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Development and Application of Biochemical Characterization Methods for Protein Structure Analysis
January 2021
Yoshihiko HIROZANE
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Development and Application of Biochemical Characterization Methods for Protein Structure Analysis
A Dissertation Submitted to
the Graduate School of Science and Technology, University of Tsukuba
in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Science
Doctoral Program in Biology
Degree Programs in Life and Earth Sciences
Yoshihiko HIROZANE
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Table of Contents
Abstract...4
Abbreviations... 7
General Introduction...9
Chapter 1 Identification and characterization of the thermostabilized GPR40 mutant protein Abstract... ...19
Introduction... 21
Materials and Methods...2 4 Results...3 1 Discussion...3 4 Chapter 2 Protein structure analysis by fluorescent probe based binding analysis for a comprehensive protein kinase Abstract...56
Introduction... ...57
Materials and Methods... ...60
Results... ...64
Discussion...65
General Discussion... ...86
Acknowledgements...91
References... ...93
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Abstract
In this research, I reported the details of biochemical approach platforms for protein structure analysis and their significance in studies of G-protein coupled receptors (GPCRs) and protein kinases. There are approximately 800 GPCRs in the human genome that are targets of approximately 25% known drugs in clinical use. GPCRs are responsible for recognizing unique ligands on the cell membrane and transmitting intracellular signals. In general, GPCRs are unstable after extraction from the lipid bilayer membrane using detergents. In addition, maintaining the activity of purified proteins is difficult owing to their flexibility and poor stability when solubilized from the cell membrane. Therefore, it is extremely challenging to analyze the structure of GPCRs as compared to other proteins. To address this issue, I have constructed a platform to obtain thermostable GPCR mutants and perform binding assays for these mutants and their ligands using liquid-chromatography mass spectrometry (LCMS) analysis. In this research, I focused on the discovery of a novel thermostabilized mutant G-protein coupled receptor 40 (GPR40) and its 12 novel thermostabilized mutations.
The highly stabilized GPR40 mutant includes four of these mutations. Using this four- point mutant, I successfully performed, for the first time, crystal structure analysis of GPR40 and fasiglifam, a GPR40 agonist. To discuss the generality of 12 amino acid variants with enhanced thermostability in GPR40, I aligned its amino acid sequence with that of other thermostabilized GPCR variants and analyzed the amino acid loci that could contribute to enhanced thermostability. Phylogenetic analysis confirmed the exact same amino acid locus as the thermostable mutation site in protease-activated receptor 2 (PAR2), which exhibits high sequence similarity with GPR40. Thus, the amino acid loci that contribute to thermostability are similar in amino acid sequence but are recognized
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by different ligands. In other words, identification of thermostable mutation sites from amino acid sequences may allow successful crystal structure analysis of GPCRs.
Protein kinases have been discovered in approximately 520 types of that in the human genome, most of which are potential therapeutic targets. Protein kinases are responsible for transmitting signals into cells to catalyze phosphorylation of target proteins using ATP as a substrate at the membrane and in the cytoplasm. Although the structures of many kinases are resolved by crystal structure analysis, it is extremely difficult to identify differences in the microstructure of the ATP-binding catalytic region because the amino acid sequences of the regions are often very similar between protein kinases.
I synthesized two novel fluorescent probes based on staurosporine, which binds to the ATP catalytic region of many protein kinases, as a ‘scale’ to measure differences in protein structure. I analyzed the affinity of these probes toward comprehensive protein kinases. The two fluorescent probes differed in the structure of 4'-methylamine as a secondary or tertiary amine. A saturation binding assay using 280 recombinant human protein kinases was performed. The results indicated higher affinity of the probe with secondary amine toward several kinases than that of the probe with tertiary amine. In the kinases that differed in the affinity toward two probes, their binding region of 4'- methylamine had a co-crystal structure with staurosporine. The kinase with higher affinity for the secondary amine probe could bind with the protein through electrostatic interactions. To characterize kinases with different affinities toward the two probes, I aligned the primary amino acid sequences of those that showed a more than two-fold difference in dissociation constants (Kd) for the two probes. The results indicated that the kinase with a hydroxyl group at the amino acid locus and interacting with the side chain tended to be highly selective for fluorescent probes with secondary amines.
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Conversely, most kinases selective for the fluorescent probe with a ternary amine had acidic amino acids. Considering the amino acid loci interacting with the 4'-methylamine of staurosporine in the main chain, basic amino acids were predominant in the kinases selective for secondary amines.
Protein structure analysis is an important technology that not only accelerates the understanding of protein function but also facilitates the efficient development of novel drugs. Structure analysis of GPCRs is very difficult owing to their low stability, thus hindering drug discovery research. In addition, it is challenging to create selective inhibitors for targeted protein kinases because the structure of the ATP catalytic region is very similar among protein kinases and may restrict structural analysis. I developed experimental biochemical platforms that complement general protein structure analysis and succeeded in obtaining novel protein structural information that could not be derived by traditional methods. This research is extremely important not only for providing a new insight into protein structure but also for the development of efficient drugs.
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Abbreviations
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Abbreviations
Bmax maximum binding FFAR1 free fatty acid receptor 1
FACS Fluorescence-activated cell sorting GPCR G-protein coupled receptor
GPR40 G-protein coupled receptor 40 Kd dissociation constant
LC-MS liquid chromatography mass-spectrometry PAR2 protease-activated receptor 2
SEC size exclusion chromatography Tb terbium
TR-FRET time-resolved fluorescence resonance energy transfer TM transmembrane region
Tmapp apparent melting temperature VLP virus-like particle
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General Introduction
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General Introduction
Proteins are biological macromolecules that perform important functions by serving such as enzymes, antibodies, receptors, and signal transmitters. Proteins are composed of amino acids, the sequence of which is specified by the gene encoding specific protein (the primary structure) [1]. During protein synthesis, genetic information is copied in the nucleus and transcribed into mRNA. The mRNA is then transported to the
cytoplasm, and serves as a template on which amino acids bound to a ribosome are assembled and connected by a peptide bond to synthesize a polymer. Amino acid polymers are called polypeptides. Polypeptides interact between amino acids to form alpha-helix and beta-sheet structures through electrostatic interactions such as hydrogen and ionic bonds and hydrophobic interactions such as van der Waals forces to maintain the protein structure and function (secondary structure) [2]. Furthermore, polypeptide chains undergo folding into a specific form to produce a functional protein (tertiary structure). Depending on their types, proteins interact to form a complex structure, which allows them to perform specific functions (quadratic structure). Structural biology, which elucidates the tertiary and quaternary structures of proteins, is an extremely important research field that can be applied to understand biological phenomena and develop efficient drugs [3].
Methods for analyzing protein structures, such as X-ray crystallography, nuclear magnetic resonance (NMR), and more recently developed cryo-electron microscopy, are well known [4]. Information on protein structure is available in the Protein Data Bank (PDB) database (http://www.rcsb.org). As of October 2020, 166,569 different protein structures have been registered in the PDB database, of which 148,451 were determined by X-ray crystallography (Figure 1). Approximately 90% of the total protein structure
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information in PDB database has been obtained by X-ray crystallography. Nevertheless, NMR and cryo-electron microscopy techniques are gaining popularity in recent years, X-ray crystallography still accounts for the majority of protein structure analysis [5].
Protein X-ray crystallography was first introduced in 1958 to determine the
conformation of myoglobin [6]. The schematic procedure of X-ray crystallography for protein structure analysis is shown in Figure 2 [7]. First, recombinant proteins
overexpressed in Escherichia coli or cultured cells are obtained at high purity. Purified protein molecules adopt the same conformation and form protein crystals, wherein adjacent molecules are appropriately arranged under the crystal growth condition. When the protein crystal is irradiated with X-rays, the atoms in the protein molecule scatter the incident X-rays and exhibit a diffraction pattern, which varies with the structure of the protein. The three-dimensional structure of proteins is determined by analyzing the diffraction data. It was recently understood that X-ray crystal structure analysis cannot be adapted for some types of proteins such as those that are difficult to purify. Protein X-ray crystal structure analysis requires a large amount of highly purified protein (depending on the type of protein, tens of milligrams of purified protein). Therefore, proteins that are less stable upon purification cannot be prepared to retain the correct structure. Such proteins are generally expressed in low amounts in in vitro protein expression systems. This limits preparation of protein samples of good quality and quantity for crystal structural analysis. The most prominent examples are a group of membrane proteins such as G-protein coupled receptors (GPCRs), ion channels, and transporters [8] [9]. Membrane proteins are embedded in the lipid bilayer of the cell membrane and mostly perform intracellular and extracellular signaling functions [10].
Membrane proteins are stable in the lipid bilayer; hence, the transmembrane region of
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the proteins is characterized with lipid-soluble properties. When membrane proteins are isolated and purified from the lipid bilayer, they often aggregate with each other and become insoluble [11]. Hence, the crystal structure analysis of membrane proteins is less successful than that of solubilized proteins (Figure 1). Another problem with X-ray crystallography is that despite the availability of protein structural information, the structure of the region of interest is very similar among some proteins. For example, enzymes that use the same biomolecule as a substrate tend to be very similar in their substrate recognition regions. In such cases, high-resolution protein structural
information can be obtained, but distinctive structural information may be difficult to achieve. One reason is that the results of protein crystal structure analysis excise one conformational snapshot that is stable in the protein crystal but not the dynamic structure of the protein in an aqueous solution.
GPCRs are responsible for cell signal responses to neurotransmitters, hormones, and ambient conditions such as olfaction, light, and taste. In the human genome,
approximately 800 different GPCRs are known that form the largest protein superfamily [12]. GPCRs are embedded in the lipid bilayer of the cell membrane and function to transmit specific signals into the cell, mainly by recognizing unique ligands outside the cell [13]. GPCRs have been classified into six classes, class A, B, C, D, E, and F, based on similarities in amino acid sequences and their functions [14]. The structure of GPCRs is divided into extracellular region, intracellular region, and transmembrane region. The N-terminus amino acid is located in the extracellular region and penetrates the cell membrane seven times as an alpha-helix structure, while the C-terminus amino acid is located in the intracellular region. The majority of GPCRs have a ligand-binding region in the extracellular region of the bundle structure created by this transmembrane
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alpha-helix. Binding of a signaling molecule to the ligand-binding region results in structural changes in the transmembrane alpha-helix region, which in turn changes the structure of the intracellular region [15]. This structural change triggers the recruitment of the trimeric G-protein, which initiates the transmission of information into the cell.
To perform GPCR crystal structure analysis, it is necessary to purify proteins from the lipid bilayer while retaining the above structure and then solubilize them in artificial detergents [16]. In addition, it is necessary to prepare large quantities of purified proteins that retain their structures.
These challenges precluded the determination of GPCR structures until 2000, when the structure of bovine rhodopsin was solved [17]. Later, it took another 7 years to solve the structure of the adrenergic receptor b2AR, the first human GPCR with a diffusible ligand [18]. Since then, 488 different structures of GPCRs have been registered in PDB to date. Many platforms have been developed to prepare and efficiently crystallize large quantities of purified GPCRs. Partial truncation of the amino acid sequence or
introduction of point mutations in GPCRs have been performed to improve protein expression, increase its solubility and crystallization efficiency, crystallize the
complexes of GPCRs with the Fab domain of antibodies stabilizing target GPCR, and prepare GPCRs with T4-lysozyme protein inserts in the intracellular region and facilitate the formation of a three-dimensional reticular structure of the lipid bilayer (lipidic cubic phase method). In recent years, many successful examples of GPCR crystal structure analysis have been reported with the combination of these strategies. I particularly focused on the methods to improve the thermostability of GPCRs through the introduction of point mutations. Crystal structure analysis of GPCRs using
thermostable mutants was first performed for Turkey beta 1 androgenic receptor [19].
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However, this method uses a radioisotope-labeled ligand-based binding assay to measure thermostability. Such assays are expensive. Subsequently, a novel platform to discover new thermostabilized mutant GPCRs using surface plasmon resonance (SPR)- based protein-ligand binding assay was developed [20]. However, it is impossible to adapt this method for all GPCRs because it is difficult to discover thermo-stabilized mutants unless wild-type GPCRs can be purified for SPR. These problems are the bottlenecks in the application of GPCR crystal structure analysis to applied research, including drug discovery research. To solve this problem, it is necessary to develop a platform to obtain thermostable mutants of GPCRs that do not require ligand labeling or purification.
Protein kinases belong to a family of proteins that add phosphate groups to target proteins. In general, protein activities, localization, and functions are controlled by phosphorylation and dephosphorylation; hence, protein kinases play integral roles in signal transduction, cellular processes, maintenance of cellular homeostasis, and communication. Furthermore, approximately 520 protein kinases are known in the human genome and these are the second largest protein superfamily after GPCRs [21].
Therefore, protein kinases are considered as potential therapeutic targets for many diseases [22]. In principle, protein kinases are classified into those that phosphorylate serine and threonine residues (STK family) or tyrosine residues (TK family) of target proteins [21]. There are diverse subcellular distributions of cytoplasmic and receptor- type kinases on the cell membrane as well as of tissue-specific kinases throughout the body. Despite the high diversity of protein kinases, their structure at the ATP-binding region is similar because almost all protein kinases recognize ATP and the target protein as substrates and catalyze phosphorylation reaction.
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As described above, protein kinases play important roles in cell signaling pathways.
To date, more than 30 small molecule kinase inhibitors have been approved by the Food and Drug Administration (FDA), and there is no doubt that protein kinases have a considerable value as targets for drug discovery to treat various disease [23]. Many types of small-molecule kinase inhibitors bind to the ATP-binding region, inhibit ATP binding to the kinase, and prevent protein signaling [24]. However, multiple inhibition of protein kinases induces toxicities in organisms; therefore, it is extremely important to discover selective inhibitors of target kinases. The structures of many types of protein kinases have been solved by crystal structure analysis with the aim of creating selective inhibitors. On the other hand, the high similarity in the ATP-binding pocket across kinase families has posed a challenge for the development of such inhibitors.
Crystal structure analysis of protein kinases is often less difficult than that of GPCRs because it mainly requires preparation of purified proteins with truncated sequences near the ATP-binding region. The ATP-binding regions are often very similar among protein kinases, making it almost impossible to design selective inhibitors by simply analyzing their crystal structures. One of the reasons for this is that the protein structural information obtained from crystal structures is a snapshot of ridged protein formation and that it is impossible to analyze the fluctuations in protein structure in an aqueous solution. Therefore, the only way to gain a detailed understanding of the structure of protein kinases in the ATP-binding region is by obtaining high-resolution structural information on co-crystals with various ligands and compare them with each other.
However, the research flow is very time-consuming and expensive.
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In this thesis, I have focused on the structural analysis of GPCRs and protein kinases using novel biochemical analysis platforms. In the first chapter, I describe the analysis of GPCR thermostable mutants from the perspective of the biological function of GPR40. Furthermore, the general features of the amino acid loci among thermostable mutant GPCRs evaluated by amino acid sequence alignment and phylogenetic analyses are described. In the second chapter, I discuss the biochemical analysis of structural differences in the ATP catalytic region of protein kinases using novel designed fluorescent probes. The results of these studies provide an insight into the structural biology of these proteins that could not be elucidated using existing platforms.
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Figures (a)
(b)
Figure 1 Number of registered protein structure in PDB as of November 2020.
(a) Comparison of registered numbers in PDB by protein structure analysis method. (b) Comparison of registered numbers in PDB by protein type.
149066
11726 6138 585
0 20000 40000 60000 80000 100000 120000 140000 160000
X-lay
diffraction Solution NMR Electron
microscopy Others
Number of registered protein structure in PDB
3691
167361
0 20000 40000 60000 80000 100000 120000 140000 160000 180000
Membrane protein Others
Number of registered protein structure in PDB
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Figure 2 The outline of experimental and analytical flow of X-ray crystal structure analysis.
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Chapter 1 Identification and characterization of the thermostabilized GPR40 mutant protein
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Chapter 1 Identification and characterization of the thermostabilized GPR40 mutant protein
Abstract
Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein- coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify
thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles (VLP) was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam. In this research, twelve kinds of amino acid loci that are contributed for increasing the thermo-stability, and the thermo-stabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.
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Introduction
Membrane proteins are important drug targets due to their wide variety of biogenic roles. One of the largest membrane protein superfamilies is the G-protein-coupled receptors (GPCRs), which are the targets of approximately 25% of all known drugs in clinical use [12]. In recent years, structural insights into GPCRs that have revealed the mechanism of receptor activation and its ligand binding have successfully been used for structure-based drug discovery [25] [26] [27]. GPCRs generally become unstable when extracted from the lipid bilayer membrane by detergent. In addition, maintaining the activity of the purified proteins is difficult because of their flexibility and poor stability when solubilized from the cell membrane. One of the main characteristics of GPCR proteins is the equilibrium between the active and inactive states [28]. These
characteristics of GPCRs make X-ray crystallographic studies challenging compared with soluble proteins. Nevertheless, determination of the crystal structure of membrane proteins such as drug-targeted GPCRs has become feasible through the use of recent protein engineering techniques [27]. Increasing their thermostability by introducing point mutations is one such technique, which has substantially contributed to the
elucidation of several GPCR protein structures [19] [20] [29] [30] [31]. It is known that increased thermostability of GPCRs tends to decrease their flexibility [28] [32].
Nevertheless, protein crystal structure analysis using thermostabilized mutant GPCRs has improved our understanding of structural aspects of mechanisms of GPCR
activation and ligand binding modes [33] [34] [35] [36]. As the methods for selecting thermostabilized mutants for crystallographic studies reported so far require solubilized membrane protein samples, universal application of this technique to GPCRs is often hindered by a low expression level and protein instability of the wildtype. Existing
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methods generally require a radiolabeled ligand binding assay, which is a major hurdle for many GPCRs due to the unavailability of high-affinity radiolabeled ligands for the receptor. These limitations can be resolved by combining a size-exclusion
chromatography/liquid chromatography mass spectroscopy (SEC/LC-MS)-based binding assay with transient overexpression in mammalian virus-like particles (VLP) [37] [38] [39]. A major advantage of this approach is that the VLP system does not require solubilization of the membrane fraction (Figure 3). Furthermore, the expression of the target membrane protein is higher than that of proteins prepared from the
membrane fraction. The SEC/LC-MS-based binding assay is a well-established technique that reveals the interaction between the ligand and the protein of interest (Figure 4) [40] [41] [42]. This technique has potential for broader use mainly because it does not require a radiolabeled ligand. Here, I report that this approach can produce a thermostabilized GPCR that is suitable for crystal structure analysis. As a case study, I examined G-protein-coupled receptor 40 [GPR40; also known as free fatty acid receptor 1 (FFAR1)]. GPR40 is activated by medium and long chain free fatty acids and
mediates free fatty acid (FFA)-induced glucose-dependent insulin secretion from pancreatic b cells (Figure 5) [43]. A GPR40-selective agonist, fasiglifam, was
demonstrated to have robust glucose-lowering effects in patients with type 2 diabetes mellitus (Figure 5) [44] [45] [46] [47] [48] [49]. Recent research has indicated that fasiglifam acts as an ago-allosteric modulator of GPR40 [50]. However, the binding mode of fasiglifam to GPR40 is not fully understood. For better understanding of its binding mode by crystal structure analysis, thermostabilized mutant GPR40 constructs were generated using the SEC/LC-MS-based binding assay combined with a
mammalian VLP system. I also determined agonistic activities and affinities of GPR40
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ligands compared with selected thermostabilized mutants to explore the relationship between ligand binding affinity and receptor function. In addition, thermo-stabilized GPCRs for crystal structure analysis have been reported in 26 different human GPCRs to date (Table 1). These researches have been conducted almost entirely as independent studies, so with little discussion of the generality of the amino acid loci that contribute to thermal stabilization. Several efforts to estimate the thermo-stabilized amino acid loci of GPCRs by machine learning based on the reported amino acid loci that contribute to thermal stabilization, but the accuracy is quite low [51]. I aligned the amino acid sequences of the GPCRs with reported thermo-stabilized variants and estimated the generality of amino acid loci contributing to thermo-stabilization in GPCRs with high and low sequence homology using GPR40 as a reference.
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Materials & Methods Materials
Fasiglifam was synthesized at Takeda Pharmaceutical Company Limited (Kanagawa, Japan). Docosahexaenoic acid (DHA) was purchased from Tocris Bioscience (Bristol, UK).
Mutagenesis
GPR40 was amplified from genomic DNA using a standard polymerase chain reaction (PCR) method and cloned into pcDNA3.1. The human GPR40 expression vector
pcDNA3.1/hGPR40 was used as a template for in vitro site-directed mutagenesis. Point mutant vectors were created using an In-Fusion® Advantage PCR Cloning kit
(Clontech Laboratories) [52]. First, pcDNA3.1 was linearized with the restriction enzymes NheⅠ (Takara Bio Inc.) and PmeⅠ (BioLabs Inc.). Second, to generate the mutation, DNA fragments were generated by PCR using pcDNA3.1/GPR40 as the template. Mutagenic sense and antisense primers were designed as 5’-6 mer-XXX-20 mer-3’, where XXX represents the amino acid codon of the desired mutation. The primer pairs overlapped by 15 bp at their 5’ ends. A sense primer overlapping with the NheⅠ restriction site was designed as
5’-ACCCAAGCTGGCTAGATGGACCTGCCCCCGCAGCT-3’, and an antisense primer overlapping with the PmeⅠ restriction site was designed as
5’-CTGATCAGCGGGTTTTTACTTCTGGGACTTGCCCCCTTGCGT-3’. Pairs of DNA fragments – a DNA fragment from the sense primer with mutagenic antisense primer, and a DNA fragment from the antisense primer with mutagenic sense primer – were generated by PCR. Finally, the generated DNA fragments pairs were joined with
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linearized pcDNA3.1 by In-Fusion. In-Fusion samples were transformed into
ECOScompetent E. coli JM109 (Nippon Gene), and full-ORF sequences of individual clones were verified using a Genetic Analyzer (Life Technologies). Residues predicted as N-terminal, C-terminal, and intracellular loop regions were excluded from the
mutagenesis study for the below reasons. First, these regions were used to fuse proteins, such as T-4 lysozyme and BRIL and to increase protein hydrophilicity and stability for crystal structure analysis [18] [53]. Second, based on previous literature, the mutation sites effective in increasing GPCR thermostability may be difficult to find in these regions [54]. A total of 265 mutated GPR40 expression vectors were prepared by changing amino acid residues to alanine; ordinal alanine was changed to valine. In addition, tyrosine was changed to alanine and phenylalanine based on in-house mutagenesis data from the compound binding assay.
Protein expression
FreeStyle293 (Life Technologies) cells were used to express wild-type and mutant GPR40 constructs in VLPs. FreeStyle293 cells were maintained in culture in
FreeStyle293 expression medium (Life Technologies) with 1 mg/ml of gentamycin in an 8% CO2 incubator with shaking at 37 ℃ and were passaged twice weekly. The GPR40 expression vector and HIV-1 GAG protein expression vector were co- transfected into FreeStyle293 cells using NeoFectin reagent (Astec) according to the manufacturer’s instructions. The cells were incubated in an 8% CO2 incubator with shaking at 37 ℃; 48 h after transfection, cells were centrifuged at 500 g for 5 min, and supernatants were pooled. The supernatants containing GPR40-expressing VLPs were centrifuged at 32,000 g for 60 min. The supernatant was discarded, and the pellet was
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resuspended in dilution buffer [50mM Tris–HCl (pH 7.5) 5mM EDTA, and Complete Protease Inhibitor Cocktail (Roche Diagnostics)]. Diluted samples were stored at - 80 ℃. Using this protocol, I obtained about 9 mg of GPR40-expressing VLPs from 2.5
×108 cells. To prepare GPR40 expressed from the cell membrane, the GPR40
expression vector was transfected into FreeStyle293 cells and cultured under the same conditions as those used for VLP preparation. Cultured cells were centrifuged at 500 g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in distilled water with Complete Protease Inhibitor Cocktail and centrifuged at 32,000 g for 60 min to remove cytoplasmic proteins. After the supernatant was discarded, the pellet was resuspended in dilution buffer and homogenized on ice. The homogenized sample was centrifuged at 6,000 g for 30 min, and a supernatant sample was collected as the cytoplasmic membrane sample.
Saturation binding experiments using the SEC/LC-MS-based binding assay
The binding affinities of fasiglifam for GPR40 expressed by VLPs and cytoplasmic membrane samples were measured by the SEC/LC-MS-based binding assay. VLP samples were diluted to 3 mg/ml, and membrane samples were diluted to 50 mg/ml with the binding assay buffer [50 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 5 mM EDTA, and 0.005% Tween 20]. Diluted samples were dispensed into a 96-well plate. Fasiglifam in DMSO was added to the wells at final concentrations of 250, 100, 40, 16, 6.4, 2.5, 1.0, and 0.4 nM, and the plate was incubated at 4 ℃. Unbound compound in 25 ml of the reaction sample was separated by SEC based on a 96-well plate [41]. Subsequently, 50 ml of 70% acetonitrile/0.2% formic acid was added to each well to dissociate the bound compound from the receptor protein. The dissociated ligand was applied to a C18,
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3.0 mm i.d.×20 mm column (Intakt) by ultrafast liquid chromatography (Shimadzu), followed by identification and quantification with an API5000 electrospray-ionization mass spectrometer (AB SCIEX). The apparent dissociation constant (Kdapp) value and maximum binding (Bmax) of fasiglifam were calculated using the one site-specific binding curve equation of Prism 5.03 software (GraphPad Software). Data points are from at least quadruplicate measurements.
Thermostabilized mutant screening
Wild-type and mutant GPR40-expressing VLPs were diluted with assay buffer [10 mM Tris–HCl (pH 7.5), 5 mM EDTA, 5 mM MgCl2, 0.005% Tween 20] to 20 mg/ml in the presence of 100nM fasiglifam (assay format with fasiglifam, hereafter called format A) or in the absence of fasiglifam (assay format without fasiglifam, hereafter called format B). After incubation at 4 ℃ for 30 min, samples were again incubated at three different temperatures for 30 min. Format A samples were incubated at 4, 41, and 55 ℃ and format B samples were incubated at 4, 38, and 55 ℃. After the heating process, samples were immediately placed on ice for 5 min. Fasiglifam (100 nM) was added to the format B samples, which were further incubated at 4 ℃ for 30 min. Receptor activity remaining after heat treatment was assessed by measuring the amount of receptor-bound fasiglifam using the SEC/LC-MS-based binding assay. Before thermostabilized mutant screening, I determined the apparent melting temperature (Tmapp) value of wild-type GPR40 in formats A and B (details of the method are given in the next section). Then, the screening temperature was set based on each apparent Tm
value.
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Tmapp value measurements
The Tmapp value of the receptor was determined by measuring the amount of receptor- bound fasiglifam using the SEC/LC-MS-based binding assay with eight-point heating temperature under format A and B conditions. The Tmapp value was calculated by nonlinear regression analysis using the dose–response curve equation in Prism 5.03 software.
Ca2+ flux assay (FLIPR)
Human Embryonic Kidney 293T (HEK293T) cells were seeded in a 25 cm2 flask at 1.5 ×106 cells/flask and incubated overnight in 5% CO2 at 37 ℃. The next day, GPR40 expression vector was transfected into HEK293T cells using Fugene® HD Reagent (Promega) according to the manufacturer’s instructions. The cells were seeded 1 day after transfection and assayed the next day. Cells were seeded onto poly-D-lysine- coated 384-well plates (BD) at 15,000 cells/well and incubated overnight in 5% CO2 at 37 ℃. Cells were incubated in loading buffer [Hanks’ buffered saline solution
supplemented with 20mM HEPES (pH 7.5), 2.5 mg/ml fluorescent calcium indicator Fluo4-AM (Dojindo Laboratories), 2.5 mM probenecid (Dojindo Laboratories) and 0.01% fatty acid-free BSA (Sigma Aldrich)] for 60 min at room temperature. Various concentrations of fasiglifam or DHA were added to cells, and the increase in
intracellular Ca2+ concentration was monitored using the FLIPR Tetra system (Molecular Devices) for 90 sec. Agonist-stimulated responses in wild-type GPR40, thermostabilized mutant GPR40, and pcDNA3.1 vector-transfected cells as a negative control were determined as the maximum value minus the minimum value after subtracting the baseline response. The dose-response relationship was analyzed by
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fitting to sigmoid dose-response curves. The agonist EC50 values were calculated using Prism 5.03 software. Data points were from quadruplicate measurements.
Fluorescence-activated cell sorting (FACS) analysis
HEK293T cells were seeded in six-well plates at 6.0×105 cells/well and incubated overnight in 5% CO2 at 37 ℃. Next day, GPR40 expression vector was transfected into the cultured cells using Fugene® HD (Promega) reagent according to the
manufacturer’s instructions. After 48 h of incubation, cells were harvested with Cell Dissociation Buffer, enzyme-free, PBS-based (Life Technologies). After the PBS wash, cells were diluted to 1.0×107 cells/well in 50 ml of FACS assay buffer [TBS (pH 7.4) with 2% FBS]. Allophycocyanin (APC)-conjugated anti-FLAG Surelight antibody (Perkin Elmer) at 10 mg/ml was added on ice for 30 min, followed by washing with 1ml FACS assay buffer and centrifuging at 500 g for 5 min. The supernatant was discarded, and samples were resuspended in 500 ml of FACS assay buffer and filtered prior to analysis of the mean values of APC area signal using FACSAria II (BD). Data points are from triplicate measurements.
Amino acid sequence alignment and phylogenetic analysis of GPCRs
The amino acid sequences of 27 human GPCRs with known thremo-stabilized
mutations, including GPR40, were downloaded from the Uniprot database (uniprot.org).
Sequences with the exception of 5 GPCRs which are different GPCR class were
discarded and 22 class A GPCR sequences were examined for the comparative analyses using SeaView package version 5.0.4 [55] [56]. The GPCRs were aligned using Muscle program. Unambiguously aligned amino acid positions were selected by the use of
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Gblocks program and resultant 133 positions were subjected for phylogenetic analysis.
The maximum likelihood (ML) tree was inferred using PhyML program with the LG+
F+4 model for amino acid substitution process. A hundred bootstrap replicates were produced and used for non-parametric bootstrap analysis to evaluate the reliability of each internal branch of the ML tree.
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Results
Screening of thermostabilized mutant
A total of 265 single-point mutants of GPR40 were expressed in VLPs. The thermostabilities of the mutants were determined under format A and B conditions (Figure 6). In the mutants that exhibited significantly lower ligand binding at 4 ℃ compared with the wild-type, the reduced binding resulted from a lower expression level or reduced ligand binding affinity. The mutants in which the signal level was
>50% lower compared with the wild-type were excluded from the subsequent thermostability analysis because they were not suitable for crystal structure analysis.
Thermostabilized mutants were selected based on the percentage of remaining binding signal, which was calculated as follows:
% of remaining binding signal =
100 ×(binding signal at screeing tempurture − bining signal at 55℃) (binding signal at 4 ℃ − binding signal at 55℃)
The percentage of remaining binding signal in the wildtype was 58% under the format A condition and 53% under the format B condition. These results were obtained by averaging six independent experiments. The percentages of remaining binding signal in the prepared mutants were determined, and thermostabilized single-point mutants were selected using the following criteria: The percentage of activity was greater than +2 SD of the percentage of activity in the wild-type (84% under format A and 74% under format B).
32
Tmapp determination for thermostabilized mutants
To confirm the thermostability of selected single-point mutants, Tmapp values were measured for the selected mutants, and some single-point mutants showed increased thermostability (Table 2). Assuming that combining most stable single-point mutants could further increase thermostability, I conducted multiple rounds of mutation
combinations that generated a four-point mutant GPR40 (L42A/F88A/G103A/Y202F).
The Tmapp value of the four-point mutant was increased by approximately 17 ℃ when compared with that of the wild-type (Figure 7).
Binding affinity of fasiglifam for wild-type GPR40 and thermostabilized mutants The binding affinities of fasiglifam for the wild-type, thermostabilized four-point mutant, FLAG-tagged wild-type, and FLAG-tagged thermostabilized four-point mutant of GPR40 expressed in VLPs and cytoplasmic membrane were determined using the SEC/LC-MS-based binding assay. Total compound binding in the presence of GPR40 was measured, while non-GPR40-expressing VLPs were used to estimate non-specific binding. Specific binding was calculated by subtracting nonspecific binding from total binding. Wild-type GPR40 and the thermostabilized four-point mutant GPR40 had Kdapp
values of 11.5 and 10.9 nM, respectively (Figure 8), which confirmed that insertion of these point mutations into the GPR40 receptor retains the ligand-binding properties of the wild-type receptor. Kdapp values of fasiglifam and Bmax of the GPR40 expressed by VLPs and cytoplasmic membrane are shown in Table 3.
Agonist activity of fasiglifam for wild-type and thermostabilized four-point mutant GPR40 and cell surface receptor expression level
33
Three (L42A, G103A, and Y202F) of four thermostabilized point mutations were close to the intracellular face of the transmembrane helices, and it is an intriguing question whether these mutations hinder the downstream signaling of GPR40 receptor. The effect of mutations on Ca2+ signaling was studied in the presence of both fasiglifam and DHA for FLAG-tagged wild-type and the four-point thermostabilized mutant GPR40 (Figure 9). The thermostabilized mutants appeared to decrease Ca2+ signaling while maintaining tight ligand binding. FACS analysis revealed that the thermostabilized four- point mutant had a higher expression level than the wild-type (Figure 10).
Phylogenetic analysis of GPCRs
Phylogenetic tree of GPCRs with known thermo-stabilized mutants was inferred by ML method (Figure 11). The amino acid loci that contribute to thermal stabilization are plotted on the amino acid sequence alignment (Figure 12). Closely related sequences in the phylogenetic tree (Figure 11) with bootstrap support values > 80 are combined by brackets. It is confirmed that the same or near amino acid loci tended to contribute to thermal stabilization within closely supported groups.
34
Discussion
I developed a versatile assay platform to obtain thermostabilized GPCRs. I selected a total of 13 single-point mutants showing an increase of 41 ℃ in thermostability (Table 2). It is noteworthy that several single-point mutants gave a shift of 45 ℃. Combining these mutations resulted in a further increase in thermostability. Our protocol provides a high-throughput parallel preparation of thermostabilized mutants because of the
miniaturized format, which takes advantage of VLP expression and label-free ligand.
GPCR expression in VLPs is superior to expression in the cytoplasmic membrane, particularly in terms of throughput and receptor density per unit of total protein. The use of VLPs transiently expressing the target GPCR streamlines the process by eliminating the membrane solubilization step. Regarding receptor density per unit of total protein, a higher density of VLP expressing GPR40 can be obtained when compared with
FreeStyle293 cell membranes, resulting in lower non-specific binding signals.
Moreover, cell surface receptor expression levels of thermostabilized mutants also increase when compared with those of the wildtype. As large amounts of the receptor protein are required for crystal structure analysis, the VLP expression system may be a good choice for binding assay-based screening of thermostabilized mutants. The SEC/LC-MS-based binding assay technique also offers the advantage of using label- free compounds to evaluate binding affinity and to identify single-point
thermostabilized mutations that can be combined, resulting in a thermostabilized receptor suitable for crystallization. It has been suggested that a unique set of thermostabilized mutants are required for each ligand of interest due to potential conformational changes in the receptor on ligand binding [30] . This was confirmed by measurement of thermostability under two conditions and the agonistic activity of the
35
two ligands fasiglifam and DHA. I determined the thermostability of GPR40 under two conditions: Format A and format B. The thermostability of the active-state receptor was determined under the format A condition. The active state is generally unstable
compared with the inactive state [57]. I anticipated different thermostabilized mutants between formats A and B because of the difference in receptor stability between the screening conditions. However, thermostabilized receptors that shared the same
mutation sites were selected by screening under conditions A and B. The Tmapp value of the wild-type under format A was 3 ℃ higher than that under format B, implying the binding of fasiglifam, known to be an ago-allosteric modulator of GPR40, to the receptor in the intermediate active state conformation of GPR40 and only a small
contribution to the stability of GPR40. The FLIPR assay revealed that the Ca2+ signaling of fasiglifam decreased over 1000-fold for the four-point thermostabilized mutant, with little impact on binding affinity. On the other hand, Ca2+ signaling of DHA, a natural full agonist, decreased only approximately 40-fold. These results indicate that
fasiglifam does not alter the conformation of the four-point mutant GPR40 to the active state. Therefore, the four-point thermostabilized mutant GPR40 is suitable for crystal structure analysis with fasiglifam. In the recent research of the group including me, this fourpoint mutant in combination with a T4 lysozyme fusion strategy was successfully used to determine the crystal structure of GPR40 bound to fasiglifam [58] . However, for crystal structure analysis, it may be necessary to decrease receptor flexibility in the full agonist-bound state.
Each of the GPCR groups in the upper and lower halves of Figure 12 forms a monophyletic group (bootstrap value = 81). In general, a tendency to show similar thermostabilized mutant amino acid loci was observed within each of the two separate
36
groups. Especially, the results on transmembrane region 3 (TM3) showed that GPCRs close to each other in the phylogenetic tree analysis tended to be stabilized by
mutagenesis at similar amino acid loci, whereas transmembrane region 6 (TM6) tended to be stabilized by mutagenesis at similar amino acid loci, irrespective of the
phylogenetic position in the tree (Figure 12 and Table 4). It has been suggested that TM3 is responsible for transmitting the conformational changes caused by ligand binding to TM6. Therefore, GPCRs in close proximity in the phylogenetic tree have similar activating conformational changes due to ligand interactions, and therefore, I speculate that they have similar amino acid loci at which mutations are introduced to fix them in the inactive conformation.
On the other hand, TM6 is known to be a region with its conformation extremely changed during the inactive state to the active state of GPCRs and vice versa [15].
Assuming that the function of TM6 is conserved across GPCRs, it is suggested that the mutation introduction sites for anchoring to the inactive conformation are similar between the GPCRs. However, there are limitations to this analysis. The analysis of amino acid loci contributing to thermal stabilization used in this discussion was a meta- analysis, and the methods analyzed by each GPCR were different. Therefore, it should be noted that even if the phylogenetic tree analysis shows that GPCRs in close
proximity to each other do not contribute to thermal stabilization at the same amino acid locus, the data provided for the analysis may be insufficient.
Conclusions
A versatile assay platform that can be used to obtain a thermostabilized mutant GPCR for the purpose of crystal structure analysis was developed. Several thermostabilized
37
single amino acid mutants of GPR40 were identified using this platform. Combining these single-point mutations resulted in a further increase in thermostability in the four- point GPR40 mutant L42A/F88A/G103A/Y202F. The four-point mutant GPR40 had lower Ca2+ signaling for fasiglifam, whereas the mutant GPR40 did not show altered binding affinity for fasiglifam. These results indicate that the four-point
thermostabilized mutant was suitable for crystal structure analysis of the
GPR40/fasiglifam complex. In conclusion, it was demonstrated that this platform can be used to identify thermostabilized mutant GPCRs optimized for particular ligands, and further trials with the other membrane protein targets are under way.
38
Figures and Tables
Figure 3 Left; Schematic view of VLP expression system. HIV; Human
Immunodeficiency Virus, Gag proteins have structural domains involved in retrovirus particle formation. Right; VLP formation by electron microscopy [59].
39
Figure 4 Schematic view of SEC/LC-MS-based binding assay experimental flow.
40
(a)
(b)
Figure 5 (a) Schematic view of the function of GPR40 in beta pancreatic cell. GIP:
glucose-dependent insulinotropic polypeptide, GLP-1: glucagon-like peptide 1.
(b) Molecular structure of fasiglifam, GPR40 selective agonist.
O
O O
O S OH
O O
41
Figure 6 Snake plot of the amino acid sequence of human GPR40 showing the thermostabilized sites.
Blue, increase (%) in activity in the fasiglifam binding condition (G29, T31, L38, C127, A128, A129, W131, A132, L133, V134, E145, G149 and V225); yellow, increase (%) in activity in the compound-free condition (V64, A109, Y122, L135, A199, F200, G204, V237, G265, T286 and V287); red, increase (%) in activity in both conditions (L42, F88, G103, R104, Y202 and K285); gray, not tested or fasiglifam binding signal for the mutant < 25% of the signal for the wild-type; * binding signal of fasiglifam not determined for both tyrosine-to-alanine and tyrosine-to-phenylalanine mutants;
† binding signal of fasiglifam not determined for tyrosine-to-alanine mutant but
determined for tyrosine-to-phenylalanine mutant; ‡ increase (%) in activity for tyrosine- to-phenylalanine mutant; ¶ increase (%) in activity for both tyrosine-to-alanine and tyrosine-to-phenylalanine mutants.
42
Figure 7 Apparent melting temperature (Tmapp, ℃) of the wild-type and the thermostabilized four-point mutant of GPR40.
Binding signals at each temperature were normalized as percentages of the binding signal at 4 ℃. This experiment was performed in sextuplet. Data are expressed in means
±SEM and were fitted using the sigmoidal dose-response curve equation of Prism 5.03 software. Closed circles, the wild-type under format A condition; open circles the wild- type under format B condition; closed triangles, thermostabilized four-point mutant under format A condition; open triangles, thermostabilized four-point mutant under format B condition.
43
Figure 8 Fasiglifam binding affinity for the wild-type and the thermostabilized four- point mutant of GPR40.
Saturation binding curves of fasiglifam for the wild-type (a) and the thermostabilized four-point mutant (b) were determined by the SEC/LC-MS-based binding assay. The specific binding signal for the receptor (open triangles) was calculated as the difference between the total binding signal (solid circles) and the non-specific binding signal (solid squares). This experiment was performed in sextuplet Data are expressed in means±
SEM and were fitted using the one site-specific binding curve equation of Prism 5.03 software.
44
Figure 9 Agonistic activity of fasiglifam (a) and DHA (b) in wild-type (circles) and the thermostabilized four-point mutant of GPR40 (squares). The experiment was performed in quadruplicate. Data are expressed in means±SEM and were fitted using the dose- response equation of Prism 5.03 software. (c) Agonistic activity (EC50) of fasiglifam and DHA for the FLAG-tagged wild-type and FLAG-tagged thermostabilized four- point mutant of GPR40.
45
Figure 10 Transient expression level of wild-type and the thermostabilized four-point mutant GPR40 on the HEK-293T cell surface detected by FACS. The experiment was performed in triplicate. Data are expressed in means±SEM.
46
Figure 11 Phylogenetic tree analysis of GPCRs with known thermo-stabilized mutants.
47
Figure 12 Aligned amino acid sequences in transmembrane region, from TM1 to TM7, of GPCRs where thermo-stabilized mutants have been reported. In the left side of the alignment closely related sequences are combined by brackets with bootstrap values (80<) in the tree in Figure 11. Thermo-stabilized mutations are shown in the right side.
Red square: thermo-stabilized mutation amino acid locus TM1
TM2
48
TM3
TM4
49
TM5
TM6
50
TM7
51
Table. 1 Reported thermo-stabilized mutant human GPCRs for crystal structure analysis.
Protein name Full name Class Reference
GPR40/FFAR1 Free fatty acid receptor 1 Class A [58]
PAR2 Proteinase-activated receptor 2 Class A [60]
P2RY1 P2Y purinoceptor 1 Class A [61]
P2Y12 P2Y purinoceptor 12 Class A [62]
CCR9 C-C chemokine receptor type 9 Class A [63]
CXCR4 C-X-C chemokine receptor type 4 Class A [64] [65]
CCR5 C-C chemokine receptor type 5 Class A [66]
APJ Apelin receptor Class A [67]
EDNRB Endothelin receptor type B Class A [68] [69]
NTR1 Neurotensin receptor type 1 Class A [70]
OPSD Rhodopsin Class A [71] [72] [73]
DRD2 Dopamine D2 receptor Class A [74]
DRD3 Dopamine D3 receptor Class A [75]
5HT1B 5-hydroxytryptamine receptor 1B Class A [76]
ADRB2 Beta-2 adrenergic receptor Class A [77]
AA1R Adenosine receptor A1 Class A [78]
AA2AR Adenosine receptor A2a Class A [35] [79]
CNR1 Cannabinoid receptor 1 Class A [80]
LPAR1 Lysophosphatidic acid receptor 1 Class A [81]
5HT2B 5-hydroxytryptamine receptor 2B Class A [82]
5HT2C 5-hydroxytryptamine receptor 2C Class A [83]
CRFR1 Corticotropin-releasing factor receptor 1 Class B1 [84]
GLP1R Glucagon-like peptide 1 receptor Class B1 [85] [86]
GLR Glucagon receptor Class B1 [87]
GRM5 Metabotropic glutamate receptor 5 Class C [88]
SMO Smoothened homolog Class F [89]
52
Table. 2 Apparent Tm (Tmapp, ℃ ) values of wild-type and thermostabilized mutants of GPR40.
53
Table. 3 Kd values of fasiglifam and Bmax for wild-type and the thermostabilized four- point mutant of GPR40.
54
Table.4 Thermo-stabilized amino acid mutations in GPCRs for amino acid sequence alignment and phylogenetic tree analysis.
Protein TM1 TM2 TM3 TM4 TM5 TM6 TM7
GPR40 T31A
L42A, V64A
F88A, G103A, R104A
Y122F, E145A
A199V, Y202F, Y202A
V225A G265A
PAR2 G89A H108A
G157A, M166L, Y174A, V176E
M268A
I289A, L293A
P2RY1 D320N
P2Y12 D294N
CCR9 M82A S141C T216A V255A
N294A, T304A
CXCR4 L125W T240P
CCR5 C58Y G163N A233D
APJ V117A W261K
EDNRB R124Y D154A K270A S342A I381A
NTR1 A86L E166A I253A L310A V360A
OPSD T94I E113Q M257Y
DRD2 I122A L375A,
L379A
DRD3 L119W
5HT1B L138W
ADRB2 H93C E122W
AA1R A57L T91A Y205A
L236A L240A
T277A
AA2AR
L48A, D52N, A54L, T65A
T88A, Q89A, S91A, R107A
K122A L202A
L235A, V239A
S277A
CNR1 T210A E273K R340E
LPAR1 D204C V282C
5HT2B M144W
5HT2C C360N
55
Chapter 2 : Protein structure analysis by fluorescent probe based binding analysis for a comprehensive protein kinase
56
Chapter 2 : Protein structure analysis by fluorescent probe based binding analysis for a comprehensive protein kinase
Abstract
Selectivity profiling of compounds is important for kinase drug discovery. To this end, I aimed to analyze the protein structure of broad-range protein kinases using synthesized novel staurosporine-derived fluorescent probes based on staurosporine and performed to estimate broad-range protein kinases by time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Upon structural analysis of staurosporine with kinases, the 4'-methylamine moiety of staurosporine was found to be located on the solvent side of the kinases. However, reported fluorescent probes were suggested to reduce the binding affinities of the modified compound for several kinases, owing to the elimination of hydrogen bond donor moiety of NH-group from 4'-methylamine and/or steric hindrance by acyl moiety. Based on this structural information, I designed and synthesized a novel staurosporine-based probe without methyl group in order to retain the hydrogen bond donor, similar to unmodified staurosporine. The broad range of the kinase binding assay demonstrated that our novel fluorescent probe is an excellent tool for understanding the detail protein structure to interact with 4'-methylamine of staurosporine.
57
Introduction
More than 520 species of human protein kinases have been discovered thus far, most of which are potential therapeutic targets [21]. Protein kinases play integral roles in signal transduction, cellular processes, and maintenance of cellular homeostasis and communication. Structurally, protein kinases possess a highly conserved ATP-binding pocket that can be exploited for binding small compounds [22]. Owing to high
similarity of the ATP binding pocket across kinase families, development of selective kinase inhibitors is considerably challenging [90]. To overcome this challenge,
development of a high-throughput and wide-range protein kinase structure information is necessary. X-ray protein crystal structure analysis is essential platform to understand the three dimensional protein structure. However, it is not enough to understand the protein structure existing in the aqueous solution. For the purpose of the detail
information of the interaction with the amino acid residues and the ligands in aqueous solution, it is necessary to develop the complemental assay system with protein crystal structure analysis.
Fluorescent probes interacting with a wide range of protein kinases are an essential tool for the development of a wide-range protein kinase assay. Staurosporine (Figure 13a) is a natural product originally isolated from Streptomyces staurosporeus, and is well known as a wide-range human protein kinase inhibitor [91] [92] [93].
Staurosporine binds to the ATP-binding site of various protein kinases with similar orientation. Its lactam and indolocarbazole rings interact with hinge region and its 4'- methylamine moiety interacts with the hinge region and/or catalytic loop by a hydrogen bond and ionic interaction in the solvent accessible region [93] [94]. Because of these biological and structural characteristics, several chemical probes have been synthesized
58
based on staurosporine, conjugating it with a fluorescent core through a linker extended from the 4'-methylamine position (Figure 13b). Generally, conjugation of a fluorescent substance and a linker unit reduces the binding affinity of the resulting probe to the target protein compared with that of the parent compound, because of steric hindrance and change in the polarity of the compound. Staurosporine based chemical probes have been conjugated with a linker unit by alkylation or acylation utilizing the
nucleophilicity of its 4'-methylamine position [95] [96] [97] [98] [99]. However, these chemical probes had reduced binding affinity for several kinds of protein kinases
presumably due to the elimination of hydrogen bond donor moiety from 4'-methylamine moiety and/or steric hindrance by acyl moiety [94]. On the basis of these structural information, I designed a novel fluorescent probe (compound 8a, Figure 13b) that would retain hydrogen bond and ionic interaction of staurosporine with kinases by eliminating methyl group of other known staurosporine-based chemical probes. The compound 8a is predicted to retain its binding affinity for many types of protein kinases compared to the known staurosporine-based chemical probes. On the other hand, the binding mode of the proteins that have not been co-crystallized with staurosporine is completely unknown. As described above, because protein crystal structure analysis is a snapshot of proteins, it is difficult to determine exactly whether the ligand and the amino acid are interacting with each other in an aqueous solution, even if they are in close proximity to each other in structural information. Therefore, I developed a method for the synthesis of staurosporine based fluorescent probe in which the structure of the 4-methylamin of the probe has secondary amine as the same of staurosporine. In addition, I synthesized a fluorescent probe whose structure has a tertiary amine of 4'- methylamin and verified the interaction between the kinase and 4'-methylamin by
59
confirming the difference in the affinity of the two fluorescent probes for the kinase in a comprehensive manner. To evaluate the effect of the difference of the 4'-methylamine moiety, I also synthesized 8b, which is a methyl capped derivative of 8a (Figure 13b).
In this study, I estimated the detail protein structure to interact with the 4'-methylamine moiety of staurosporine using above two different types of probes. For the purpose, I focused on the TR-FRET based binding assay for comprehensive protein kinases since it has strong advantages including high-throughput performance and low false-positive signals from intrinsic fluorescence (Figure 13c) [100] [101]. TR-FRET based binding assay requires two fluorophores, a donor fluorophore, terbium (Tb) conjugated target protein tag antibody, and an acceptor fluorophore, BodipyFL conjugated staurosporine, are used as fluorescent probe. Excitation of the donor by laser occurs an energy transfer to the acceptor fluorophores if the donor and the acceptor exist within a given
proximity. Then the acceptor fluorophores produce emission light at specific wavelengths of fluorophores. TR-FRET based binding assay is not required highly purified protein. Additionally, TR-FRET based binding assay is high-throughput assay system because this is homogeneous assay. In the reason, it is suitable for accurate assessment of affinity for comprehensive protein kinases.
I evaluated the binding affinity of probes based on 8a and 8b for 288 kinases by TR- FRET based saturation binding assay and estimate the difference of the affinity between the two probes.