Surface plasmon resonance analysis of seven-transmembrane receptors.

CHAPTER TWENTY-THREE

Surface Plasmon Resonance Analysis of SevenTransmembrane Receptors Tonia Aristotelous, Andrew L. Hopkins, Iva Navratilova1 Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Surface Plasmon Resonance 3. SPR Approaches to FBDD 4. SPR Applications for GPCRs 5. SPR Applications for Allosteric Compounds 6. SPR Fragment Screening of Thermostabilized GPCRs 7. SPR Fragment Screening of Fully Functional GPCRs 8. Confirmation of SPR Fragment Hits 9. Conclusion References

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Abstract G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for around a third of all drug targets of approved medicines. However, almost all the screening efforts for novel ligand discovery rely exclusively on cellular systems overexpressing the receptors. Current receptor assay systems are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding. Issues of allosteric modulation, probe dependence, and functional selectivity create challenges in selecting suitable assay formats. Therefore, a method that directly measures GPCR–ligand interactions, independent of binding site, probe, and signaling pathway would be a useful primary and orthogonal screening method. An alternative ligand discovery strategy would be the direct measurement of GPCR–ligand interactions by label-free technologies, such as surface plasmon resonance (SPR). In this chapter, we summarize overview of the SPR technology and development of applications for detection of ligand binding to GPCRs using wild-type and thermostabilized receptors. We discuss the utilization of SPR as a biophysical screening method for fragment-based drug discovery for GPCRs. In particular, we show how SPR screening can detect both orthosteric and allosteric ligands with the appropriate experimental design. Methods in Enzymology, Volume 556 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2015.01.016

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2015 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Membrane proteins are attractive drug targets as many are implicated in a wide spectrum of pathological conditions. They account for 30% of the human genome (Wallin & von Heijne, 1998). Their study has been limited due to their sophisticated nature that makes their expression and purification a very challenging task. Difficulties include the low abundance of the membrane proteins in the biological membranes (Drew, Froderberg, Baars, & de Gier, 2003), the membrane-anchored structure in the lipid bilayer, and the insolubility in aqueous fractions due to the differences in hydrophobicity of the protein components (Seddon, Curnow, & Booth, 2004). The challenge is to express and purify the membrane protein in a way that promotes stability and functionality outside the lipid bilayer. Progress on the experimental practicalities of conducting biophysical analysis on membrane proteins is advancing and more membrane proteins are being successfully expressed, but still problems are faced when it comes to purification especially due to the extraction from the natural lipid environment (Thomsen, Frazer, & Unett, 2005). Solubilization of membrane proteins engineers precise selection of solubilizing reagents including detergents or combined lipid–detergent systems (Seddon et al., 2004). The nature of the detergent as well as the concentration used can influence solubilization and in consequence, the stability and activity of the receptor (le Maire, Champeil, & Moller, 2000). An ideal system should consist of solubilizing components, maintaining the physiological properties of both the transmembrane and loop regions of membrane proteins that are hydrophobic and hydrophilic, respectively (Seddon et al., 2004). Membrane proteins are usually soluble in detergent micelles which act by mimicking the natural lipid bilayer environment (Garavito & Ferguson-Miller, 2001). Various approaches have been established to overcome some of the obstacles to improve the stability of purified membrane receptors, including the introduction of thermostabilizing mutations in the amino acid sequence of the receptor. These mutations affect the receptors flexibility and as a consequence, some aspects of their functionality (Robertson et al., 2011). Advances in using mutantstabilized membrane protein for biophysical screening studies will be discussed below. Despite the advances to date in the biophysical study of membrane receptors, new approaches are required which retain the functionality of the receptors. The class of membrane proteins we shall focus our attention

Surface Plasmon Resonance Analysis of Seven-Transmembrane Receptors

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on in this chapter are seven-transmembrane (7-TM) G-protein coupled receptors (GPCRs). GPCRs are a large diverse superfamily of proteins consisting of approximately 800 members. A diverse range of GPCRs have been shown to be highly druggable and account for the most attractive drug target class. The medical importance of GPCRs as drug targets has focused the development of novel screening technologies to discovery of new ligands. An attractive approach to the discovery of new molecules targeting these receptors is the recent advances in biophysical fragment screening. Generally, popular biophysical methods for fragment-based drug discovery (FBDD) include surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), and X-ray crystallography when targeting a soluble protein. However, application of these techniques has been limited for GPCRs due to physical difficulties that are faced when working with membrane proteins, as discussed above. In this chapter, we will outline the development of SPR methods to analyze GPCR interactions, which has resulted in methods sensitive enough to be employed for fragment screening.

2. SURFACE PLASMON RESONANCE SPR is a sensitive and quantitative biophysical technique that enables measurement of binding affinity and kinetics (Khan, Farkas, Kumar, & Ling, 2012). The benefits of applying SPR to fragment screening include the exclusion of false positives (Giannetti, Koch, & Browner, 2008), the label-free nature of SPR assays (Gopinath, 2010), and the low amount of target required (Giannetti, 2011). SPR experiments involve the capture of the target on a chip surface and monitoring its interaction with an analyte in solution in real time. SPR biosensors measure the change in refractive index of the solvent near the assay surface occurring during complex formation and dissociation (Rich & Myszka, 2000). The assay surface is typically a thin film of gold which coats a glass surface (Fig. 1). The detection of biomolecular interaction between a ligand and an analyte molecule requires the immobilization of the ligand on the sensor surface and subsequent exposure of the immobilized ligand to the analyte by injection in the aqueous solution passed through the flow cells. Polarized light is directed to the glass surface under the gold film layer through a prism. The polarized light causes the generation of surface plasmons at a critical angle of the incident light, resulting in a decrease in intensity of the reflected light. The critical angle depends on the refractive index of the medium near the gold surface. Consequently, during the formation of the ligand:analyte complex on the surface

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Figure 1 Schematic representation of Surface plasmon resonance (SPR) phenomenon for the measurement of analyte binding to immobilized ligand. (A) The ligand is immobilized on the sensor chip which is composed on a gold thin layer on a glass slide via different chemistries. Analyte diluted in running buffer is then injected over the immobilized ligand and if binding occurs, complexes of immobilized ligand:analyte are formed. For the detection of binding, polarized light generated from a laser source is directed to the back of the gold layer causing the generation of surface plasmons at a critical angle of the incident light. The light is then reflected to the detector in an angle depending on the refractive index of the medium near the gold surface. Upon binding event, change in the refractive index results in change of the angle of the reflected light from a to b, as shown in the picture, which is detected as a shift in the reflected light intensity. (B) Reflected light intensity shifts upon critical angle change from a to b, resulting from a binding interaction event. (C) Light intensity shifts are transformed into sensorgrams, a plot of Response units (RU) versus time. During association phase, analyte is injected over sensor surface and immobilized ligand:analyte complexes are formed. The sensorgram curve can be used for the measurement of association rate (kon). When analyte-free buffer is injected, dissociation phase is monitored which can be used for the measurement of dissociation rate (koff).

when the analyte binds to the immobilized ligand, there is a change in the refractive index due to change in the mass present on the analysis surface. This refractive index change is observed as an increase in the signal intensity and is denoted as resonance or response units (RU). A critical angle shift of 104 degree corresponds to 1 RU. The RU response is directly proportional to the change of mass on the sensor surface (Merwe, 2001). SPR is the most popular biophysics technique for the measurement of accurate affinity and kinetic parameters. The shape of the binding curve when the ligand is exposed to the analyte molecules can be used for the measurement of association rate (kon). After association phase, dissociation phase


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occurs when the analyte molecules are removed from the ligand due to continuous flow of analyte-free buffer. Similarly, the shape of the dissociation phase curve is used for the measurement of dissociation rate (koff). Binding affinity (KD) can then be calculated using the kon and koff values calculated during the association and dissociation phase, respectively, or using steadystate analysis. SPR technology was pioneered by Pharmacia Biosensor AB with Biacore as the first commercial instrument sold in 1990s and BIAlite followed few years later with a manual handling system (Liedberg, Nylander, & Lundstrom, 1995). Since then great advances have been performed in the technology with constant upgrading of the systems. Initially, most of the instruments launched had low capabilities in aspects regarding the throughput and detection limits. More specialized and sensitive machines of this series to follow included Biacore X, 2000, 3000, and Q for quality control. Further improvements involved the handling of liquid with hydrodynamic addressing. Biacore S51 and A100, array-based biosensors, were released with much wider flow cells and five detection spots in each compared to one that was present before (Safsten, Klakamp, Drake, Karlsson, & Myszka, 2006), which were later upgraded to Biacore 4000. Another widely used Biacore instrument is T100 (recently updated to the more sensitive version, T200). Variability in biosensor studies was explored by Rich et al. with a collaborative work between many biosensor users in which comparison of the performance of different biosensors can be found (Rich, Papalia, et al., 2009).

3. SPR APPROACHES TO FBDD Fragment-based drug design is an approach that is increasingly being used in drug discovery projects over the last decade. The FBDD approach involves the screening of only few hundreds to few thousands of compounds of low molecular weight, typically between 100 and 300 Da (Murray & Rees, 2009). The advantage of fragment screening over conventional high-throughput screening (HTS) is its ability to discover low affinity, low-molecular weight starting points for optimization from screening a relatively small number of compounds. The most important aspect of FBDD is the fact that the amplitude of potential chemical space explored through a small fragment library is greater than that of an actual HTS library, which is usually 2 to 3 orders of magnitude larger, in terms of population size, and typically composed of large compounds with molecular weight over

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300 Da and usually less than 500 Da (Murray & Rees, 2009). During highthroughput biochemical screen, not only the hit rate is low but also there is high rate of false positives due to the setup of such screens (Keseru & Makara, 2009). HTS of GPCRs is often performed in cellular assays with overexpression of the receptor. Fragment hits can be valuable starting points for lead optimization by medicinal chemists and that has been proven by many examples in the literature to date (Bollag et al., 2012; Tsai et al., 2008). Usually the resulting affinities from the screening of fragments range from low μM to mM values due to weak binding compared to HTS hits whose affinities are mostly in μM range (Keseru & Makara, 2009). Biosensor sensitivity is therefore required for such screenings (Erlanson, 2012). Thus, SPR has emerged in recent years as a sophisticated tool of choice in FBDD campaigns. Fragment-based drug discovery has stringent requirements for sensitivity and HTS that not all marketed instruments meet (Kim Retra & van Muijlwijk-Koezen, 2010). Manufacturers including Biacore (T200 and 4000), Sierra Sensors (MASS-1), and Sensi Q (Pioneer FE) have recently designed and developed powerful SPR biosensors that meet most of these requirements. The specifications of these four SPR instruments are compared in Table 1. Instruments differ in many technical aspects as well as practical. As seen from the table, the instruments differ in their molecular weight detection limit as well as their association and dissociation rate detection limit. Applying SPR to FBDD projects requires an instrument with high sensitivity as the detection of small-molecules binding can be challenging due to lower affinities expected for small fragments. All the instruments listed in Table 1 represent high-sensitivity SPR biosensors with capabilities of detection of compounds with molecular weight as low as 100 Da, which is highly preferable when conducting a fragment screening. The detectable association and dissociation rates also differ in the instruments specifications. Kinetic analysis (association (ka) and dissociation (kd) rates) detection threshold is an important aspect during fragment screening because fragments tend to have fast off rates due to low affinity. Another important parameter is the high throughput of the instrument for the conduction of fragment screening as it enables the screening of more populous fragment libraries. Both the number of detectable sensors and sample capacity contribute to high throughput. Biacore T200 and Sensi Q have four and three single-sensor flow cells, respectively. On the other hand, Biacore 4000 and MASS-1 have four five-sensor flow cells and simultaneous processing of up to four samples per cycle and eight dual-sensor flow cells and simultaneous processing of up to eight samples per cycle, respectively.

Table 1 Examples of SPR instruments and their specifications MASS-1 Biacore 4000

Biacore T200

Sensi Q Pioneer FE

Molecular weight detection limit

>100 Da

>50 Da

No limits

>70 Da

Sample concentration

50 pM

>100 pM

10 pM

103 to 1011 M

Kinetic analysis

ka: 103 to 107 M1 s1

ka: 103 to 109 M1 s1 LMW: 5 106 M1 s1

kd: 106 to 101 s1

kd: 5 105 to 1 s1

ka: 103 to 3 109 M1 s1 ka: 102 to 108 M1 s1 LMW: 103 to 5 107 M1 s1 kd: 105 to 1 s1 kd: 106 to 0.1 s1

Number of flow cells Eight dual-sensor flow cells

Four five-sensor flow cells

Four single-sensor flow cells

Three single-sensor flow cells

Number of sensors

20

4

3

440 °C

445 °C (max. 20 °C below ambient)


Sample temperature

440 °C Ambient or temperature control with external cooling unit



Cycle throughput

Simultaneous processing of Simultaneous processing of up to up to eight samples per cycle four samples per cycle

Simultaneous processing of Gradient injections: up to one sample per cycle FastStep™, OneStep®, injections

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Analysis temperature 440 °C (or 15 °C below ambient)

Continued

Table 1 Examples of SPR instruments and their specifications—cont'd MASS-1 Biacore 4000

Biacore T200

Sensi Q Pioneer FE

Sample capacity

2 96- or 384-well microplates + 24-vial rack (+36 96- or 384-well microplates with robotics integration)

10 rack trays with 384- or 96-well Max. 1 96- or 384-well microplate + up to microplate and one 24-well 33 reagent vials reagent plate

Automated buffer exchange

Up to five different buffer solutions

Up to four different buffer solutions

Up to four different buffer solutions

Unattended operation

Up to 120 h

Up to 60 h

Up to 48 h

>72 h

Source

www.sierrasensors.com

www.biacore.com

www.biacore.com

www.sensiqtech.com

Two sample racks


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This enables the study of more targets at the same time. Biacore 4000 accommodates 10 rack trays that can implement 96-well or 384-well microplates and a 24-well reagent plate empowering the screening of relatively large fragment libraries. MASS-1 accommodates two 96-well or 384-well microplates; however, additional robotics integration is available increasing the standard sample capacity to 36 microplates. SPR biosensors, due to their sensitivity, can serve as a sophisticated tool for the binding measurement of the low-molecular-weight fragments (Navratilova, Besnard, & Hopkins, 2011; Navratilova & Hopkins, 2010, 2011). Importantly, SPR screenings benefit from the exclusion of promiscuous binders early in the procedure, thus reducing the false-positive hits, often discovered by other screening methods such as HTS (Giannetti et al., 2008). Being a label-free technique, SPR enables the routine study of most drug targets without the risk of false information about molecular interactions that might be obtained by other labeling techniques (Gopinath, 2010). The low amount of receptor needed for such screenings and the option to work with crude samples makes this method even more powerful as membrane receptors are hard to purify (Navratilova, Dioszegi & Myszka, 2006; Navratilova, Sodroski, & Myszka, 2005). SPR biosensor assays represent a great advance in the field of GPCRs study, since the label-free character and sensitivity of these biosensors provide an alternative approach to the current assays that mostly rely on secondary responses that occur downstream. In contrast, SPR-biosensor assays directly measure GPCR–ligand interaction and provide biomolecular insights of the interaction. Kinetic parameters and affinities can be accurately measured. The kinetic measurement is not limited to known ligands, but also small fragments that usually have low affinities. Current receptor assays measure only ligand displacement or downstream secondary responses rather than direct observation of ligand binding. A method that directly measures GPCR–ligand interactions, independent of binding site, probe, and signaling pathway would be a useful screening and analysis method.

4. SPR APPLICATIONS FOR GPCRs Appreciation of SPR for the study of membrane proteins and in particular GPCRs started in the 1990s and continues to advance today. Advances in expression, purification, and characterization of GPCRs as well as the advances in the SPR technology itself account for the main reasons for

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the improvement of the SPR biosensor assays developed for GPCRs. To date, several GPCRs have been subject of partial or extensive study with SPR (Locatelli-Hoops, Yeliseev, Gawrisch, & Gorshkova, 2013; Patching, 2014). These include, in chronological order, the Rhodopsin (Bieri, Ernst, Heyse, Hofmann, & Vogel, 1999; Karlsson & Lofas, 2002; Salamon, Wang, Soulages, Brown, & Tollin, 1996), chemokine receptors CCR5 and CXCR4 (Huttenrauch, Nitzki, Lin, Honing, & Oppermann, 2002; Navratilova, Dioszegi, et al., 2006; Navratilova, Pancera, Wyatt & Myszka, 2006; Navratilova et al., 2005, 2011; Rich, Miles, Gale, & Myszka, 2009; Safsten et al., 2006; Silin, Karlik, Ridge, & Vanderah, 2006; Stenlund, Babcock, Sodroski, & Myszka, 2003), Adenosine-A2A receptor (Congreve et al., 2011), β1-adrenergic (Christopher et al., 2013), and β2-adrenergic (Aristotelous et al., 2013) receptors, to which the following discussion will concentrate. The light-activated receptor Rhodopsin, was the first GPCR that was subject of SPR analysis. In their attempt to detect binding to a GPCR, Salamon et al. used bovine rhodopsin that was incorporated into an egg phosphatidylcholine bilayer which was deposited on a thin metal film. In this particular study, the metal used was silver. Following immobilization of the receptor on the surface, the group has investigated the binding and activation of G-protein to the immobilized receptor. Interestingly, they were able to monitor and quantify G-protein binding to saturation concentrations and subsequently, monitor the effects of light-induced conformational change and the binding profile of GTP once added to the complex (Salamon et al., 1996). Following applications of SPR on the same receptor included a micropatterned immobilization technique (Bieri et al., 1999). In this case, Rhodopsin was immobilized on the sensor surface in a system utilizing a GPCR-conserved glycosylation site present at the N-terminus and application of carbohydrate-specific chemistry for biotinylation. Using a gold metal film they created mixed self-assembled monolayer of biotinylated thiols with an excess of ω-hydroxy-undecanethiol onto which streptavidin was bound. The receptor then bound to streptavidin directed by the carbohydratespecific biotinylation site mentioned above. Finally, detergent injection caused the formation of a supported lipid bilayer around the receptor, promoting proper folding of the receptor. The binding of agonists 11-cis-retinal and 9-cis-retinal was measured to opsin, after a series of biomolecular events stimulated by light illumination.


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Another application of SPR to rhodopsin involved a method in which the sensor chip carboxylated dextran was modified with long alkyl groups and purified receptor was immobilized by amine coupling. The receptor was detergent-solubilized. Mixed micelles composed of POPC/ octylglucoside (lipid/detergent) were used to wash the surface. Elution of the detergent allowed the lipids to form a bilayer on the sensor surface. The formed bilayer facilitated the reconstitution of the receptor, whose activity was monitored by rhodopsin-mediated dissociation of G-protein (Karlsson & Lofas, 2002). Validation of the chemokine receptors CCR5 and CXCR4 as drug targets attracted the attention to the development of SPR assays for their study. These receptors have been used for the development of a variety of SPR methods including the purification, solubilization, reconstitution, and functional analysis of GPCRs. Initially, β-arrestin 1 binding to CCR5 was investigated by two different approaches. In one, C-terminalderived peptides of CCR5 were immobilized on a CM5 sensor chip via the N-terminal cysteine thiol group and the other involved a cytoplasmic loop of CCR5 immobilized on a Sa5 streptavidin sensor chip via an N-terminal biotin moiety. This approach showed binding of β-arrestin 1 to a conserved Asp-Arg-Tyr motif present in the second intracellular loop (Huttenrauch et al., 2002). With purification being a limiting factor for the study of this class of receptors, the SPR method developed by Stenlund et al. involved the “capture and reconstitution” of both human chemokine receptors CXCR4 and CCR5 for SPR analysis from crude sample preparations on Biacore 2000 and 3000 instruments. CCR5 served as a negative control for the experiments. This approach was addressed advantageous for three reasons: (a) purification of the receptor is unnecessary prior to capture, (b) orientation of the immobilized receptor is homogeneous due to directed capture through a probe, and (c) high densities were achieved enabling the study of medium-size ligands. In this approach, 1D4 monoclonal antibody immobilized through aldehyde coupling chemistry to a hydrazide-modified L1 sensor chip was used as a probe for the capture of GPCR. C-terminally C9 peptide tagged receptors were solubilized in detergent CHAPSO from crude cell lysates and the solubilized receptors were captured over the immobilized antibody molecules. The tag was present at a distinct site than the orthosteric binding site of the receptor orienting the receptor in a way that the ligand binding site is exposed. Following capture, POPC/

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CHAPSO (lipid/detergent) mixed micelles were injected resulting in the reconstitution of the receptor in a lipid bilayer. The detergent was subsequently washed from the assembly with buffer. Both conformational integrity and functionality were established for CXCR4 by interaction with the conformation-dependent anti-CXCR4 antibodies and a chemokine ligand stromal cell-derived factor 1-alpha (SDF-1α), respectively. To determine the structural integrity of CCR5, binding of a conformation-dependent anti-CCR5 antibody was monitored. In a parallel study, the receptors were captured on L1 and CM5 chip without the lipid bilayers. The binding responses for SDF-1α were similar in all three different surfaces, concluding that introduction of lipids is not a prerequisite for the functionality of the receptor and thus, detergent-solubilized receptor could be adequate (Stenlund et al., 2003). Extending this method, Navratilova et al. developed the first measurements of SPR for monitoring small-molecule interactions with GPCRs. CCR5 and CXCR4 were captured over the same probe, 1D4 antibody, however, in this case the antibody was immobilized on a CM4 sensor chip using standard amine coupling chemistry. Native chemokine ligands were used for activity assessment; RANTES and SDF-1α for CCR5 and CXCR4, respectively. Initially, using a Biacore 2000 and a Biacore 3000 automated assay they aimed to improve the chemokine receptors activity and stability by screening a series of solubilization conditions and identifying detergent/lipid/buffer combinations. The receptor’s activity was tested by conformational-dependent antibodies, which were validated for their specificity to the correctly folded receptor by comparing binding levels with receptor solubilized in a detergent disrupting the structure of the receptor. Stability of solubilized CXCR4 over time was also tested in order to assess the method for long-term experiments like solubilization condition screening. During the solubilization buffer screen, cells expressing CCR5 and CXCR4 were suspended in different solubilization conditions composed of lipids, detergents, and cholesterol. Cell suspension was mixed with lipid/detergent mixture and incubated for a short time prior capture over 1D4 antibody surface. Response for 2D7 and 12G5 antibodies was then monitored for each condition for the corresponding receptor. Combination of detergents CHS/DDM/CHAPS with lipids DOPC/DOPS (7:3) was found to exhibit the best initial receptor activity for both chemokine receptors. In this study, it was demonstrated that HIV-1 viral surface protein gp120 binds to solubilized CCR5 only in the presence of CD4 in a ratio-dependent way. Affinity of CD4:gp120 complex to CCR5 was


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estimated to be around 10 nM, value consistent with radiolabeled gp120 studies for membrane associated CCR5. Binding of gp120 was inhibited by TAK-779, a small-molecule inhibitor, blocking gp120:CCR5 interaction opening the door to exploration of other small-molecules binding to the receptors. Moving from the binding of high-molecular weight ligands such as monoclonal antibodies, to binding of proteins such as gp120, to the binding of small molecule, TAK-779, was a great advance in the SPR biosensor assays for GPCRs (Navratilova et al., 2005). Therefore, using Biacore 2000 and S51 biosensors, 19 small-molecule inhibitors of average molecular weight of 550 Da were tested against CCR5 resulting in affinities in good agreement with whole-cell-based assay inhibition constants. In this study, RANTES/CCR5 and SDF-1α/CXCR4 interactions were optimized and small-molecule inhibitors interactions with the solubilized receptors were measured. Binding of JM-2987 and TAK-779 inhibitors was monitored for CXCR4 and CCR5, respectively. Binding affinity of JM-2987 correlated well with literature values; however, binding of TAK-779 was found to be weaker than the expected literature values (Navratilova, Dioszegi, et al., 2006). Other methods were demonstrated by Silin et al. for the selective immobilization of functional CCR5. In their method, the initial template was a protein-resistant surface containing a low percentage of surfacebound biotin on gold. The template was activated by sequential immobilization of avidin, binding specifically to biotin already present at the initial template. Biotin was then used for directed binding of biotinylated goat anti-mouse immunoglobulin G (Bt-IgG) through the avidin binding sites. Finally, Rho 1D4 antibody bound the Fab region(s) of Bt-IgG through its Fc portion. CCR5-containing cell membrane vesicles from immunoprecipitation-enriched vesicle preparations were then injected over the assembly, with C9-tag present in CCR5 binding the 1D4 antibody (Silin et al., 2006). CCR5 was also used as a model for the development of an affinity purification method and screening against cocrystallization conditions. A truncated gp120 construct of nine variants tested was found to bind CCR5 independently of CD4. The identified variant was used in an affinity purification step which was aiming to improve the receptor’s activity. The achieved activity improvement accounted for around 300%. Fifty crystallization conditions varying in pH, salts, and molecular weight of polyethylene glycol (PEG) were screened against CCR5 investigating their effect on CCR5 complex formation. The method involved capturing of CCR5

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and then monitoring gp120:CD4 binding. The gp120 used was the truncated one mentioned above. It was demonstrated that high-molecular weight PEGs enhance CCR5 receptor activity and stability as well as complex formation with gp120 and conformation-dependent antibodies (Navratilova, Pancera, et al., 2006). Detergent screening SPR assay was also developed using CCR5. Using serial (Biacore 2000, Biacore 3000, or Biacore T100) and array (Biacore Flexchip) biosensor technologies, Rich et al. compared and contrasted two biosensor approaches for the screening of solubilization conditions. Briefly, the screening involved solubilization of the receptor in the presence of different detergents, capture on the 1D4 antibody, and monitoring of the binding of 2D7 Fab fragment. In the set up using Biacore 2000, three detergent conditions were tested per cycle, each captured on a different flow cell. The fourth flow cell was used as reference. 1D4 surface regeneration enabled the efficient screening of 96 detergent conditions. On the other hand, Biacore Flexchip technology enabled the capture of all 96 solubilized CCR5 preparations at 96 positions on a Flexchip slide coated with 1D4 mAb. 2D7 antibody binding level was then monitored over the 96 spots. Results from both instruments correlated well, with most of the best detergents being maltosides with C9 to C13 alkyl chain, validating the reliability of both approaches (Rich, Miles, et al., 2009). In the development of SPR analysis of GPCRs, as described above, researchers have employed different immobilization/capture methods for the study of either reconstituted or solubilized receptors. Figure 2 shows some examples of the timeline of the GPCR SPR-based assays and the reduction in the size of the molecules investigated for GPCR–ligand interaction. Binding of large molecular weight ligands initially used by different groups has advanced to smaller molecular size ligands and fragments, expanding the application of SPR in a drug discovery perspective. Interestingly, the molecular size reduction with increasing detection limit for ligands that were detectable by SPR was improved within few years enabling the biosensor technology to become sophisticated tool for measuring smallmolecule kinetics to GPCRs. The gap between detection of small molecules toward obtaining optimized assays for fragments binding to membrane proteins is much larger. This is however not as dependent on instrument sensitivity or lack of membrane protein activity on sensor surface as on the throughput of SPR technologies and establishment of robust assays for soluble proteins and screening of larger libraries of small molecules and fragments.

Figure 2 See legend on next page.

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5. SPR APPLICATIONS FOR ALLOSTERIC COMPOUNDS GPCRs share high percentage of similarity in their orthosteric binding sites and selectivity in targeting the receptors can be challenging in many cases. The SPR assay for β2 adrenergic receptor described later in this chapter elaborated three screening surfaces. Among them was the receptor captured at its inactive state; the orthosteric binding site was occupied by BI-167107, a very slow-off rate compound (Rasmussen, Choi, et al., 2011). In addition to the reduction of nonspecific binding, this negative control can serve as a template for the discovery of allosteric compounds. Such compounds bind to binding sites that are topologically distinct from the classical orthosteric binding site and they are termed “allosteric modulators.” Allosteric GPCR ligands have three possible ways of action. They can either be positive or negative modulators for the activity of the endogenous agonists, employ agonist activity on their own or can bind to the receptor without influencing the receptor activity. Allosterism is much desirable as it is shown to improve selectivity among members of a family of transmembrane proteins which share high percentage of similarity in their orthosteric binding site (Reiter, Ahn, Shukla, & Lefkowitz, 2012). Allosteric binding sites in GPCRs may be accessible from either the extracellular or intracellular sides of the receptor. Figure 2 Timeline of SPR–GPCR assays sensitivity development and improvement over years. Example shows binding of conformation-dependent antibodies, gp120:CD4 complex, chemokines, and small molecules to detergent/lipid-solubilized CXCR4 and/or CCR5 captured on 1D4 antibody, and fragment binding to purified wild-type β2 adrenergic receptor. Left figure: 1D4 monoclonal antibody is immobilized on CM4 chip via amine coupling. Detergent/lipid solubilized CXCR4 or CCR5 is captured on 1D4 antibody via the C-terminal C9 peptide tag present on the receptor. The captured receptor can then be tested for stability and activity. Sensorgrams from the left: Binding of 12G5 (156 nM) to CXCR4 during time-dependent loss of receptor activity test (10 min intervals). The sensorgram represents four repeated cycles. Binding studies for gp120:CD4 complex on CCR5. BAL gp120 binding with and without CD4 on CCR5 (Navratilova et al., 2005). Binding of chemokine RANTES to CCR5, screened at threefold concentration series of 33 nM highest concentration. Threefold concentrations series of small molecule TAK-779 (30 μM highest concentration) to CCR5 (Navratilova, Dioszegi, et al., 2006). Right figure: Purified β2 adrenoceptor is captured on NTA chip via decahistidine tag (His-10) and binding of fragment injected at threefold concentration series (0.45 nM to 1 μM) is monitored (Aristotelous et al., 2013). Adapted from Navratilova et al. (2005) and Navratilova, Dioszegi, et al. (2006) with permission from Elsevier and Aristotelous et al. (2013).


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A number of chemokine receptor antagonists and inverse agonists belong to distinct chemical classes in terms of molecular properties. Mutagenesis evidence suggests these may bind to distinct allosteric sites on some chemokine receptors. To determine the suitability of SPR as a method to detect the binding of compounds to distinct allosteric binding sites on membrane proteins, Navratilova et al. developed a GPCR biosensor assay protocol for high-throughput label-free screening measuring GPCR–ligand interactions using Biacore 4000 instrument. Both orthosteric and allosteric ligands were identified using solubilized, native CCR5 captured as previously described (Fig. 3). 1D4 antibody was immobilized on all detection spots of all four cells and solubilized receptor was captured via the C-terminal C9 tag on two of the five spots in each flow cell. For the discovery of allosteric compounds CCR5 receptor on one spot was blocked with maraviroc over the course of the screen. Two hundred compounds with average MW of 362 Da were screened against the active and inactive form of the receptor at three concentrations. Five hits were identified as real binders for the active form of the receptor which were confirmed by screening on Biacore T100. Most of these binders were found to show some response to inactive CCR5 at higher concentrations. The affinities of the fragments for the active form of the receptor ranged from 8.2 to 49 μM with ligand efficiencies (LE) 0.24–0.335 kcal/mol/nonhydrogen atoms. To determine whether SPR is a suitable method to detect the binding of compounds to distinct allosteric binding sites on membrane proteins, two pyrazinyl sulfonamides

Figure 3 Example of SPR assay setup for detection of allosteric molecules: Active receptor is captured on one flow cell, receptor with blocked binding site is captured on another flow cell. Responses are then collected on both receptor surfaces and reference surface and compared to determine whether the binding of ligand is orthosteric or allosteric (Navratilova et al., 2011).

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compounds were included in the screening library. The pyrazinyl sulphonamides are known allosteric CCR4 antagonists that are reported to be weak CCR5 ligands (Andrews, Jones, & Wreggett, 2008). Affinities of two pyrazinyl sulphonamide compounds to CCR5 were detected and measured in the screen. The compounds were shown to bind the blocked CCR5 with twofold higher response values (Rmax) than the active state receptor (Navratilova et al., 2011).

6. SPR FRAGMENT SCREENING OF THERMOSTABILIZED GPCRs Applications of SPR assays on GPCRs have been described for fragment screening of thermostabilized receptors, known as StaRs (Christopher et al., 2013; Congreve et al., 2012). As demonstrated by Navratilova et al., receptors can be solubilized from cells with the appropriate detergent and lipids and used directly for capture on the sensor chip. The ability to work with crude samples makes SPR assays powerful for the study of membrane proteins since purification is not a prerequisite (Navratilova et al., 2005). This in fact elevates the study of wild-type receptors, which are hard to purify. In contrast, StaRs are stabilized receptors that are engineered with a series of point mutations in their sequence. The StaR generation procedure stabilizes a receptor by restraining the conformation in a trapped pharmacological state, such as an agonist or antagonist form, depending on the ligand used in the selection process (Congreve et al., 2011). Thermostabilized receptors have been shown previously to be appropriate for biophysical fragment screening (Christopher et al., 2013; Congreve et al., 2012). Two examples of fragment screenings against thermostabilized receptors StaRs have been published so far, for Adenosine A2A receptor (A2AR) and β1-adrenoceptor (β1AR). Mutations introduced to A2AR promoted the stabilization of the receptor, allowing the application of fragment screening by SPR due to the high density captured on the sensor chip. The confirmed hits from this screen were eight compounds with affinities ranging from 10 μM to 5 mM for A2AR. The library screened against this target was a small focused fragment library based on xanthine structures, including caffeine, a known ligand for this receptor. The molecules in the library exhibited very low-molecular weight ranging from 136 to 94 Da (Congreve et al., 2012). Christopher et al. published their work on thermostabilized β1AR. In the case of β1AR, six point mutations were introduced resulting in an antagonist functional receptor. Biophysical


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fragment screening against β1AR led to the discovery of a new class of compounds selective to β-adrenergic receptors, the substituted arylpiperazine hits. Heptares library of approximately 650 fragments was screened against both β1AR and A2AR. The latter was used as a reference for the exclusion of nonspecific binders. Two of the initial hits identified to be selective, 1-[3-(trifluoromethyl)phenyl]-piperazine and 2-(piperazin-1yl)quinolone, were found to exhibit both good affinities and LE; KD ¼ 16 μM/LE ¼ 0.41 kcal/mol/nonhydrogen atom and KD ¼ 5.6 μM/ LE ¼ 0.48 kcal/mol/nonhydrogen atom, respectively. Thermostabilization has boosted the study of GPCRs due to the enhanced stability of the receptors, but thermostability is gained at some cost to the full pharmacological function of the mutated receptors. Thermostabilized receptors although they obtain the stability required for the capture and analysis by SPR, they are not pharmacologically flexible. These receptors are biased toward the binding of a certain class of ligands either agonist or antagonist with increased affinities, to which they were selected for. Depending on which ligand is used during the preparation of these targets the pharmacology is altered. Many studies support that the pharmacology of the receptor is altered once mutations are introduced (Magnani, Shibata, Serrano-Vega, & Tate, 2008; Robertson et al., 2011; Serrano-Vega, Magnani, Shibata, & Tate, 2008). In contrast, an SPR biosensor assay developed for a wild-type, nonthermostabilized GPCR approves the capability for the application of such screenings to solubilized and stable wild-type receptors. A study with a wild-type receptor, when full pharmacology is retained, increases the chances of any novel binding sites to be discovered (Navratilova et al., 2011). Therefore, application of fragment screening by SPR on a wild-type, nonthermostabilized receptor is a breakthrough, with which not only the direct measurement of the interaction of the receptor with its ligands will be measured but also potential hits will be identified with pharmacological integrity.

7. SPR FRAGMENT SCREENING OF FULLY FUNCTIONAL GPCRs More recently, wild-type double-tagged β2 adrenergic receptor (β2AR) was used as a model system for the development of SPR-based biosensor assays for nonthermostabilized GPCRs (Aristotelous et al., 2013). Such a well-studied receptor represents a valuable candidate for the development of novel assays for GPCRs. β2AR has been the most thoroughly

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investigated receptor in the family of GPCRs and has been used as a model system for the development and application of a variety of techniques. It was the first to be cloned and sequenced (Dixon et al., 1986), to be the subject of crystallography (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007), virtual screening (Kolb et al., 2009), and study of the complex with G-protein (Rasmussen, DeVree, et al., 2011). The receptor has extensively been under study for many decades and that is the reason why the expression and purification protocols have been optimized, giving fractions of active and fully functional receptor. Working with a receptor so well studied can be the starting point for applying any novel approaches to many other GPCRs and to further extend to other membrane proteins. As a proofof-concept, the SPR assay for β2AR was developed followed by fragment screening. As with any technique or set of experiments a key point to a reliable set of data is the assay development. In the case of SPR, many aspects can contribute to the reliability of the data set as well as the appearance of the sensorgrams; from both the maintenance and performance of the biosensor instrument to components of the running buffer. The importance of the assay buffers has been demonstrated over the years by users of SPR (Giannetti, 2011). The buffer components can contribute to both the target’s stability and activity once immobilized or captured on the sensor chip. The activity of a target is ideally assessed by screening against known ligands, for example, substrates or inhibitors. Literature values of the corresponding affinities of the ligands tested are always useful to evaluate reliability of the SPR assay. Therefore, the choice of the ligands used during assay development should be made wisely. When working with membrane proteins one of the most important buffer components is the detergent. Many detergents differ in nature and percentage of their critical micelle concentration. Each membrane protein can respond differently to the presence of detergents (Rich, Miles, et al., 2009). Finding the detergent that works best is usually time-consuming but careful selection can improve stability drastically. The receptor was expressed in baculovirus system, in particular Sf9 cells. A double-tagged construct of the human β2AR was designed for the expression. The construct exhibited a tag at either terminus, both facilitating purification of the expressed receptor. The N-terminus was engineered with a FLAG tag, while the C-terminus had a decahistidine (His-10) tag. The expressed receptor was solubilized and purified in either dodecyl maltoside (DDM) or Lauryl Maltose Neopentyl Glycol (MNG) detergent as described previously (Kobilka, 1995). The presence of a histidine tag on the receptor


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facilitated the study of the receptor by SPR on NTA chip via Nickel chelation. In the case of β2AR, the nature of the detergents that keeps the receptor stable and functional has been established during the purification trials, thus the same detergents were used in the assay buffer for the study of β2AR by SPR. Throughout the assay development the activity of the captured receptor was monitored by the binding of fenoterol (agonist) and alprenolol (antagonist). Since the receptor retains the “wild-type” 7-TM amino acid sequence, binding of both agonist and antagonist is feasible, unlike other studies using conformation-biased receptors (Robertson et al., 2011). As described above, it is a prerequisite to establish the activity of a captured protein on the chip and thus, pharmacology of the receptor was investigated by the binding of an agonist and antagonist. The receptor was found to be pharmacologically active binding both the ligands with affinities close to the reported literature values for the studies with affinities measured by radioligand binding. Alprenolol was injected at concentration series 0.46–111 nM and fenoterol was injected at concentration series 0.6 nM to 3.7 μM, in increasing concentrations from low to high. The affinity measured for fenoterol was KD ¼ 139 nM, whereas alprenolol revealed affinity of KD ¼ 790 pM, compared to the reported ki values ki ¼ 126 nM and 1 nM, respectively. The comparison to the literature values strengthens the confidence of the developed assay for the study of the receptor. The data suggested that immobilized β2AR on the SPR surface maintains its pharmacological properties. As a conclusion, in vitro drug screening of these receptors by SPR is practicable. Correct development of the SPR biosensor assay enabled the successful conduction of the first biophysical fragment screening against a wild-type, nonthermostabilized GPCR: human β2AR. The activity and stability of the receptor, both required for achieving a reliable fragment screening, were well assessed during the assay development stage. Fragment screening involved the screening of a small in-house fragment library composed of 656 fragments with average molecular weight (MW) of 187 Da (equating to 13 nonhydrogen (heavy) atoms). The fragment sizes varied between 94 and 341 Da. For the detection of small molecules binding to the target, high density of the receptor was required on the surface. The receptor was therefore immobilized at density around 10,000–11,000 RU on NTA chip in buffer composed of 50 mM HEPES (pH 7.4), 150 mM NaCl, 50 μM EDTA, 3% DMSO, 0.1% DDM, or 0.01% MNG. All the experiments were performed on Biacore T100 SPR biosensor and for increased stability of β2AR on the sensor chip the analysis temperature used was 10 °C.

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Typical example of fragment screening assay run using SPR is shown in Fig. 4. Initially, the fragments were screened at single concentration; 50 μM. In this way, only the fragments with potential binding profile were further confirmed at concentration series and false positives were possible to be excluded early in the procedure. Referencing is an important parameter in SPR. The rate of false positives is relatively high when proper referencing is not used in the screening. Working with low-molecular-weight small molecules is a challenge in terms of data reliability since being small the fragments tend to bind to many targets nonspecifically, especially when screened at high concentrations. For that reason, a negative control target surface was required. Such surface serves as a reference surface for the reduction of nonspecific binding and to further assist exclusion of false positives. The receptor’s orthosteric binding site was preoccupied by a slow-off rate compound BI-167107 before capture on a different flow cell in the same capture conditions as the active receptor. A blank nonactivated flow cell was used as further reference. The use of the negative control surface and the blank channel confers more reliable and easily interpretable data analysis as nonspecific binding can be detected. The negative control surface was the receptor itself but at the inactive state. This approach has been previously described in literature as an appropriate way of referencing in GPCR analysis by SPR (Navratilova & Hopkins, 2010). Potential hits were selected by inspecting the sensorgram of each fragment against all three surfaces: the active receptor, inactive receptor, and the blank channel, and the selected hits were subject of further confirmation. The data were double-referenced against both reference surface and blank injections collected throughout the screen. Out of the 656 fragments

Figure 4 Example of typical fragment screening data collected using SPR. (A) Fragments are injected at one concentration over sensor surface and binding response is collected. Overlay shows individual binding responses of fragments along with positive and negative control. Gray vertical line shows time point at which binding response is read for all fragment and data are then transformed in binding plot B. (B) The binding plot shows responses for all fragments including controls. Horizontal lines show area of possible hits from fragment screen.


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composing the fragment library screened 81 fragments were selected as potential hits. During the first confirmation round of potential hits, the fragments were screened at concentration series at 300 μM highest concentration and then concentrations were adjusted individually depending on the affinities. Five fragments were confirmed to be β2AR hits by SPR. These fragments were screened at threefold concentrations series in duplicates. The concentrations as well as the dissociation time studied varied depending on the fragment. Both DDM and MNG have shown to be appropriate for the extraction of β2AR from the membrane, thus the binding profile of the five confirmed hits was investigated in the presence of both detergents in order to monitor any differences. The fragment screening and confirmation of the hits was performed in DDM. For comparison, the fragments were screened at the same concentrations in the presence of MNG. The binding activities revealed minimal difference in affinities for both screening setups, showing that both detergents can result in functional receptor and that the fragments do not have any detergent preference.

8. CONFIRMATION OF SPR FRAGMENT HITS Extending the confirmation from in vitro to the cellular level, the affinities for the five fragments were measured in radioligand competition binding assays to not only confirm them as hits but also determine if they bind to the orthosteric binding pocket of the receptor. Functional assays further confirmed the fragments as β2AR binders but also characterized them as functional antagonists. The functional assays were conducted by the Lefkowitz laboratory at Duke University in North Carolina. All five fragments showed specific inhibition of [125]-cyanopindolol (CYP) with ki values close to the affinities observed by SPR. The data suggests that they all are orthosteric binders. Due to high similarity of β2AR binding pocket with β1AR, the fragments were tested against both receptors. Only fragment A showed around eightfold selectivity for β2AR over β1AR while the rest did not show much selectivity. Selectivity studies were enhanced by profiling screening of the five fragments against a whole panel of 27 GPCRs in the groups of serotonin, adrenoceptors, histamine, and dopamine receptors. The profiling screen was performed by the Roth’s laboratory at the University of North Carolina. Fragments A, B, and E showed off-target activity with ki below 1 μM only for three receptors. In particular, fragment A exhibited off-target activity against 5-HT2B (ki ¼ 407 nM, 23-fold selectivity), 5-HT2C (ki ¼ 965 nM, 55-fold selectivity), and H1 (ki ¼ 399 nM, 23-fold selectivity). Whether the ligands are agonists or antagonists was

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determined by G-protein coupling detection in cell-based signaling assays, in both cAMP level elevation and β-arrestin recruitment assays. None of the fragments activated G-protein signaling. Instead, they all inhibited isoproterenol-induced response in both cAMP production and β-arrestin recruitment assays, leading to the conclusion that the fragments are functional antagonists. The relative inhibition of these fragments corresponded to their affinities for the β2AR. All the fragments are novel β2AR ligands with 4-piperazine-quinoline scaffold showing high affinity for the receptor. This scaffold is similar to the fragment discovered for β1AR by Heptares (Christopher et al., 2013). It is proposed that the initial fragment hits with highest LE should be selected for further optimization (Keseru & Makara, 2009). Although fragment E has the highest LE, fragments A and B were the two selected for additional study and rounds of synthetic chemistry. The latter fragments were those showing the highest selectivity for β2AR. These two hits, particularly fragment A, exhibited high affinity and optimization would be a challenging task. However, fragment analogs with alternations of the functional groups would reveal the positions on the fragment that are responsible for the high affinity and the strong interactions in the binding pocket of the receptor. Thirtynine compounds in total were synthesized and screened at concentration series. The rest were screened at concentration series with the highest concentration adjusted to their affinities. From the analogs screened, it is concluded that the NH group at the 4-position of the piperazine ring is a major contributor to affinity.

9. CONCLUSION SPR biosensors have recently become very popular and necessary technology in drug discovery in both pharmaceutical industry and academia. Even though majority of manufacturers offer wide range of built-in assay wizards and analysis tools to help guide SPR users during experimental setup and simplify data analysis process, SPR technology still requires large amount of experience and accuracy in both assay preparation as well as data interpretation as any other biophysical technique in order to obtain trustable results. However by implementing all necessary requirements, this technology is showing high potential to be one of the most powerful tools in GPCR drug discovery.


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