieved through the use of new instruments and systems – means that researchers can crystallise and analyse new structures in a more time and cost-efficient manner. Using these new systems has meant that even proteins that have been notoriously difficult to crystallise due to their complex structure, poor availability or unique characteristics, can now be studied. Whether using hanging drop, microbatch, vapour 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.

Technology has allowed us to progress from manually setting up crystallography screens and conducting multiple experiments in 24-well plates. The result is a screen using minimal amounts of valuable protein samples/reagents and a throughput that allows the crystallographer to more rapidly screen for the optimal conditions. This has proven essential in taking the challenge of crystallising membrane proteins to a relatively routine procedure.

Crystallography has clearly made many contributions to science over the last century. As technology continues to develop and more researchers have access to equipment such as advanced robotics and particle accelerators like the Diamond Light Source, crystallography looks set to continue to drive our understanding of molecular structure and help to push drug discovery forward.

References

1.        Cherezov, V. Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struct. Biol. 21, 559–566 (2011).

2.        Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–43 (2013).

3.        Doré, A. S. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature (2014). doi:10.1038/nature13396

4.        DIABETES PREVALENCE 2013 (FEBRUARY 2014). Diabetes UK (2014). at <http://www.diabetes.org.uk/About_us/What-we-say/Statistics/Diabetes-prevalence-2013/>

5.        Itoh, Y. et al. Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature 422, 173–176 (2003).

6.        Negoro, N. et al. Discovery of TAK-875: A potent, selective, and orally bioavailable GPR40 agonist. ACS Med. Chem. Lett. 1, 290–294 (2010).

7.        Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature (2014). doi:10.1038/nature13494

8.        Zhang, J. et al. Agonist-bound structure of the human P2Y12 receptor. Nature 509, 119–22 (2014).

Author:

Sarah Burl, PhD, is responsible for scientific communications at TTP Labtech and editor of the labCrystal journal.