Elsevier

Neuropharmacology

Volume 60, Issue 1, January 2011, Pages 36-44
Neuropharmacology

The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery

https://doi.org/10.1016/j.neuropharm.2010.07.001Get rights and content

Abstract

G protein-coupled receptors (GPCRs) are one of the most important target classes in the central nervous system (CNS) drug discovery, however the fact they are integral membrane proteins and are unstable when purified out of the cell precludes them from a wide range of structural and biophysical techniques that are used for soluble proteins. In this study we demonstrate how protein engineering methods can be used to identify mutations which can both increase the thermostability of receptors, when purified in detergent, as well as biasing the receptor towards a specific physiologically relevant conformational state. We demonstrate this method for the adenosine A2A receptor and muscarinic M1 receptor. The resultant stabilised receptors (known as StaRs) have a pharmacological profile consistent with the inverse agonist conformation. The stabilised receptors can be purified in large quantities, whilst retaining correct folding, thus generating reagents suitable for a broad range of structural and biophysical studies. In the case of the A2A–StaR we demonstrate that surface plasmon resonance can be used to profile the association and dissociation rates of a range of antagonists, a technique that can be used to improve the in vivo efficacy of receptor antagonists.

Introduction

G protein-coupled receptors (GPCRs) play a central role in cellular responses to a diverse array of signals including neurotransmitters, hormones, nutrients, metabolites, odours and tastes. Many diseases are associated with GPCRs or can be treated by modifying GPCR function. Within the central nervous system (CNS) in particular, GPCRs are located both pre- and post-synaptically where they regulate neurotransmitter release and post-synaptic action potentials through regulation of ion channel signalling (Stephens, 2009). They are also located on astrocytes and glia as well as the vasculature and immune cells. For this reason they are one of the most important families of proteins in drug discovery and a high proportion of CNS drugs for diseases such as depression, schizophrenia, anxiety, Parkinson’s disease and pain act via GPCRs (Conn et al., 2009).

Many of the existing drugs on the market directed at GPCRs are simple analogues of the natural neurotransmitter, for example, beta-adrenoceptor agonists and antagonists are analogues of adrenaline. Many of the current GPCR targets of interest have more complex ligands such as peptides, proteins and lipids and these require a different approach. High throughput screening has been the route of choice for pharmaceutical companies to identify starting points for GPCR drug discovery programmes however these have a poor success rate in getting drugs to the market since many of the molecules identified by high throughput screening have a high molecular weight, high lipophilicity and are difficult to optimise with respect to drug like properties (Keseru and Makara, 2009).

Structural and biophysical methods have successfully been applied to the design of drugs targeting soluble targets such as kinases and proteases and there is a rapid increase in the proportion of drugs identified by these approaches successfully reaching the market (Hegde and Schmidt, 2007, Congreve and Marshall, 2010). The ability to obtain X-ray crystal structures, nuclear magnetic resonance or surface plasmon resonance (SPR) information of ligand–receptor interactions requires the availability of purified protein which retains its native ligand binding capability. In the case of membrane proteins this is hindered by their flexibility and poor stability when extracted from the cell membrane using detergents, a necessary prerequisite for receptor purification.

Mutagenesis to identify conformationally selective point mutations that increase the thermostability of GPCRs (Magnani et al., 2008, Serrano-Vega et al., 2008, Shibata et al., 2009) is a new method which enables the purification of correctly folded receptors in detergent providing a useful reagent for structural and biophysical studies. This approach has been used successfully to obtain a high resolution structure of the β1-adrenergic receptor (β1AR) in complex with cyanopindol (Warne et al., 2008, Warne et al., 2009). The method to engineer thermostable receptors involves systematic scanning mutagenesis and optimal combination of thermostabilising mutations identified from the screens. This approach has successfully been applied across a diverse range of receptors includes both peptide and non-peptide receptors (Magnani et al., 2008, Serrano-Vega et al., 2008, Shibata et al., 2009). These stabilised receptors are known as StaRs™. An important aspect of the method is that the receptors are stabilised in a specific and chosen conformation which is part of the natural range of conformations occupied by the GPCRs during their transition from ground state to fully active G protein-coupled state. This conformational selection derives from the ligand chosen to assay the thermostabilising mutations. Selecting an antagonist conformation results in a StaR which has antagonist pharmacology similar to that which would be seen in the presence of high concentrations of guanine nucleotides added to a membrane preparation (Lefkowitz et al., 1976), namely a reduced agonist affinity, whereas selecting an agonist conformation results in an increased agonist affinity compared to the wild-type receptor and a reduced antagonist affinity. The first StaR made was to the β1AR, known as m23, (Serrano-Vega et al., 2008) which was stabilised in an antagonist conformation and as a result had reduced agonist affinity whilst maintaining antagonist binding. Transfer of these stabilising mutations into β2AR resulted in a thermostabilised β2AR (Serrano-Vega and Tate, 2009). StaRs have been made in both agonist and antagonist conformation to the adenosine A2A receptor (Magnani et al., 2008) whilst the neurotensin receptor was stabilised in an agonist conformation (Shibata et al., 2009).

In this paper we describe the generation of a new set of StaRs to GPCRs of high interest as targets in CNS diseases. The adenosine A2A receptor is a target for the treatment of Parkinson’s disease (Morelli et al., 2009) and the M1 muscarinic receptor is a target for cognitive disorders such as Alzheimer’s disease (Langmead et al., 2008). We explore the pharmacology of these StaRs in comparison to the native receptors and evaluate their properties as reagents for structural and biophysical studies. In particular we show that StaRs can be immobilised on chips enabling the detection of binding kinetics of small-molecule ligands using SPR. The importance of receptor kinetics and, in particular, the residence time or half-life of a drug on the receptor is now recognised as a critically important component in drug discovery. Changes in residence time can alter the properties of a drug with respect to disease efficacy, selectivity, side effect profile and duration of action (Copeland et al., 2006).

Section snippets

Materials

Compounds were purchased from Sigma (UK) and Tocris (UK), detergents from Anatrace, NHS and 1-ethyl 3-(3-dimethylaminpropyl)-carbodiimde hydrochloride (EDC) from GE Healthcare, streptavidin (SA) and EZ-link sulfo-NHS-LC-LC-biotin from Pierce.

Cell culture

HEK293T cells were maintained in culture in DMEM + 10% FBS and passaged twice weekly. Cells were transfected with plasmids containing wild-type or StaR constructs using GeneJuice according to manufacturer’s instructions and harvested after 48 h with Cell

Adenosine A2A and muscarinic M1 receptors can be engineered to increase thermostability in a range of detergents

For the stabilisation of the A2A receptor, the tritiated inverse agonist ZM241385 was used for binding experiments. The stabilised A2A–StaR1, also referred to as Rant22 (Magnani et al., 2008) was used as starting point for further stabilisation. Initial experiments revealed the Tm of the purified wild-type A2A to be 31 °C and StaR1 to be 42 °C in DDM. In order to identify further stabilising mutations, HEK293T cells transfected with mutated StaR1 constructs were solubilised using 1% DDM. The

Discussion

The discovery of new effective drugs for the treatment of CNS diseases presents an enormous challenge to the pharmaceutical industry. Unfortunately the success rate is poor with failures occurring through all stages of research and development. Although the medical need and commercial opportunity remains high, many large pharma companies are reducing their drug discovery efforts in this area. Reducing the attrition rate of drug candidates during discovery and development is an essential

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