Chapter 20

Characterization of ~3Adrenergic receptor: Homology modeling, ligand binding and activation studies

This chapter has been extracted from: P. Prathipati and A.K. Saxena. Characterization of ~3 Adrenergic receptor: Homology modeling, ligand binding and activation studies. J. of Computer Aided Molecular Design, 2005, under communication

Part of this work was presented as invited lectures at -o 2nd International symposium on Computational methods in toxicology and

pharmacology integrating internet resources (CMTPI-2003) (Sept. 17-19, 2003), Aristotelian University of Thessaloniki, Dept. of Pharmaceutical Chemistry, 54006, Thessaloniki, Greece.

o Universitat Konstanz, Fachbereich Chemie,UniversiHit Konstanz, Germany (29th Sept., 2003).

o Rheinische Friedrich-Wilhelms-Universitat Bonn, University of Bonn, Germany (9th Oct. 2003)

o 2nd International Symposium on Current Trends in Drug Discovery Research (17-20th Feb. 2004), Central Drug Research Institute, Lucknow.

As a poster presentation at-o International Symposium on Drug Discovery and Process Research

(Jan.23-25, 2003), Shivaji University, Kolhapur.

Chapter 20

Characterization of r13 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Chapter 20 Characterization of ~3 Adrenergic receptor: Homology modeling, ligand binding and activation studies Contents

20.1 20.2 20.2.1 20.2.2 20.2.3 20.3 20.3.1 20.3.1a 20.3.1b 20.3.2 20.3.2a 20.3.2b 20.3.3 20.3.3a 20.3.3b 20.3.4 20.3.4a

Abstract Introduction Structure based drug design of GPCRs Relevance of b3AR as a drug target Structural models of b3AR-Iigand receptor interactions Methodology Homology Modelling Sequence and Structures Sequence and Structure comparisons Structure refinement Minimization Simulated annealing molecular dynamics Model validation Structural validation Ligand docking Results And Discussion Use of rhodopsin as a template for GPCRs in general and adrenergic receptor in particular.

20.3.4b Ligand receptor molecular recognition theories: beyond specific recognition 20.3.4b.1 Ligand-Macromolecule Complex: specific recognition 20.3.4b.2 Ligand-Macromolecule Complex: beyond specific recognition 20.3.4b.2a Occupancy Theory 20.3.4b.2b Rate Theory 20.3.4b.2c Induced-Fit Theory 20.3.4b.2c.1 Macromolecular Perturbation Theory 20.3.4b.2c.2 Activation-Aggregation Theory 20.3.4b.2d Receptor dimers or dimerization 20.3.4c Sequence and Structure comparisons

413

Chapter 20

20.3.4d 20.3.4e 20.3.4f

- 20.3.4g

Characterization of r13 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Modelling the TMRs of b3AR Structural comparison Conservation of the microdomain interactions Conformational c~anges in the activated con!ort:n~t!on of the P!otein~

20.1 Abstract 03 adrenergic receptor is an important drug target for a diverse array of diseases of current interest viz. obesity, NIDDM, etc. drug discovery efforts against this target were plagued by the unfavorable side effects of the existing ligands which include, non-selectivity with respect to the closely related !32AR and an unfavorable ADME profile. Thus the identification of novel prototypes that circumvent these problems are highly desirable. Thus based on the recent crystal structure of a GPCR, bovine rhodposin, we have built atomic models of ~3AR both in its inactive and active forms and docked agonists like adrenaline and L-222 222 to its active site. While the site-directed mutagenesis analysis of the related ~2AR helped in identifying the most likely active site residues, the pharmacophore and QSAR studies helped in the identification of a potential bioactive conformer and important functional groups of the molecules that form a pharmacophore. The structures of the docked ligands correlate with available biochemical data and our previous phamacophore and QSAR studies, and reveal that the determinants for agonist activity and subtype selectivity are relying essentially on the residues of the micro-domains some which were inaccessible to ligands in ~2AR. The b3AR-agonist models should be useful for the design and identification of novel b3AR-agonistic prototypes molecules with improved profile and could provide pointers for a site-directed mutageneis study of the b3AR. The overall study can be used a case study for the modeling of ligand-GPCR interactions, using an integration of ligand based- and receptor based- approaches.

20.2 Introduction

20.2.1 Structure based drug design of GPCRs Structure-Guided Drug Design has_ become an integral part of modern drug discovery as it

investigates the molecular recognition between the three dimensional (3-D) drug target structures and the integrating small molecules.1 Over the past few years, the 3-D structures of protein targets and their co-crystals have been made readily available, enabling structure-guided drug design to become a revolutionary new tool for lead generation andoptimization.2 Thus the cornerstone of any rational drug design program requires that a 3D model of the target protein be available. Yet, however desirable they may be to model, GPCRs have certain characteristics that make them extremely difficult to determine their 3D structures using the standard approaches such as X -ray crystallography and nuclear magnetic resonance (NMR).3 The determination of the crystal structure of the G-protein-coupled receptor (GPCR) rhodopsin in 2000 was a landmark in the

414

Chapter 20

Characterization of f)3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

study of GPCRs.4 However, given the considerable challenges inherent in repeating this feat with other GPCRs, the GPCR-based drug discovery has focused on how well this structural . information can be extrapolated to other GPCRs of therapeutic interest using the homology modeling techniques. 5-

7 Homology modeling describes an extended collection of techniques with the goal of predicting the 3D details of biomolecules of unknown structure, relying heavily on resources such as pattern-to-function relationship predictors and sequence-to-structure determination predictions. 8•

9

20.2.2 Relevance of ~3AR as a drug target In recent years, the activation of human ~3 adrenergic receptor (~3 AR) has attracted much attention [1-13] as a potential approach towards the treatment of obesity [14-20] and non-insulin dependent diabetes mellitus.(NIDDM) [19-21 ], which is increasing at an alarming rate in western countries. The ~3AR, which was initially (1980's) was referred as atypical because of the characterization of only ~1 and ~2AR at that time, also belongs to the seven transmembrane G­protein coupled receptor, present in white and brown adipose tissues [22], gastrointestinal tract [23], stomach [23] and some heart tissues [24]. It is implicated in the regulation of lipid metabolism. Stimulation of this receptor elevates cyclic AMP levels thereby stimulating lipolysis [25] and upregulation of adipose specific genes. The increased expression of uncoupling protein (UCP-1 ), a brown adipose tissue specific mitochondrial protein, uncouples fatty acid oxidation from oxidative phosphorylation. The process increases heat production with a commensurate boost in energy consumption rendering ~3-AR as potential antiobesity agents. Besides this, ~3-AR has also been shown to mediate various pharmacological and physiological effects such as intestinal smooth muscle relaxation [23], urinary bladder detrusor muscle relaxation [26-28] and · is thought to play an important role in glucose homeostasis and energy balance in humans. Thus ~3-AR agonist therapy represents a novel approach to alter energy utilization and thus ameliorate obesity, NIDDM, intestinal hypermotility and urinary bladder dysfunction such as urinary frequency and in continence.

20.2.3 Structural models of f3sAR-Iigand receptor interactions

Most of the information on the active site of ~JAR has been gained from site directed mutagenesis and chimeric fuand ~~adrenoreceptor studies. Only one amino acid residue 'Asp117' has been shown to be involved in the ligand binding as identified by site directed mutagenesis study. 11 A homology model of ~;) AR aligned upon bacteriorhodospin and ~7AR helped, in assigning the specific amino acid residues to individual helices and in defining their particular roles with respect to structure and function of the receptor.12 However some of the drawbacks of these site directed mutagenesis studies is that they were based on the computer modelling of bacterorhodpsin that is not a true GPCR inspite of its heptaheical structure, secondly only limited number of substrates and activators of receptor, were used to interact with the mutant receptor. As yet there is no x-ray structure of a ligand-receptor complex except a few reports of an initial

415

Chapter 20

Characterization of f·h Adrenergic Receptor: Homology modeling, ligand binding and activation studies

hypothesis on potential binding conformation 13 and ligand receptor interactions. 14'

15 One of these papers describing the P3-AR: ligand interactions14 deals mainly with P1- and PrAR and makes casual remarks about P3AR. Although the other recent paper15 which appeared during the course of this work describes the 3D model for P3-AR complex with agonists and antagonists in terms of · direct design where the P3AR ligands are shown to share most of the structural features common to P1- and P2-AR agonists. In this paper the selectivity of P3-AR agonists has been attributed predominantly to the interactions between theN-substituted carboxylate or sulphonamide group of the ligands and Arg315 (TM6). However it neither quantifies the results in terms of scores nor corroborates the results with the binding site information derived experimentally from site directed mutagenesis studies. It also does not provide the plausible mechanisms for the agonist­induced activation. In continuation of our studies to elucidate the essential structure and physicochemical properties responsible for the P3AR agonistic activity homology modeling, ligand induced activation and docking studies were undertaken. While the previous work focused on the identification of the pharmacophore and 3D QSAR models at the ligand level, 10 wherein the pharmacophore and QSAR model explained well the observed P3AR agonistic activities of diverse structural classes viz. aryloxypropanolamines, arylethonolamines of P3AR agonists, including six b3AR agonists (AJ-9677) currently in clinical trails. The present work focused on the identification of the complementary pharmacophore at the receptor level, using an integrated ligand based- and structure based- approaches. Also since the present research objective is to understand the binding orientations of compounds that behave as human P3-AR receptor agonists -compounds that alter the equilibrium between the inactive and active receptor state in favor of the active form, the receptor activation studies were also performed and the resulting model assessed for its validity. Finally a hypothesis for the P3-AR activation was deduced. This work apart from serving as a useful tool in structure based drug design could provide useful pointers in the design future site directed mutagenesis experiments of the P3AR and also enables a consistency check search for spatial complementarities between the ligand-based and receptor structure-based pharmacophores based on favorable physicochemical interactions.

20.3 20.3.1

Methodology Homology Modelling

20.3.1 a Sequence and Structures The primary sequences of the three adrenergic proteins (human P1-AR, P2-AR and P3-AR) were obtained from GenBank, available from the National Centre of Biotechnology (NCBI), accession numbers 4557265, 114765 and 4557267 respetively. Structural data for bovinerhodpsin were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB-PDB) and the sequence was extracted from its crystal co-ordinates.

416

Chapter20

Characterization of [33 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

20.3.1 b Sequence and Structure comparisons Since Bovinerhodpsin is the only available template, no sequence searches were conducted using neither the BlastP or the SwissProt database available at NCBI. Direct alignments were performed using PAM _120 matrix34 implemented in the homology module of insight!! with the default parameters and structurally conserved regions defined (Scheme 20.1). Various parametric adjustments were made in an effort to enhance the alignment; however, no significant improvements could be obtained beyond that resulting from default inputs. In order to validate the sequence alignments particularly the transmemerane helical regions, the same were compared with those obtained from another software HMMTOP independently.35

-38 (fig 1, table 1)

Coordinates were assigned to the residues of the ~3-AR from the regions of greatest structural conservation (the transmembrane domains and the loop region between IV and V) among the bovinerhodpsin as well as the three beta-adrenergic receptors. Those regions which do not retain significant conservation across receptor (specifically, loop regions) were not modeled. This is for two reasons, first, most of the cytosolic extra-membrane parts could not be seen in the electron density map, and the extra-cellular parts seem to have structure induced by crystal packing forces.

20.3.2 Structure refinement

20.3.2a Minimization The discover module of Insight!I was used for energy minimization using the CVFF force field parameters for the ligands and protein models.39

•40 Various structure refinements were performed

for the side chains of mutated residues and later for the side chains of all the residues. They were selectively minimized using the steepest descentO followed by conjugate gradients, with a convergence value of 0.005 and number iterations set at 10, 000. Later the whole protein model was subjected to energy minization using the steepest followed by conjugate gradient and other algorithms of insight!! for 5000 iterations or until the maximum derivative was< 0.01 kcal/(mol A).

20.3.2b Simulated annealing molecular dynamics The structure of the b3Ar adrenaline complex was subjected to unconstrained minimization using the Steep descents to Newton Raphson algorithm as implemented in insight!I. The CVFF force field set was used for all protein residues. All minimization and simulations employed a switch non-bond smoothing with a nonbonded cutoff of 14.0 and dielectric equal to 1.0. A simulated annealing protocol incorporating the distance restraints of the Metall form of rhodpsin, was used to refold the starting complex into a structure that was consistent with the experimental distance constraints characterizing the Metall form. Using molecular dynamics within insight!I, the construct with the distance restraints of metall was heated to 1000 °K for 1000 fs and then cooled to 300°K for 1500 fs. The default cvff force field and charges within insight!I were used and the calculations were performed on a SGI 02 WS.

417

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

20.3.3 Model validation

20.3.3a Structural validation Several geometrical criteria such as bond lengths, bond angles, and torsions of all the residues were compared against established knowledge base using ProStal0•

41 and in addition ramachandran plots were computed using the SPDV software.43

20.3.3b Ligand docking The molecules adrenaline molecule (I) and the most active agonist molecule (II) considered in the current investigation were manually docked into the active site in their presumed pharmacologically active conformation: in a conformation mapped to the pharmacophore model # 1 for molecule (II) and the global minimum energy conformation of adrenaline. The ligands were initially positioned in such a way that the interactions between their functional groups and the appropriate amino acid side chains, primarily involved in the binding process, could be visualized asH-bonds. The interaction energies between the receptor and the ligand were then minimized by manually adjusting the positions of the ligands in the binding sites, while attempting to maintain the primary interactions displayed as H-bonds, using the docking procedure as implemented in insight!!. Finally the whole ligand-receptor complex was minimized using the discover module of insight!!.

20.3.4 Results And Discussion

20.3.4a Use of rhodopsin as a template for GPCRs in general and adrenergic receptor in particular.

Based on the rhodpopsin structure, most of the cognate residues that were shown to be involved in intracellular receptor-specific ligand binding in other receptors, such as P2-Adrenergic receptor, are found pointing toward the interior of the seven transmembrane helices. 16

-19 The high

resolution X-ray structure of bovine rhodpsin (BR), a GPCR, is regarded as the only protein with sequential and functional homology (template) with GPCRs. 16

•20 In recent times there were

several reviews that have proposed a correspondence of the basic architecture and a common activation mechanism of GPCRs. 19

'21

-24 However there are several issues surrounding the use of

rhodpsin as a template that must be considered. First and foremost, the crystal strucuture is a snap shot of rhodpsin in its inactive, non-:signaling state.4'

25 The ligand is in the ground-state structure, 11-cis-retinal, functions as an inverse agonist to diminish activity levels below that of retinal-free opsin, which is crucial for rhospsin's competent function as a single-photon detector.25 The details of the conformational changes that rhodopsin or other GPCRs undergo upon activation were unclear till recently, but biophysical studies suggest that nontrivial conformational changes do occur.26

-30 Additionally, a number of structural features of rhodopsin were somewhat

surprising, such as the highly ordered extra-cellular region, including a structured globular amino

418

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

terminus and ~-sheet moieties in the loops, and striking bends, bulges, kinks, and stretches of 310

helix in the transmembrane domains.4 Since there is a little sequence similarity between rhodopsin and most other GPCRs in the extracellular domains, and only limited sequence similari ty in most transmembrane regions (e.g. ~ 22% sequence identity between rhodopsin and ~rreceptor in the trans-membrane domains), it is not clear which of the there structural features may be unique to rhodpsin and the opsins, and which may be more general. To address these concerns, several different strategies have been used. One approach, applied recently in several GPCR modeling studies, used the rhodopsin crystal structure in conventional homology modeling exercises to generate 3D models for GPCRs in their ground state, with subsequent structural refinements to model the conformational changes that accompany receptor activation.31

•32 While

this is an inherently attractive strategy, it was problematic at that time because there was not enough experimental data regarding the detailed conformational changes that lead to receptor activation to guide the modeling process. Subsequently a recent paper chose to use the available experimental data of GPCR-agonist interactions as spatial constraints in refinement of plausible agonist complexes also called 'de nevo' models. 15 We therefore in the present study used both the recently available constraints from a detailed NMR study of conformational changes that accompany receptor activation33 as well as the GPCR-agonist interactions as spatial constraints in the subsequent structural refinement of the model of ~rAR obtained by conventional homology modeling using the rhodopsin crystal structure.

20.3.4b Ligand receptor molecular recognition theories: beyond specific recognition

20.3.4b.1 Ligand-Macromolecule Complex: specific recognition A majority of pharmacologic responses are mediated through Macromolecular proteins: receptors and enzymes (note exceptions: such as anesthetics and some diuretics function based on their physical properties alone) (figure 20.1 ). Macromolecules recognize specific ligands, based on the complimentary structure of the ligand and a binding pocket on the macromolecular target, imparting the necessary specificity required for physiological function. This specificity is exploited for pharmacological intervention. (Figure 20.2 and table 20.1)

Molecular recognition -the specific interaction between two or more biological molecules

Interaction "Change" <,

• ?%'

+ 4 ..

.. - . ~ . •'

Association rat~ ~1 Dissociation rate ~ Affinity constant

"Action"

Signalling Altered state Metabolism

419

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Figure 20.1: Ligand-protein molecular recognition theory

Emil Fischerls 1894 model of a lock-and-key

0 The most dominant model to explain the origin of biological specificity 0 Molecules whose shape, i.e. whose steric characteristics, perfectly complements the

shape of the receptor will be active. 0 Later the concept of extended to include many other complementarities like

electrostatics, hydrogen bonds, solubility, etc.

Limitations of the Lock and theory

0 Non-consideration of the role of water in biological reactions fails to explain the noncompetitive inhibition"

Lock Key Enzyme-substrate

complementarity

Substrate

Figure 20.2: Emil Fisher's 1894 model of 'Jock and key' .

.,., bl 20 1 s fth t't' ' j' d t . t t' a e . omeo e camp emen an 1es m 1gan -recep or m erac 1ons .. Physical property

Shape or volume Surface potential Hydrogen bonds Solubility

20.3.4b.2

Receptor Ligand Interaction Distance

Concave Convex Induced diQole Short (r-6) Convex Concave Charge Longer(r-1 ) Positive Negative Dipole Shortish (r-3)

egative Positive Induced dipole - Short (r-6) dipole

Ligand-Macromolecule Complex: beyond specific recognition

Beyond specific recognition alone, many molecules may bind to the same protein and yet they may elicit different responses. Several theories which have evolved over the years, were fonnulated to rationalize their actions of eliciting different responses. (Figure 20.3)

420

Chapter 20

Characterization of P>3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

o Agonist: agents that elicit a maximal biological response. o Partial agonist: agents that elicit a response, however, the maximum response obtained is

less than that of an agonist (e.g. the physiological ligand). o Antagonist: agent that binds (occupies the receptor) but does NOT elicit a response

(antagonists can block an agonist from binding to the receptor); as with an enzyme inhibitor.

The drug-receptor molecular recognition

1) Occupancy Theory

2) Rate Theory

3) Induced-Fit Theory

4) Macromolecular Perturbation Theo ---· m,ctivation- Aggregation Theory ---6) Receptor dimers or dimerization

Figure 20.3: Ligand-protein molecular recognition theory beyond specific recognition

20.3.4b.2a Occupancy Theory

The Occupancy theory states that the intensity of the pharmacological effect is directly proportional to the number of receptors occupied by the drug and the response ceases when the drug dissociates from the receptor. Some of the concepts associated with the occupancy theory. Affinity: capacity of a receptor to bind a drug (molecular complimentarity) Intrinsic activity/efficacy: ability of drug-receptor complex to initiate a response (considered to be constantly on or off in original theory) Agonist: high affinity, high intrinsic activity/efficacy) Antagonist: high affinity, low intrinsic activity/efficacy Limitation: doesn't account for partial agonists

20.3.4b.2b Rate Theory The rate theory states that the intensity of the pharmacological effect is directly proportional to the total number of encounters of the drug with its receptor per unit time. Therefore, the intensity depends not on the number of receptors that are occupied, but on the rate on which, a drug associates and dissociates from the receptor: each encounter elicits a response.

421

Chapter 20

Characterization of [-13 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Some concepts associated with the rate theory -Agonist: association is fast and dissociation is fast. Antagonist: association is fast, dissociation is slow (i.e. drug stays bound to the receptor). Limitations: i) Doesn't rationalize differences in biological activity between structurally similar compounds. ii) Relationship between binding strength and biological activity fails: based on the above

definition an agonist with have bind less favorably than an antagonist, however, this is often not accurate.

20.3.4b.2c Induced-Fit Theory The induced fit theory states that the binding of a drug to its receptor induces a conformational change of the active (binding) site of the receptor. It is the conformational change that initiates the pharmacological response. Further the drug itself can also change conformation (as with acetylcholine). According to this theory, the concepts of agonist and antagonist are formalized as follows -Agonist: binds and induces the correct conformational change. Antagonist: binds but doesn't induce the correct conformational change. The induced-fit theory further evolved into the following theories: Macromolecular Perturbation Theory and Activation-Aggregation Theory.

20.3.4b.2c.1 Macromolecular Perturbation Theory According to the macromolecular perturbation theory-Agonists: Are ligands that cause specific conformational perturbations that lead to a pharmacological response. Antagonist: Are ligands that cause nonspecific conformational perturbations, which do NOT lead to a response. Consequences of the macromolecular perturbation theory, the establishment of the relevance and validity of common pharmacophore concept in agonists.

20.3.4b.2c.2 Activation-Aggregation Theory This theory states that a dynamic equilibrium exists between an activated (R, relaxed) and inactive (T, tense) forms of the receptor and that agonist shifts equilibrium to the R state while an antagonists shift equilibrium to the T state and a partial agonist favors both states to different extents. Some of its advantages:

o Allows for the agonist binding site in the R state to be different from the antagonist binding site in the T state

o Allows for an understanding oflarge structural differences which may occur between agonists and antagonists

422

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

The activation-aggregation theory is today widely used for explaining the different binding orientiations of agonists and antagonists and the further rationalizing the structure mechanism of proteins.

20.3.4b.2 d Receptor dimers or dimerization Recent studies have shown that many peptide hormones activate receptors including GPCRs via receptor dimerization or alteration of receptors comprised of dimer. An example is the erythropoietin receptor (Livnah et al., Science, 1999, 283:987 and Remy et al., Science, 1999, 283:990). In this system, the receptor homodimer in the inactive state assumes a conformation where the intracellular kinase domains are spatially separated. Upon binding of erythropoietin a conformational change occurs that brings the intracellular domains together. This allows for autophorphorylation of the kinase domains leading to activation of their kinase activity followed by phosphorylation of cellular proteins (signal transduction).

20.3.4c Sequence and Structure comparisons The first, and arguably the most critical step in protein homology modeling, is the use of appropriate alignment between the template and experimental sequences. Optimal multiple sequence alignments were obtained for all the ~-adrenergic receptors to study the consistency of alignments with the experimental data (Scheme 20.1 ). Some of the conserved resides in the entire GPCR family and also observed in the sequence alignment of l3z-AR, ~1-AR, ~3-AR and bovine rhodposin respectively are: Asn51, Asn76, Asn55 and Asn55 in TM1; Leu53, Leu78, Leu57 and Leu57 in TM1; Asn69, Asn94, Asn73 and Asn73 in TM2; Leu75, Leu100, Leu79 and Leu79 in TM2; Ala76, Ala101, Asp80 and Asp80 in TM2; Ala78, Ala102, Ala82 and Ala82 in TM2; Asp79, Asp104, Asp83 and Asp83 in TM2; Met82, Met107, Met86 and Met86 in TM2; Cys106, Cys131, Cys110 and Cys110 in TM3; Leu115, Leu140, Leu119 and Leu119 in TM3; Ala128, Ala153~ Ala133 and Ala133 in TM3; Arg130, Arg155, Arg135 and Arg135 in TM3; Tyr131, Tyr156, Tyr136 and Tyr136 in TM3; Trp158, Trp182, Trp162 and Trp162 in TM4; Pro168, Pro202, Pro182 and Pro182 in TM4; Val206, Val231, Val211 and Val210 in TM5; Phe208, Phe233, Phe213 and Phe212 in TM5; Pro211, Pro236, Pro216 and Pro215 in TM5; Leu212, Leu23 7, Leu217 and Leu216 in TM5; Phe217, Phe24 2, Phe222 and Phe221 in TM5; Tyr219, Tyr244, Tyr224 and Tyr223 in TM5; Phe282, Phe334, Phe301 and Phe273 in TM6; Cys285, Cys337, Cys304 and Cys276 in TM6; Trp286, Trp238, Trp307 and Trp279 in TM6; Lue287, Lue241, Lue310 and Lue282 in TM6; Pro288, Pro242, Pro311 and Pro283 in TM6; Asn322, Asn266, Asn235 and Asn307 in TM7; Pro323, Pro267, Pro236 and Pro308 in TM7. While the residues conserved in ~-adrenergic receptors which are in involved in binding to their common agonist adrenaline are- Asp 113, Asp 138 and Asp 117 in TM3; Ser204, Ser229 and Ser209 in TM5; Ser207, Ser232 and Ser211 in TM5; Phe289, Phe243 and Phe312 in TM6 of ~2-AR, ~1-AR and ~3-AR respectively. Further the alignments obtained have also shown a good conservation of the transmembrance regions, which is also consistent with the HMMTOP predictions (table 20.2).

423

Chapter 20

Characterization of[~: Adrenergic Receptor Homology modeling, ligand binding and activation studies

The sequence alignments presented in scheme 20.1 seem to fulfill perfectly the characteristics of GPCRs in general and ~-ARs in particular.

Scheme 20.1: Alignment of the seven selected regions putatively included in transmembrane helices (TMH) for the human {JtAR, [J2AR and {JJAR. Residues of the active sffe are boxed in blue and other conserved residues are boxed in green.

B2AR : ( l BlAR : ( 1 B3AR : ( l Bov rh : ( l

MGQPGNGSAFLLAPNRSHAPDHDVT MGAGVLVLGASEPGNLSSAAPLPDGAATAARLLVPASPPASLLPPASESP

MAPWPHENSSLAPWPDLPTLAPNTANTSG MNGTEGPNFYVPFSNKTGVVRSPFEAPQY

B2AR : ( 26 BlAR :(51 B3AR : (30 Bov_ rh : (30

B2AR : ( 76 BlAR : ( 101) B3AR : ( 8 0 ) Bov_ rh : ( 80 )

B2AR : (126) BlAR : (151) B3AR : ( 13 0) Bov_ rh: (130)

TM3

TMl

LMKMWT FGNFW C VWGRW EYGSF F C LTGHWPLGATG C LHGYFVFGPTG C

RYFAI TSPFKYQSLLT RYLAITSPFRY QSLLT RYLAVTNPLRYGALVT RYVVVC KPM-SNFRFG

B2AR : (176 ) ATHQ - EAINCYA NETCCDFFTNQA BlAR : (201 ) AESD - EARRCYNDPKCCDFVT B3AR : ( 18 0 ) VGADAEAQRCHSNPRCCAFASNMP

Bov rh : ( 17 9) I PEGMQCSCGIDYYTPHEETNNES

-TM4

TMS

B2AR : (225) EAKRQLQKIDKSEGRF - ----- - - - ----------------- HVQ NL SQV BlAR : (250) EAQKQVKKIDSCERRFLGGPARPPSPSPSPVPAPAPPPGPPR PAAAAATA B3AR : (230) VATRQ LRLLRGELGRF -- - --------- PP EE SPPAPSRSLAPA PVGTCA Bov rh: (gap)

B2AR : (249 ) EQDGRTGHGLRRSSKFC BlAR : (300) PLANGR AG KRRPSRLVA B3AR : (268) PP EGVPACGRRPARLLP Bov_ rh : (gap) -- VFTVK EAAA I SATTQ

B2AR : ( 298) IQD BlAR : (349) FHR B3AR : (318 ) GGP Bov_ rh : (278 ) HQG

TM7

TM6 NIV HV NVVK A VLRAL FYIFT -

P I Y - CRSPDFRIAFQELLCLRRSS P IY - CRSPDFRKAFQGLLCCARRA P IY - CRSPDFRSAFRRLLCRCGRR P IYIMMNKQFRNCMVTTLCCGKNP

B2AR : (3 47 ) LKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPS BlAR : ( 398 ) ARRRHATHGDRPRASGCLARPGPPPSPGAASDDDDDDVVGATPPARLLEP B3AR : ( 367) LPPEPCAAARPALFPSGVPAARSSPAQPRLCQRLDGASWGVS Bov _ rh: ( 3 2 9) I STTVSKTETSQVAPA

B2AR : ( 3 9 7 ) DNIDSQGRNCSTNDSLL BlAR : ( 448 ) WAGCNGGAAADSDSSLDEPCRPGFASESKV B3AR Bov rh:

Table 20.2. HMMTOP predictions TMJ TM2 TM3 TM4 TM5 TM6 TM7

Bovine 39-61 (23) 74-95(22) 114- 148- 197- 232- 265-Rhodpsin 133(20) 170(23) 219(23) 256(25) 287(24)

424

I Cha~ter 20

Characterization of r.13 Adrenergic Receptor: Homology modeling, ligand binding and activation studies .

B1AR 59-83(25) 96-120(25) 133- 177- 223- 327- 359-152(20) 196(20) 243(21) 346(20) 378(20)

B2AR 34-58(25) 71-95(25) 110- 152- 200- 275- 307-129(20) 169(18) 219(20) 294(20) 326(20)

B3AR 38-62(25) 75-99(25) 112- 156- 205- 290- 327-131(20) 174(19) 224(20) 314(25) 346(20)

20.3.4d Consideration of loops in ~3AR In the current studies only the trans-membrane (TM) regions of b3AR were modeled: the extra cellular loops of bovine rhodpsin to a large degree are determined by crystal contacts and hence the loop regions of bovine-rhodpsin cannot be used as template to model the loop regions of ~3-AR. Moreover the work of Yeagle45 has also made it clear that the determination of the structure of the loops independently for the rest of the structure is not likely to be a successful option either. Hence no attempt was made to model the loop regions based the on the corresponding coordinates of bovine rhodopsin or through a search of the crystallographic databases either.

20.3.4e Structural Validation A comparison of the 13;AR model with the template rhodpsin structure shows that the TM helices are conserved and their global arrangement is roughly maintained (Figure 20.7). However the differences between the primary sequences of the ~3-AR and rhodpsin cause some minor differences between the two 3D structures such as differences in the position of kinks in the transmembrane domains. This difference is due to the non-conservation of proline residues, which are predominantly implicated, in the inducing kinks in a helix. Today it is widely accepted that the distortion of the helical conformation produced by a proline residue at position 'i' is brought about by two mechanisms: a steric clash between the proline ring and the backbone carbonyl group at position (i-4) has to be avoided; and the backbone hydrogen bonds for the carbonyl groups at positions (i-3) and (i-4) cannot form. (Macarthur et al. 1991, Woolfson and Williams. 1990, Visiers et al. 2000).

425

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

(a) Rho~sin model (b) (33AR model

igure20.7. Comparison of the transmembrane domains of bovine rhodpsin (on left) and ~3AR (on right)

F

The structural quality of the unconstrained minimized model was assessed using the ProStat software implemented within insightii which indicates 92.7 % percentage Phi, Psi core region occupancy(. Also a comparison of the distances between some of conserved amino acid in crystal structure of bovine rhodpsin and the unconstrained optimized structure of the ~3AR reveals significant conservation of the structure as most of the interhelical distances (table 20.3) are retained which is shown by the small differences between the beta carbon atoms some of the conserved residues in the entire GPCR fami ly of each transmembrane domain, viz. B (Cp of Asn55 ofTMl- C~ of Asn83 ofTM2): -0.01 ; B (C.p of Asn55 ofTMl- C~ of Ala132 ofTM3):-1.36; B (C.p of Asn55 of TMl- C~ of Trp 161 of TM4 ): -0 .05; 0 (C p of Asn55 of TMl - Cp of Pro215 ofTM5): -1.12, B (C.pof Asn55 ofTM1- Cpof Phe261 ofTM6): -0.8, B (C.p of Asn55 of TMl- Cp of Asn302 of TM7): -1.42, B (C.pof Ala 132 of TM3- C.p. of Phe261 of TM6): -0.58, further validating the model. (Table 20.3).

20.3.4f Functionally important micro-domain interactions in GPCRs An other powerful approach in the process of validating molecular models of GPCRs has evolved from the strong cooperation between experimental exploitations of structure-function relations and computational simulation approaches (e.g. , see Sealfon et al.,46 Ballesteros et al. ,47 and Ebersole and Sealfon48

) . This approach is based on parsing the receptor sequence into groups of residues that correspond to "microdomains". The inactive state of the receptor is constrained by

426

Chapter20

Characterization of [13 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

these various microdomains, groups of residues that are characterized by specific structural motifs not necessarily contiguous but exhibit a very high degree of conservation. In most cases these microdomains participate in receptor mechanisms through conformational changes in backbone or side chain interactions, which are propagated into large segments of the receptor protein. The identification of such structural and functional microdomains48

'49 has been based

upon (1) sequence analysis for correlated mutations and mutagenesis, (2) biophysical principles and mutagenesis and (3) the substituted cysteine accessibility method (SCAM) and computational simulation. The best structural motifs (SM) characterized as functional microdomains include the ligand-binding site, the aromatic cluster in TM6 the "arginine cage" comprising resides in the cytoplasmic end of TM3 and TM6.20

-24

'50

'51 The crystal structure of bovine rhodopsin,

demonstrates the ionic interaction of Arg135 with Glu247, part of theE/DRY motif conserved in · TM3 of all GPCRs, as well as with a conserved 'E' residue in TM6. These residues, E/DRY in TM3 and E247 in TM6, are part of the conserved set of amino acids that define the fingerprint of the GPCR super-family.

Table 20.3. The distances between the C~-C~ atoms of some highly conserved amino acids in the crystal structure of rhodopsin and the energy minimized homology model of the ~3 Adrenergic Receptor and activated conformation induced by the molecular dynamics of p3 AR-adrenaline complex. Amino acids Distance Amino acids Distance Distance

Asn55(TM1)­Asp83(TM2) Asn55(TM1)­Ala132(TM3) Asn55(TM1)­Trp161(TM4) Asn55(TM1)­Pro215(TM5) Asn55(TM1)­Phe261 (TM6) Asn55(TM1 )­Asn302(TM7) Asp132(TM3)­Phe261(TM6)

(Ca-Ca) (Ca-Ca) (Ca-Ca) [crystal [inactive [active state structure] state] induced by

adrenaline] 6.48

21.85

17.46

24.11

16.37

8.78

13.52

Asn55(TM1)­Asp83(TM2) Asn55(TM1 )­Ala132(TM3) Asn55(TM1 )­Trp162(TM4) Asn55(TM1)­Pro216(TM5) Asn55(TM1)­Phe30 1 (TM6) Asn55(TM1 )­Asn342(TM7) Asp 132(TM3)­Phe301(TM6)

427

6.49 9.02

23.21 23.62

17.51 18.14

25.23 25.38

17.17 17.78

10.20 9.37

14.10 14.0

Chapter 20 I

Characterization of f·IJ Adrenergic Receptor: Homology modeling, ligand binding and activation studies

20.3.4g Conformational changes in the micro-domains of the GPCRs upon activation

Activation of GPCRs is accompanied by rigid domain motions and rotations of transmembrane helices (TMHs) 3 and 6. At their intracellular ends, trans-membrane helices (TMH) '3' and '6' in bovine rhodposin are constrained in a salt bridge by a Glu134 I Arg135/ Glu247 that limits the relative mobility of the cytoplasmic ends of TMH3 and TMH6 in the inactive state and acts like an "ionic lock".20

-24 During activation, Pro267 of the highly conserved CWXP motif in TMH6 of

GPCRs, may act as a flexible hinge, permitting TMH6 to straighten upon activation, moving its intracellular end away from TMH3 and upwards towards the lipid bilayer. 20

-24

•50

•51 A further

investigation of the crystal structure4 demonstrates an cluster of hydrogen bonding interactions between Thr51 and Asn55 in TMl and Asp83 in TM2, this interaction is also known to be involved with the receptor activation mechanism of the related ~2-AR receptor. 51

Since such micro-domains are most likely to have a specific role in the function of the proteins, the identification of such structural and functional microdomains of the query protein was accomplished using the sequence comparisons obtained from the multiple sequence alignments. Consistent with these observations (microdomains) described above, the query ~3-AR model, retained the salt bridge interaction at the "arginine cage" (figure 20.8) comprising resides in the cytoplasmic end of TM3 (Aspl35) and TM6 (Glu287) and another hydrogen bonding cluster of residues (figure 20.8) located between TMl (Thr51, Asn55) and TM2 (Asp 83). Thus the model satisfies structural criteria pertaining to the inactive state of the receptor, further validating the model.

428

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin has revealed the structural changes that accompany activation of a G-protein coupled receptor based on NMR and modeling studies.33 A number of experimental long-range distance constraints are avai lable from a variety of studies that describe the spatial relationship of the elements of secondary structure in the Meta II state of rhodpsin. So accordingly in the present work we arrived at a potentially activated form of the receptor using simulated annealing via a restrained molecular dynamics (SA/MD). The model of the ligand substrate complexes (figure 20.9) was generated by Molecular Dynamics incorporating the distance constraints of the Metaii state of rhodpsin. A simulated annealing protocol was used to refold the starting structure into a structure that was consistent with both the experimental distance constraints characterizing Metaii (table 20.4) and the spatial constraints of the GPCR-agonist interactions.

430

Chapter 20

Characterization of f~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

:~ Figure 20.8: The major hydrogen bonding microdomains of p3-AR in the inactive form.

20.3.4h In silico activation of ~3AR

Our research objective is to understand the binding orientations of compounds that behave as human P3AR receptor agonists -compounds that alter the equilibrium between the inactive and active receptor state in favor of the active form . However the development of a homology model of our target receptor (p3AR) based solely on the inactive Rh crystal structure as the template would result in a model of the inactive state of the receptor. The structural changes that accompany activation of a G-protein coupled receptor (GPCR) were not well understood until recently. Several workers reported different strategies to circumvent this problem, for example, Chambers and ichols32 used an activated form of the bovine rhodpsin (crystal structure) as a template for modeling the 5HT2A receptor structure, wherein the in silica activation of the rhodopsin structure was accomplished by isomerization of the 11 -cis-retinal chromophore, followed by a constrained weighted mass molecular dynamics technique to relax the high energy structure. Furse and Lybamd reported the activated model of P1-, P2- and P3- ARs wherein the available experimental data of GPCR-agonist interactions were used as a spatial constraints in refinement of plausible agonist complexes also called 'de nevo' models. 15 However, the recent

429

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

(

Figure 20.9: Stereo-view of the interactions between the fJJAR and one of its most potent agonist.

Table 20.4. Experimental Long-range constraints for activated Rhodpsin and the distances between the corresponding residues in R

15-20 17.86

15-20 15.95

12-14 10

15-20 29

-10

431

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Chapter20

.. :r·· .. . v • • . '

Charactenzation of f~3 Adrenergic Receptor: Homology modeling, ligand binding and activation stud1es

~- \

\· J .·. -\ \

\

.. . :~.i~:

' . .. . · . .' ''-J~ ., l

r--1 ' \

\ \

l

. \ ..

i

\ . \ ' \ \.

Figure 20.10. Ramachandran plots for (JJAR inactive (Jeff plot indicating 92 % core [yellow] region occupancy) and activated forms (right plot indicating 72 % core region occupancy)

432

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Figure 20.11: The cytoplasmic view of the relative positions of the various transmembrane domains of the (JJ-AR, in the active (blue) and inactive forms (red).

The complex was structurally stable during the MD simulation, since within the highly conserved regions of GPCRs, most of the distances (Ca - Ca) were preserved, expect for the distance between Asn55 and Asp83 (Table 2). This is consistent with the observation that these residues are a part of the microdomain that are involved in activation mechanism of GPCR, hence lose the constraining interactions of the inactive state. Further this model also retains some of the experimental constraints (Table 20.4). However the microdomain involving the arginine cage was disrupted as expected, since some of the experimental constraints involved the "arginine cage" miocrodomain [(Vall39 (TM3)- Thr25 1(TM6); Va1139 (TM3)-Arg252 (TM6)] , no further effort was made to compare the hydrogen bonding microdomains of the metaiJ form of rhodpsin and activated form of rhodpsin. A Pro Stat analysis of the activated structure of ~3AR reveals 75% of the residues lie with the core regions of the Ramachanran plot further validating the model (Figure 20.10). A comparison of the conformational changes upon activation in the micro-domains indicate big conformational changes corresponding to the resides of the micro-domains (Table 20.4). Consistent with experimental observations, helical translation motions were observed towards the cytoplasmic side which were accompanied by the disruption of the inter-helical hydrogen bonding between TM3 and TM6 (arginene cage) and between TM1 and TM2 (figure 20.1 0, table 20.4). Further the secondary structure representations of the activated ~3-AR also reveals the cytoplasmic end of TM6 and the TM8 no longer retains their helical form, also corroborating with the experimental observations (figure 20.1 0).22

'21 The fluorescence spectroscopy analysis of

cysteines labeled with IANBD of a series of mutant ~rAR suggested an agonistic induced rigid movement of TM6 involving a counterclockwise rotation and movement of the cytoplasmic end of the helix away from TM3 (figure 20.11 ), would cause Cys285 (Cys304 in ~3AR) to be exposed

433

Chapter 20

Characterization of [13 Adrenergic Receptor: Homology modeling ligand binding and activation studies

to a more polar environment in the interior of the protein. Similarly, a counterclockwise rotation and/or tilting of TM3 would cause Cys 125-NBD to be exposed to a more polar interior of receptor. 52

•53 These movements were observed in the activated form of ~3AR (figure 20.11) and

hence also corroborate with the fluorescence and with spin labeling studies in rhodpsin.

Table 20.5:Distances of residues in the microdomains responsible for the inactive state of Rh ~3AR and the corresponding distances in the activated ~3AR.

Amino acids Distance Amino Distance !Amino Distance Rh (Ca-Ca) acids (C~-C~) [inactive acids (C~-C~)

[inactive ~3AR state] IB3AR [active state induced by adrenaline state] and MD]

051(TM1)-A55(TM2) 5.79 T51-A55 5.99 lf51-A55 6.54 051 (TM1 )-A83(TM2) 7.08 T51-A83 6.51 fr51-A83 25 .97 A55(TM1 )-A83(TM2) 6.48 T55-A83 6.49 fr55-A83 25 .58 0134(TM3)- 3.80 T134- 3.91 fr134- 3.93 A135(TM3) A135 ~135 0134 (TM3)- 0247 10.84 T134- 10.90 fr134- 17.81 (TM6) A287 IA287 A 135 (TM3)- 0247 8.54 Al35- 8.31 ~135- 14.37 (TM3) A287 IA287

Further, the information in table 20.5, based on the multiple sequence alignment involving bovine rhodopsin and human ~ 1 - , ~r, and ~3- adrenergic receptors and the ~2-AR specific mutation data, which was complied in a docking study of ~2AR by Reyonlds et. al., was used as a check on the receptor model. Indeed the activated form of ~3-AR is consistent with all the data in that all the residues implicated in · induced activation int inwards (Figure 20.12).

Figure 20.11: The rigid body rotational movement of TM3 and TM6 (view from the intracellular side) on activation (red-active and blue- inactive) are depicted by the movements of Cys205 (TM3) and the Cys283 (TM6).

434

I

I

I

I

I

I

I

I

I

I

I

I

I

I

Chapter 20

Characterization of [~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

2 0. 3. 4i ~3AR-selective agonists interactions

One of the most active ~rAR agonist in its proposed bioactive conformation from our earlier pharmacophore and 3D QSAR studies was docked into the binding site region of activated structure of ~3-AR. Herein the information of the binding site of the related ~2-AR obtained from several site directed mutagenesis studies (table 20.6), guided the initial placement of ligand. The complex was then subjected to an unconstrained energy minimization, wherein it was observed that the conformation of the ligand and the receptor were preserved to a large extent. The overall docking analysis revealed important features of the binding mechanism of agonists (i) the ring aromatic groups corresponding to phenoxide establishes aromatic stacking interactions with Phe309 of TM6, a residue part of the micro-domain that coordinates the steric trigger responsible for activation of most GPCRs , this interaction was represented by the ring aromatic feature of the pharmacophoric analysis and which was conspicuously absent in the 3D QSAR model (ii) nitrogen of the propylamino group reinforces the charge neutralizing ionic salt bridge interactions with Asp 117, also a part of the proposed micro-domains responsible for activation which was represented by the positive inonizable feature of the pharmacophore analysis and in the 3D QSAR analysis it was represented as a by one electron rich nitrogen atom of aryloxy propanolamino group capable of donating electrons as biophoric site (BS 1) (iii) the aromatic groups of benzenesulphonamide establish aromatic n-n staking interactions with Phe308(TM6) and Tyr336 (TM7), this interaction was represented by the 6p-electron cloud of the phenyl ring of phenyl ethylene part (BS2) in the 3D QSAR model and was not represented in the pharmacophore analysis (iv) the nitrogen atom of the bezenesulphoamide group establishes charge neutralizing hydrogen bonding interactions with side of Asp 83, part of an other microdomain responsible for activation of ~3AR and the oxygen establishes H-bonding interactions with side chain of Asn342 and main chain of Asp83, this interaction was represented by the hydrogen bond donor and this is represented in both the pharmacophore and 3D QSAR analysis as hydrogen bond donor and presence of hydrogen bond acceptor in the vicinity of oxygen of the sulfonamide group respectively (v) Finally N of the ureido group is in the vicinity of Asn 55, also a residue of the functionally important microdomain, and may establish H-bonding interactions with its side chain and aromatic ring of phenyl ureido group is in the vicinity of Leu 84 and Ala implicating its importance in hydrophobic interactions, while former interaction is represented by two other pharmacophoric hypothesis where HBA and HBD feature in the vicinity of Oxygen of CO group and N of the ureido group respectively was obtained, while the later interactions is represented by the ring aromatic feature in the vicinity ofbenzenesulphonamide groups and as hydrophobicity in the vicinity of phenyl ring of benzene sulphonamide group (SS4) in the 3D the QSAR model. Further consistency of the spatial and physicochemical complementarities between the ligand based and receptor-based pharmacophores was observed reinforcing our indirect and direct modeling results.

435

Chapter 20

Characterization of ~2 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Figure 20.12: The residues implicated in ligand-binding of [J:AR and related proteins are shown facing the binding site crevice.

Table 20.6: Mutations affecting the activity of the /32-Adrenergic Receptor and the corresponding residues in the

Lys Il

Asp79 Ala H2 45 Asp83

Asp79 Asn H2 57 Asp83

Aspll3 Glu H3 45,58 Aspll7

Aspl30 Asn H3 59 Asp l34

Prol38 12 56 Prol42 Ser204 H5 45 Ser209

Ser207 Ala H5 45 Ser212

Glu268 Gly I3 56 Glu287

Cys285 Ser No maximal H6 58 Cys304 stimulation Reduced st H6 60

436

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

~1 adrenergic receptor I ~3 adrenergic receptor

binding Asn312 Ala No antagonist binding H7 60 Asn332 Asn312 Thr Reduced H7 60 Asn332

antagonist/agonist binding

Asn312 The No binding of H7 60 Asn332 antagonist/agonist

Ser319 Ala Low agonistic H7 60 Ser339 binding

Tyr380 ala Lower cAMP C-term 61 production

Figure 20.13: The residues implicated in agonistic binding and activation of (JJAR.

437

Chapter 20

Characterization of p3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Figure 20.16: A close up view of the major {JJAR-agonist interactions.

20.3.4h Conclusion 439

Chapter20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

A three dimensional model of p3 adrenergic receptor (P3-AR) has been constructed by homology modeling using high resolution X-ray structure of bovine rhodpsin (BR) as a template. In order to understand the P3-AR agonist induced conformational changes/ binding orientations, the · universal P3-AR agonist adrenaline was docked using the available experimental data as spatial constraints. This model was further refined using simulated annealing and constrained molecular dynamics in terms of NMR-NOB constraints of activated from of rhodopsin. The resulting active and inactive models showed 78 and 92 % phi, psi core region occupancy, which were also consistent with the experimental data of related proteins. The structurally and functionally important residues in the four micro-domains that constrain the inactive form and involved in substrate binding site were identified from the sequence alignments and site directed mutagenesis data of P3-AR and related proteins. The conformation of the most active molecule 2 and the pharmacophoric features obtained in our previous study were used for docking 2 into the active site of P3-AR and for identifying the key residues involved in activation. The overall analysis revealed the following interactions to govern the P3-AR agonistic binding and activation (i) the ring aromatic group corresponding to phenoxide of 2 for aromatic stacking interactions with Phe309 (TM6), which is a part of a microdomain that coordinates the steric trigger responsible for activation of most GPCRs (ii) nitrogen of the propylamino group for the charge neutralizing ionic salt bridge interactions with Asp 117, a part of another microdomains responsible for activation (iii) the aromatic groups of benzenesulphonamide establish aromatic n-n staking interactions with Phe308 (TM6) and Tyr336 (TM7) (iv) the nitrogen atom of the bezenesulphoamide group for charge neutralizing hydrogen bonding interactions with side chain of Asp83 which is part of yet-an-other microdomain responsible for activation of P3AR and the oxygen atom of S02 group establishes H-bonding interactions with side chain of Asn342 and main chain of Asp83 (v) TheN of the ureido group falls in the vicinity of Asn55, a residue of the functionally important microdomain, and may establish H -bonding interactions with its side chain while the aromatic ring of phenyl ureido may in hydrophobic interactions as it falls in vicinity of Leu84 and Ala85. Thus the observed consistency with the spatial and physicochemical complementarities between the l~gand based and receptor-based pharmacophores reinforce our indirect and direct modeling results.

References 1) Wyss DF. Structure-guided applications in drug discovery. Drug Discov Today. 2003, 8, 924-6. 2) van Dongen M, Weigelt J, Uppenberg J, Schultz J, Wikstrom M. Structure-based screening and design in drug discovery. Drug Discov Today. 2002, 7, 471-8. 3) Muller G. Towards 3D structures ofG protein-coupled receptors: a multidisciplinary approach Curr Med Chern. 2000, 7, 861-88. 4) Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M Crystal structure of rhodopsin: A G protein-coupled receptor Science. 2000 289, 739-45.

440

Chapter20

Characterization of f~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

5) Becker OM, Shacham S, Marantz Y, Noiman S. Modeling the 3D structure of GPCRs: advances and application to drug discovery Curr Opin Drug Discov Devel. 2003, 6, 353-61. 6) Klabunde T, Hessler G. Drug design strategies for targeting G-protein-coupled receptors. Chembiochem. 2002,3, 928-44. 7) Stenkamp RE, Filipek S, Driessen CA, Teller DC, Palczewski K. Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors. Biochim Biophys Acta. 2002, 1565, 168-82. 8) A

9) B 10) c fl) Gros, J.; Manning, B.S.; Pietri-Rouxel, F.; Guillaume, J.L.; Drumare, M.F.; Strosberg A.D. Site-directed mutagenesis of the human beta3-adrenoceptor--transmembrane residues involved in ligand binding and signal transduction. Eur J Biochem. 1998,251, 590-6. 12) Guan, X. M.; Amend, A.; Strader, C.D.; Determination of structural domains for G-protein coupling and ligand-binding in beta3-adrenergic receptor. Molecular Pharmacology. 1995, 48, 492-498. 13) Amici, M.D.; Micheli, C. D.; Kassi, L.; Carrea,G.; Ottolina, G. and Colombo, G. 03-

Adrenergic receptor ligands: insight into structure-activity relationships using Monte-Carlo conformational analysis in water, Tetrahedron, 2001, 9, 1849-1855 14) Nagatomo, T.; Koike, K. Recent advances in structure, binding sites with ligands and pharmacological function ofbeta-adrenoceptors obtained by molecular biology and molecular modeling. Life Sci. 2000,66,2419-26 15) Furse, K. E.; Lybrand, T. P. Three-dimensional models for beta-adrenergic receptor complexes with agonists and antagonists. J Med Chern. 2003, 46, 4450-62. 16) Ballesteros JA, Shi L, Javitch JA. Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors. Mol Pharmacal. 2001, 60, 1-19. 17) Oprian DD. The ligand~binding domain of rhodopsin and other G protein-linked receptors. J Bioenerg Biomembr. 1992,24,211-7. 18) Donnelly D, Findlay JB, Blundell TL. The evolution and structure of aminergic G protein­coupled receptors. Receptors Channels. 1994,2,61-78. 19) Filipek S, Teller DC, Palczewski K, Stenkamp R. The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors. Annu Rev Biophys Biomol Struct. 2003, 32, 375-97. 20) Ballesteros J, Palczewski K. G protein-coupled receptor drug discovery: implications from the crystal structure of rhodopsin. Curr Opin Drug Discov Devel. 2001, 4, 561-74. 21) Kamik SS, Gogonea C, Patil S, Saad Y, Takezako T. Activation ofG-protein-coupled receptors: a common molecular mechanism. Trends Endocrinol Metab. 2003, 14, 431-7. 22) Bissantz C.Conformational changes of G protein-coupled receptors during their activation by agonist binding. J Recept Signal Transduct Res. 2003,23, 123-53. 23) Meng EC, Bourne HR. Receptor activation: what does the rhodopsin structure tell us? Trends Pharmacal Sci. 2001, 22, 587-93.

441

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

24) Sakmar TP, Menon ST, Marin EP, A wadES. Rhodopsin: insights from recent structural studies. Annu Rev Biophys Biomol Struct. 2002, 31, 443-84. 25) Okada T, Ernst OP, Palczewski K, Hofmann KP. Activation of rhodopsin: new insights from structural and biochemical studies. Trends Biochem Sci. 2001,26, 318-24. 26) Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science. 1996, 274, 768-70. 27) Dunham TD, Farrens DL. Conformational changes in rhodopsin. Movement of helix f detected by site-specific chemical labeling and fluorescence spectroscopy. J Bioi Chem. 1999, 274, 1683-90. 28) Altenbach C, Klein-Seetharaman J, Hwa J, Khorana HG, Hubbell WL. Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: a site­directed spin-labeling study. Biochemistry.1999 38, 7945-9. Ghanouni P, Gryczynski Z, Steenhuis JJ, Lee TW, Farrens DL, Lakowicz JR, Kobilka BK. Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor. J Bioi Chem. 2001, 276, 24433-6. 29) Peleg G, Ghanouni P, Kobilka BK, Zare RN. Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. Proc Nat! Acad Sci USA. 2001,98,8469-74. 30) Bissantz C, Bernard P, Hibert M, Rognan D. Protein-based virtual screening of chemical databases. II. Are homology models of G-Protein Coupled Receptors suitable targets? Proteins. 2003, 50, 5-25. 31) Chambers JJ, Nichols DE. A homology-based model of the human 5-HT2A receptor derived from an in silica activated G-protein coupled receptor. J Comput Aided Mol Des. 2002, 16, 511-20. 32) Choi G, Landin J, Galan JF, Birge RR, Albert AD, Yeagle PL. Structural studies of metarhodopsin II, the activated form of the G-protein coupled receptor, rhodopsin. Biochemistry. 2002,41,7318-24. 33) Altschul SF. Amino acid substitution matrices from an information theoretic perspective. J Mol Bioi. 1991,219, 555-65. 34) Kall L, Sonnhammer EL. Reliability of transmembrane predictions in whole-genome data. FEES Lett. 2002,532,415-8. 35) G.E Tusnady and I. Simon. Principles Governing Amino Acid Composition oflntegral Membrane Proteins: Applications to Topology Prediction. J. Mol. Bioi. 283, 489-506 36) G.E Tusnady and I. Simon. The HMMTOP transmembrane topology prediction server. Bioinformatics 2001, 17, 849-850 3 7) http://www. enzim.hu/hmmtop/html/submit. html 38) Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D. J.; Wolff, J.; Genest, M.; Hagler, A. T. Structure and energetics of ligand binding to proteins: E. coli dihydrofolate reductase­trimethoprim, a drug-receptor system, Proteins: Structure, Function and Genetics, 1988, 4, 31-47. 39) Hobza, P.; Kabelac,M.; Sponer, J.; Mejzlik, P. and Vondrasek, J. Performance of Empirical Potentials (AMBER, CFF95, CVFF, CHARMM, OPS, POLTEV), Semiemprical Quantum

442

Chapter 20

Characterization of 113 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

Chemical Methods (AMI, MNDO/M, PM3) and ab initio Hartree-Fock Method for Interaction of DNA Bases: Comparison ofNonempirical Beyond Hartree-Fock Results, J Comp. Chern. 1997, 18, 1136-1150. 40) Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl. Crystallogr. 1993, 26, 283-291 41) Engh R A & Huber R. Accurate bond and angle parameters for X -ray protein structure refinement. Acta Cryst., 1991, A47, 392-400. 42) Neve KA, Cox BA, Henningsen RA, Spanoyannis A, Neve RL. Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenylyl cyclase. Mol Pharmacal. 1991, 39 733-9. 43) Horstman DA, Brandon S, Wilson AL, Guyer CA, Cragoe EJ Jr, Limbird LE. An aspartate conserved among G-protein receptors confers allosteric regulation of alpha 2-adrenergic receptors by sodium. J Bioi Chern. 1990, 265, 21590-5. 44) Strader, C. D.; Sigal, I. S.; Register, R. B.; Candelore, W. S.; Rands, A.; Dixon, R. A. F. Identification of residues required for ligand binding to the beta adrenergic receptor. Proc. Nat!. Acad. Sci. US.A. 1987, 84, 4384-4388.; Strader, C. D.; Sigal, I. S.; Candelore, M. R.; Rands, E.; Hill, W. S.; Dixon, R. A. F. Conserved aspartic acid residues 79 and 113 of the beta adrenergic receptor have different roles in receptor function. J Bioi. Chern. 1988,263,10267-10271. 45) Sealfon SC, Chi L, Ebersole BJ, Rodic V, Zhang D, Ballesteros JA, Weinstein H. Related contribution of specific helix 2 and 7 residues to conformational activation of the serotonin 5-HT2A receptor. J Bioi Chern. 1995, 270, 16683-8. 46) Ballesteros J, Kitanovic S, Guarnieri F, Davies P, Fromme BJ, Konvicka K, Chi L, Millar RP, Davidson JS, Weinstein H, Sealfon SC. Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J Bioi Chern. 1998, 273, 10445-53. 47) Ebersole BJ, Sealfon SC. Strategies for mapping the binding site of the serotonin 5-HT2A receptor. Methods Enzymol. 2002, 343, 123-36. 48) Flanagan CA, Zhou W, Chi L, Yuen T, Rodic V, Robertson D, Johnson M, Holland P, Millar RP, Weinstein H, Mitchell R, Sealfon SC. The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor. J Bioi Chern. 1999, 274, 28880-6. 49) Carrieri A, Centeno NB, Rodrigo J, Sanz F, Carotti A Theoretical evidence of a salt bridge disruption as the initiating process for the alphald-adrenergic receptor activation: a molecular dynamics and docking study. Proteins. 2001, 43, 382-94. 50) Ghanouni P, Steenhuis JJ, Farrens DL, Kobilka BK. Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor. Proc Nat! A cad Sci US A. 2001,98,5997-6002. 51) Gether U, Lin S, Kobilka BK Fluorescent labeling of purified beta2-adrenergic receptor: evidence for ligand-specific conformational changes. J Bioi Chern. 1995,270, 28268-28275

443

Chapter 20

Characterization of ~3 Adrenergic Receptor: Homology modeling, ligand binding and activation studies

52) Gether U, Lin S, Ghanouni P, Ballesteros JA, Weinstein H, Kobilka BK Agonists induce conformational changes in transmembrane domains III and VI of the S2 adrenoceptor. EMBO J 1997, 16:6737-6747 53) Gouldson PR, Snell CR, Reynolds CA. A new approach to docking in the beta 2-adrenergic · receptor that exploits the domain structure of G-protein-coupled receptors. J Med Chem. 1997, 40, 3871-86. 54) 55) O'Dowd, B. F.; Hantowich, M.; Regan, I. W.; Leader, W. M.; Caron, M.G.; Lefkowitz, R. Site-directed mutagenesis of the cytoplasmic domains of the human beta-2-adrenergic receptor­localisation of regions involved in G-protein-receptor coupling. J Bio. Chem. 1988,263, 15985-15992. 56) Wang, H. Y.; Lipfert, L.; Malbon, C. C.; Bahouth, S. Site-directed anti-peptide antibodies defme the topography of the beta- adrenergic-receptor. J Bio. Chem. 1989, 264, 14421-14431. 57) Strader, C. D.; Candelore, M. R.; Hill, W. S.; Irving, I. S.; Dixon, R. A F.; Sigal, I. S. Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor. J Bio. Chem. 1989, 264, 13572-13578. 58) Fraser, C. M.; Chung, F. Z.; Wang, C. D.; Venter, I. C. Site. directed mutagenesis ofhuman beta-adrenergic receptors - substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate-cyclase. Proc. Nati. A cad. Sci. USA. 1988, 85, 5478-5482. 59) Suryanarayana, S.; Kobilka, B. K Amino-acid Substitutions at position 312 in the 7th hydrophobic Segment of the beta(2)- adrenergic receptor modify ligand-binding specificity. Mol. Pharmacal. 1993, 44, 111-114. Suryanarayana, S.; Daunt, D. A.; von Zastrow, M.; Kobilka, B. K A point mutation in the seventh hydrophobic domain of the Q2-adrenergic receptor increases its affinity for a family of fJ receptor antagonists. J Bioi. Chern. 1991, 266, 15488-15492. Valiquette, M.; Bonin, H.; Bouvier, M. Mutation oftyrosine-350 impairs the coupling of the beta-2-adrenergic receptor to the stimulatory guanine-nucleotide-binding protein without interfer- ing with receptor downregulation. Biochemistry 1993, 32, 4979-4985.

444