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In silico Medicinal Chemistry

Kooistra, A.J.

2015

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citation for published version (APA)Kooistra, A. J. (2015). In silico Medicinal Chemistry: Investigating GPCRs: key regulators of signal transductionand cell function.

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125

Structure-based prediction of GPCR-ligand function

Chapter 6

126

Chapter 6 - Structure-based prediction of GPCR-ligand function

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Structure-based prediction of GPCR-ligand function - Chapter 6

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G protein-coupled receptors (GPCRs) are versatile membrane-bound switch nodes of cell function and constitute arguably one of the most important classes of proteins that is encoded in the human genome.373, 379 Next to stimulating (agonism), blocking (an-tagonism) or reducing constitutive activity (inverse agonism), GPCRs ligands are able to stim-ulate cell-signaling pathways that are completely independent of G-proteins.363, 380, 384 This new concept of GPCR biased-signaling is an intriguing phenomenon in which different ligands can promote or block multiple conformational states of the same receptor that are each linked to a different functional outcome.78, 380, 384 The first human GPCR crystal structures were only solved in the past seven years93, 176 and have further increased our understanding of the molecular details of how GPCRs work.73, 117, 176 These spectacular advances in GPCR structure determination have resulted in 109 crystal structures410 at this moment, covering 20 class A93, 176, 2 class B81, 82, 1 class C107 and 1 class F84 GPCRs. Despite these advances less than 2% of all human GPCRs have been crystallized, and most of these structures represent inactive-state conformations, with the exception of the adenosine A2A receptor, the neurotensin receptor type 1411, and the β-adrenoceptors 1 (β1R) and 2 (β2R).176 The 31 different β1R/β2R structures (while finalizing this chapter a 32nd, covalently bound, β-adrener-gic structure was released394) cover multiple receptor activation states in combination with 19 ligands (Table 6.1) with different functional effects (Table 6.1).102, 104, 105, 187, 193, 206, 302, 303, 386-388, 390-393 This unique GPCR structure-function data set offers a unique opportunity to assess the possibil-ities and limitations of the structure-based computational prediction of GPCR ligand function in virtual screening (VS) studies. GPCR crystal structures157, 158, 161, 163, 164, 173 and homology models115, 132 have been successfully used to discover new GPCR ligands. While in most VS studies new antagonists were found by docking into inverse agonist/antagonist bound GPCR crystal structures157, 158, 161, 163, 164, 173 or GPCR models based on the inactive bovine rhodopsin (bRho) structure91 and refined using known antagonists115, 132, several reports show that also agonists can be found by structure-based VS in inactive GPCR models.122, 141, 144, 146, 152, 156 In some of these studies,141, 146, 152 the initial bRho-based inactive state homology model was refined using true agonists, but in other cases122, 144 the inactive model was even refined by antagonists. The other way around, agonist-biased models have also been suc-cessfully applied to discover new antagonists.156 Previous docking-based VS studies149 against β2R crystal structures nevertheless indicated that the functional activity of in silico hits is the same as the functional activity of the co-crystallized ligand bound to the structure that is used for the docking simulations.149 While docking against the first carazolol (antagonist/inverse agonist) bound β2R crystal structure enabled the discovery of new β-adrenergic antagonists/inverse ago-nists164, 178, docking studies against the BI167107 (full agonist) bound active-state crystal structure facilitated the identification of partial/full agonists.395 Moreover, even before the determination of partial/full agonist bound β2R crystal structures104, 303, 388 several studies showed that the in-verse-agonist-bound β2R crystal structure could be used for the selective identification of ago-nists by introducing (small) conformational changes in the β2R structure and using customized scoring approaches to select docking poses.162, 276, 404, 412 In independent modeling studies162, 276, 404, 412 the position and/or orientation of the side chains of S2045.43 and S2075.46 (through rotation of

Co-authored by: Rob leurs, Iwan J.P. de Esch, and Chris de Graaf

Submitted for publication

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TM5 and/or adjustment of the rotameric states of S2045.43 and S2075.46) was customized based on ligand SAR and receptor mutation studies,248, 263, 389, 408, 409, 413 thereby enabling the preferen-tial scoring of agonists over antagonists. The selective identification of partial/full agonists was significantly increased by employing an interaction fingerprint (IFP)45 scoring method that de-termines ligand-binding-mode similarity instead of classical (energy-based) scoring functions.162 These studies illustrate how (small) differences in protein conformation can influence struc-ture-based virtual screening results, as has also been demonstrated by recent comparative VS studies against GPCR crystal structures and homology models.157, 159, 166, 173 Systematic consider-ation of alternative protein conformations/flexibility in docking simulations can, for example, be taken into account either by incorporating flexibility for (selected) binding-site residues during the docking simulations414, 415, by creating binding-site ensembles143, by using multiple crystal structures416, or refining protein models with different ligands.115, 154, 417

GPCR crystal structure-based identification of new ligands with a different functional effect than the co-crystallized ligand has been defined as one of the challenges in the current era of GPCR structural and chemical biology.117 The growing number of β-adrenoceptor crystal structures meanwhile covers multiple activation states and multiple co-crystallized ligands with different efficacy classes.379, 383, 384 This unique GPCR structure-function data set allowed us for the first time to systematically and separately investigate the effect of both the reference ligands and the receptor conformations in combination with classical (energy-based)149, 418 and IFP-scoring45, 419 approaches. In this study we have investigated whether (and to what extent) i) the crystal structures retain their preference for ligands with the same functional effect as the co-crystal-lized ligand, ii) the different binding site conformations of the crystal structures have an impact on the outcome of VS studies, iii) specific IFPs can change or amplify the preference of a crystal structure for ligands with a specific functional effect and iv) the IFP derived from the predicted docking pose of a small agonist (norepinephrine) can be used for the selective retrieval of par-tial/full agonists (p/fAGO) over antagonist/inverse agonists (ANT/iAGO) and decoys.

6.1 Computational Methods

Crystal structures retrieval and preparation

All β-adrenergic crystal structures were downloaded from the PDB (last accession date, 4th of April 2014).61 Subsequently, all structures were superposed using MOE420 based on the resi-dues surrounding the ligands. Based on the initial superposed structures all (47) residues with-in 4.5Å of any ligand were determined as being the pocket residues: V90/M822.53, V94/862.57, G98/902.61, A99/912.62, L101/H932.64, V102/I942.65, G105/K972.68, W107/99, C114/1063.25, W117/1093.28, T118/1103.29, D121/1133.32, V122/1143.33, L123/1153.34, V125/1173.36, T126/1183.37, V172/T1644.56, S173/1654.57, C198/19045.49, C199/19145.50, D200/19245.51, F201/19345.52, V202/F19445.53, T203/19545.54, N204/1965.35, Y207/1995.38, A208/2005.39, I209/2015.40, S211/2035.42, S212/2045.43, I214/V2065.45, S215/2075.46, F216/2085.47, W303/2866.48, F306/2896.51, F307/2906.52, N310/2936.55, N313/H2966.58, V314/2976.59, D322/K3057.32, F325/Y3087.35, V326/I3097.36, A327/L3107.37, N329/3127.39, W330/3137.40, G332/3157.42, and Y333/3157.43.The first and second residue number refer to the Uniprot residue number of β1R and β2R, respec-tively, while the Ballesteros-Weinstein83 and extracellular loop 2 (ECL2)160 residue numbers are reported as superscript. An in-house protocol similar to the KLIFS protocol370 was performed to align all β-adrenergic crystal structures based on the selected 47 pocket residues, protonate the structures and separately extract the ligand, pocket, protein and (if present) waters, ions, or-

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ganometallics and cofactors in MOL2 format. All structures were subsequently inspected using MOE and when necessary the protein-hydroxyl groups were adjusted for optimal H-bonding between the co-crystallized ligand and the receptor.

Consideration of alternative ligand ring conformations in 3ZPQ

The ring conformation of the fragment-like arylpiperazine ligand 19 in chain B of the β1R crystal structure (PDB-code 3ZPQ) did not allow for the secondary amine in the piperazine moiety to interact with both N329/3127.39 and D121/1133.32 due to its twist conformation. We therefore re-docked the co-crystallized ligand allowing for all possible ring conformations and selected the highest-ranking pose, which is in the more stable chair conformation421 (named 3ZPQ_dock, see Figure S6.1).

Pocket and ligand analyses

Pocket volumes were calculated using POVME422 (version 1.1.0) with a grid spacing of 0.5Å and a padding of 1.09Å. The initial inclusion volume was generated based on three spheres with a radius of 7.0Å of which the centers were based on the superposed co-crystallized ligands (blue volume in Figure S6.7). RMSD values between the pockets from the crystal structures were calculat-ed using MOE. Distance calculations within the quadruplet (formed by G98/902.61, D121/1133.32, N329/3127.39 and S211/2035.42) and the subsequent Z-score analyses were performed using an in-house script. The residues that form the quadruplet were selected based on their location and function in the binding pocket: G2.61 is opposite of S5.42 (an important interaction point for polar groups, in particular for agonists), N7.39 is opposite of D3.32 (both are key interaction points for the ligand amine group), the N7.39/D3.32 pair is perpendicular to the G2.61/S5.42 pair (Figure S6.3), and all four residues are roughly positioned at the same height in the TM helices with respect to the membrane. The 2D similarity of the co-crystalized ligands was assessed using Extended Connectivity fingerprint (ECFP-4) in the Pipeline Pilot Suite385 and EDprints30 (in-house imple-mentation).

Compound preparation and docking

26 ligands (13 f/pAGO and 13 ANT/iAGO) and 980 decoy compounds were obtained from the supporting information accompanying the publication from de Graaf et al..162 In total 24 addition-al β-adrenergic ligands (12 f/pAGO and 12 ANT/iAGO) were selected based on the classification of Baker.333, 423 Molecular (2D) structures of all ligands are shown in Figure S6.4. All compounds were prepared starting from the SMILES format by first protonating all compounds at physiologi-cal pH using Chemaxon's Calculator (version 5.1.4)364 and were subsequently converted to MOL2 format using CORINA (version 3.49).365, 424 All molecular docking simulations were carried out in fivefold using PLANTS345 (version 1.2) using the (default) ChemPLP scoring function, search speed 2, flipping of free ring corners enabled, and generating 99 poses for every compound with a clustering RMSD of 1.0. The docking experiments were performed in fivefold in order to reduce the effect of the stochastic nature of PLANTS345, as the use of a stochastic algorithm (as used in multiple docking algorithms, e.g. PLANTS345, GOLD425, and QXP426) can result in different docking poses for the same compound when performing multiple docking runs.The binding site was based on the center of carazolol in β2R (PDB-code 2RH1) with a binding-site radius of 15Å, thereby covering all pocket residues (except for G105/K972.68, which is pointing away from the binding site). In total 1030 compounds were docked in 48 β-adrenergic structures yielding almost 24.5 million docking poses in total.

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Interaction Fingerprint calculation and scoring

From the individual pockets and ligands, that were extracted from the crystal structures, in-teractions fingerprints were calculated through application of the FingerPrintLib (version 2.2, downloaded from http://bioinfo-pharma.u-strasbg.fr, using OpenEye’s OEChem Toolkit).45, 427 All docking poses were post-processed with IFP by combining the docking poses with the consistent pocket residue selection as indicated earlier. The IFPs from all docking-poses were scored using all 40 reference IFPs resulting in ~979 million IFP-scores representing their interaction profile similarity (as calculated by the Tanimoto similarity coefficient).

Virtual screening assessment

Based on the PLANTS and IFP scores of all docking poses the highest scoring docking poses according to each of the scoring methods were selected. The resulting hit lists were used to rank both the f/pAGO and the ANT/iAGO ligands as well as the decoys and determine the enrichment factor (EFx, where x is the false-positive rate) for each reference IFP (or ChemPLP score) and docking structure combination. The EFs were calculated by determining the true-positive rate (TP-rate, i.e. the percentage of f/pAGO and ANT/iAGO respectively) at a 1% false-positive rate347 (FP-rate, i.e. 1% of the decoys, see Figure 6.3 and 6.5a/b) resulting in the EF1% of 1968 scoring combinations (40 reference IFP scores and 1 PLANTS score combined with 48 structures) for both the ranking of f/pAGO and ANT/iAGO ligands (see Figure 5).

6.2 Subtle ligand-type-dependent structural changes in the receptor binding site

There currently are 31 β-adrenergic structures; 15 human β2R and 16 turkey β1R structures (Table 6.1).102-105, 187, 193, 206, 302, 303, 386-388, 390-393 While finalizing this chapter a 32nd β-adrenergic structure was released394 with a covalently-bound agonist that forms similar H-bond interactions with TM5 as the epinephrine (PDB-code 4LDO)103 and has the same covalent linker as FAUC50 (PDB-code 3PDS).388 Multiple crystallization methods have been employed to obtain stable crystals for structure determination of these receptor-ligand complexes,93 amongst them T4 lysozyme insertion at the intracellular loop (ICL) 3 or at the N-terminus, introduction of thermostabilizing mutations, introduction of covalently binding ligands, and the addition of antibody fragments.428 All crystal structures were obtained in combination with one of the 19 unique ligands (apart from the ligand-free structure,393 see Table 6.1 and 6.2); 9 (partial/full) inverse agonists, 7 (partial/full) agonists, 1 covalently-bound full agonist, and 2 unvalidated fragments (but suggested to be antagonists,392 vide infra). Throughout this manuscript we abbreviate full and partial agonists as f/pAGO, antagonists as ANT and (full/partial) inverse agonists as iAGO (see legend Table 6.1). It should be noted that we did not focus on the differences between β1R and β2R in this manuscript, as the pocket-lining residues are almost identical (only residue 7.35 differs, a phenylalanine in β1R but tyrosine in β2R) we assume that the ligand-binding modes in β1R and β2R are transferable.429 Also, in the set of ligands that we analyzed there is only one highly selective compound (ICI 118,551, which has a ~540 fold higher affinity for β2R than for β1R).423 Moreover, it is suggested that selectivity for one of the receptors333, 423, 430 is based on ligand kinetics431 (i.e. binding to and dissociation from the receptor) and receptor activation429 rather than differences in ligand binding modes.431

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Table 6.1. Overview of all β-adrenoceptor crystal structures, the co-crystallized ligands and their function.

PDB Ligand Funct-iona,p Biase PDB Ligand Funct-iona,p Bias

e

3NYA193 OOH

NH2+

Alprenolol

ANT

●●●●●

p/fAGOANT/iAGOANT (Not validated)Fab−complexLigand−freep/fAGOANT/iAGOANT (Not validated)Fab−complexLigand−free

Yesj,k

Nof,i3ZPR392

N

N+H2N

Arylpiperazine 20

ANTb●●●●●


-

4AMI105

N

OOH

NH2+

HN

Bucindolol

ANTo

●●●●●


Yesg,m3P0G303,q

3SN6104,q

4LDE103,q

O

OHNH

O

NH2+ OH

BI-167107

fAGO

●●●●●


Yesn

2YCW390

2RH1102

4GBR391

2R4R386,d

2R4S386,d

3KJ6206,d

NH

ONH2+ OH

Carazolol

iAGOo●●●●●


Noh 2Y02302

OHNH2+

O OHNH

O

Carmoterol

fAGO

●●●●●


Yesm,n

4AMJ105OH

O

NH

NH2+

O

O

Carvedilol

iAGO

●●●●●


Yesg,j,m

Nof2Y00302

2Y01302

Dobutamine

pAGO

●●●●●


Yesf

Nol

2VT4387

2YCX390

2YCY390

4BVN101

NH

ONH2+

N

OH

Cyanopindolol

iAGOo●●●●●


- 4LDO103,q OH

OH

OHNH2+

Epinephrine

fAGO

●●●●●


Nof,g,l

3NY8193 NH2+

OOH

ICI 118,551

iAGO●●●●●


Nof,h 3PDS388,c

FAUC50

fAGO

●●●●●


-

2YCZ390

N

INH

ONH2+ OH

Iodocyanopindolol

iAGO●●●●●


- 4LDL103,qOH

OH

OHNH2+

HO

Hydroxybenzyl-isoproterenol

fAGO

●●●●●


Yesn

3D4S187

O

N NS

NOOH

NH2+

Timolol

iAGO●●●●●


Nof 2Y03302 OH

OH

OHNH2+

Isoproterenol

fAGO

●●●●●


Nof,g,-h,i,j,k,l

OHOH

NH2+

HO

NH2+ OH

OO OH

S O

NH

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PDB Ligand Funct-iona,p Biase PDB Ligand Funct-iona,p Bias

e

3NY9193

OO

O

OOH

NH2+

VS hit (Kolb)

iAGO●●●●●


- 2Y04302OH

OH

OHNH2+

Salbutamol

pAGO

●●●●●


Nol

3ZPQ392 N+H2N

NH

Arylpiperazine 19

ANTb●●●●●


- - OH

OH

OH

+H3N

Norepinephrine

fAGO

●●●●●


Nog

4GPO393 Ligand-free

a) Function abbreviations: ANT – antagonist, AGO – agonist, i – inverse, p – partial, f – full. b) Not validated.392 c) covalent-ly-bound ligand. d) Ligand and extracellular part of the receptor were not resolved due to weak electron density. e) This column indicates whether a ligand has a signaling preference for β-arrestins over G-proteins or not according to f) Casella et al.430, g) Drake et al.432, h) Kahsai et al.433, i) Kaya et al.434, j) Kim et al.435, k) Liu et al.205, l) Rajagopal et al.436, m) Warne et al.105 and n) Weiss et al.395. o) Although these ligands are considered antagonists/inverse agonists, they can also have a partial agonist effect depending on the activation state of the receptor.333, 437 p) Color coding: Full/partial agonist (red), antagonist/inverse agonist (blue), or unvalidated antagonist (cyan). Ligands with a circle do not show a signaling preference or it is not known for these compounds, ligands with triangular icons have been shown to have a β-arrestin signaling bias. q) The receptor is in its active state.

Superposition of all individual monomers of the crystal structures of β1R and β2R based on their pocket residues (see Materials and Methods) shows the conserved heptahelical fold, but also indicates some structural variation, in particular in TM1 (a 60 degree kink)387, 390, TM5 and TM6 (which differ between active and inactive states104, 303 and the straight and bent inactive-state TM6390). While the structural differences in the intracellular regions have been described in sev-eral reviews73, 176, our systematic analysis will focus on the structural variations in the ligand-bind-ing site within the β-adrenergic GPCR family.Figure 6.2a/b shows that the pocket residues in all (β1R and β2R) crystal structures have largely conserved conformations. The outliers are the FAB-complexes (PDB-codes: 2R4R, 2R4S386 and 3KJ6206) in which the ECL domain, the top of the helices, and the ligand (carazolol) were not solved due to the distorted density in this region.206, 386 When comparing the pocket residues by means of the mutual RMSD values (Table S6.2, Figure 6.2c), they are generally low (average all-atom RMSD 0.9Å). By determining the average RMSD of the binding site residues of each individual structure to all f/pAGO and all ANT/iAGO structures it becomes apparent that all f/pAGO structures and all inactive-state ANT/iAGO structures cluster together (Figure 6.2c). This means that the binding site conformation within the inactive-state f/pAGO structures, but also within the ANT/iAGO structures is conserved. Moreover, the active-state structures form a sep-arate cluster compared to other f/pAGO structures, which can be ascribed to the movement of TM5 and TM6104, 303 (Table 6.1, Table S6.2, Figure S6.2).438 Outliers are the FAB-complexes (due to the distorted electron density), but also the ligand-free structure (4GPO393), which have a high RMSD value compared to the other structures. Interestingly, this apo structure does not cluster with any of the f/pAGO structures or with ANT/iAGO structures.To further quantify the differences between the pockets we measured the distances between the Cα atoms of G2.61, D3.32, N7.39, and S5.42 (Figure 6.1c, Figure S6.3, and Table S6.3). While the mea-surements show contraction of the pocket upon agonist binding302 (mainly through movement of TM5), the highest contraction of the pocket was found for the ligand-free β1R structure.393

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RMSD

a

(f/pAGO)

RMSD

a

(ANT/iAGO

)

Distanceb

Volumec

Receptor

2RH1_A 0.8 0.6 0.2 377 β22VT4_A 0.8 0.6 0.2 492 β12VT4_B 0.9 0.7 0.1 442 β12VT4_C 0.9 0.7 0.1 442 β12VT4_D 0.8 0.6 0.2 493 β12YCW_A 0.7 0.5 -0.2 411 β12YCW_B 0.8 0.6 -0.2 409 β12YCX_A 1.0 0.7 0.2 459 β12YCX_B 1.0 0.7 0.1 464 β12YCY_A 0.8 0.6 0.0 437 β12YCY_B 0.8 0.6 -0.1 441 β12YCZ_A 0.9 0.6 0.2 469 β12YCZ_B 0.9 0.6 0.2 472 β13D4S_A 0.8 0.6 0.2 278 β23NY8_A 0.9 0.7 0.8 387 β23NY9_A 0.8 0.6 0.5 299 β23NYA_A 0.9 0.7 0.5 248 β24AMI_A 0.7 0.6 -0.2 411 β14AMI_B 0.6 0.6 -0.2 396 β14AMJ_A 0.7 0.5 0.5 488 β14AMJ_B 0.7 0.5 0.3 424 β14BVN_A 0.7 0.5 0.5 302 β14GBR_A 0.9 0.7 0.8 360 β23ZPQ_A 0.7 0.5 0.0 445 β13ZPQ_B 0.7 0.5 0.0 391 β13ZPR_A 0.7 0.5 0.2 460 β13ZPR_B 0.7 0.5 0.1 414 β12Y00_A 0.6 0.6 -0.6 368 β12Y00_B 0.6 0.6 -0.7 325 β12Y01_A 0.6 0.6 -0.4 435 β12Y01_B 0.6 0.6 -0.6 319 β12Y02_A 0.6 0.7 -0.4 430 β12Y02_B 0.6 0.7 -0.5 389 β12Y03_A 0.7 0.7 -0.1 462 β12Y03_B 0.6 0.6 -0.4 348 β12Y04_A 0.6 0.6 0.0 493 β12Y04_B 0.6 0.5 -0.5 406 β13P0G_A 0.9 1.1 -0.6 270 β23PDS_A 0.9 0.7 -0.6 293 β23SN6_Rd 1.0 1.1 -0.7 388 β24LDE_A 0.9 1.2 -0.8 284 β24LDL_A 0.9 1.2 -1.0 273 β24LDO_A 1.0 1.2 -1.0 236 β24GPO_A 1.0 0.9 -1.5 282 β14GPO_B 1.0 0.9 -1.5 283 β12R4R_A 2.4 2.2 2.6 - β22R4S_A 2.4 2.2 2.7 - β23KJ6_A 2.2 2.0 1.4 - β2

H2.64

S5.42

S5.43

S5.46

D45.51

F45.52

Y5.38

D3.32

K/D7.32

Y/F7.35

H2.64

W3.28

F6.51

N6.55

F45.52

A5.39

V3.33

V3.36 W6.48 Y7.43

I7.36

b

a c

Figure 6.1 A visual (a-b) and quantitative (c) analysis of the pocket residues from all X-ray structures. Pocket residues (carbon atoms are colored blue, salmon, white and gray for ANT/iAGO, f/pAGO, FAB-complexed and ligand-free structures respectively) of all chains of all crystal structures (listed in C by their PDB-code followed by their chain identifier) are shown superimposed with the cartoon representation of β2R (2RH1102) and bucindolol (green carbon atoms, 4AMI105), as seen from the side (a) and from the top (b). An overview (c) of a) Average all-atom RMSD using the pocket residues (Å) compared to all 16 f/pAGO and all 27 ANT/iAGO structures (full overview in Table S6.2), b) the average Z-score of the distances between G2.61, D3.32, N7.39 and S5.42 (full overview in Table S6.3, Figure S6.3) and c) the pocket volume (Å3, Figure S6.4). d) The side chains of the ECL2 residues are not all fully resolved, thereby influencing the pocket volume (Figure S6.7) as well as RMSD values. The red and blue background coloring mark values associated with f/pAGO and ANT/iAGO properties, respectively. Icons: f/pAGO (red), ANT/iAGO (blue), or unvalidated ANT(cyan) with no or unknown signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

Note, however, that these differences are small and that mainly the distance between D3.32 and N7.39 to S5.42 varies between the different structures (on average 0.6Å and 1.0Å, respectively). Analysis of volumes of the individual binding pockets (Figure 6.1c, Figure S6.7) indicates that β1R generally has a bigger pocket volume than β2R (on average 415 Å3 compared to 308 Å3). This can mainly be ascribed to the residue differences at positions 7.32 (D/K in β1R/2 respectively) and 7.35 (F/Y in β1R/2 respectively) resulting in a more confined ligand-binding space in β2R (Figure

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6.1, Figure S6.7). Although the RMSD values between all pocket residues are low (Figure 6.1c, Table S6.7), the effect of all subtle changes combined is reflected by the large differences in the pocket volumes. For the β1R and β2R the pocket volumes range from 282 to 493 Å3 and from 236 to 388 Å3, respectively (only comparing structures with fully resolved pocket residues). In line with the results of the distance measurements and the RMSD calculations, the ligand-free structure has the smallest pocket volume of all β1R structures. The active-state adrenaline-bound structure (4LDO103) has the smallest pockets volume of all β2R structures, adapted to this small endogenous agonist.

6.2.1 Identification of ligand-type specific molecular interaction profiles

The binding modes and interaction profiles of the 19 different co-crystallized ligands (Table 6.1 and 6.2) were subsequently investigated using the aforementioned IFP method.45 This technique encodes 7 different types of interactions between the ligand and each of the binding pocket residues. The following interactions with the ligand are encoded: hydrophobic contact, aromatic face-to-edge and face-to-face, H-bond acceptor-donor and donor-acceptor, negative-positive and positive-negative ionic interaction (as shown in Figures 6.2 and 6.5).45 The IFPs for all ligands in all monomers were calculated for the available β-adrenergic crystal structures. The 31 crystal structures contain in total 48 monomers (43 ligand-bound). Of the 14 crystallized ligand-pro-tein complexes containing more than one monomer, only 3 structures had identical interaction patterns within different monomers of the same crystal structure (see Figure 6.2). Moreover, ligands present in multiple co-crystal structures (e.g. cyanopindolol, carazolol, BI-167107) did also not yield identical IFPs. This emphasizes once more that crystal structures are snapshots in time439 of entities that are flexible by nature. The overview of all generated reference IFPs from the ligand-bound crystal structures (Figure 6.2) shows that there are several conserved interactions within the pocket. Apart from dobu-tamine and arylpiperazines 19 and 20, all co-crystallized β1R and β2R ligands contain an etha-nolamine moiety (which is abundant in β-adrenergic ligands368) that can form a tight H-bond interaction network with the sidechains of D3.32 and N7.39 (Figures 6.2, 6.5a/b).367, 389 In chain B of the arylpiperazine 19 structure the ligand does not form H-bonds with N7.39 nor with D3.32. We therefore redocked the co-crystallized ligand into its structure considering alternative ring conformations, resulting in an additional IFP (annotated as 3ZPQ_dock, see Figure 6.2, Figure S6.1, and Materials and Methods).Most β-adrenergic ligands contain an H-bond donor with which they are able to interact with S211/2035.42 (74%, Figure 6.2, Table S6.8).429 More specific for full agonists (carmoterol, iso-proterenol, BI-167107 and FAUC50) is a catechol or a catechol-mimicking moiety with two (or more) H-bond donors that allows for an extra H-bond with another TM5 serine, namely S215/2075.46.440 Partial agonists also have two H-bond donors that can interact with TM5, but their interactions seem to be less optimal (e.g. the shortest observed distance between the H-bond donor from the catechol in dobutamine to the S5.46 acceptor is 3.8Å).429 All ligands also contain at least one aromatic ring, which allows for aromatic stacking with F307/2906.52, F306/2896.51, and F201/19345.52 in 95%, 56% and 54% of all structures respectively. We furthermore observed aromatic interactions with W117/1093.28 for many agonists (60%), ionic interactions with the key ionic anchor D121/1133.32 for all ligands and double H-bond interactions with N329/3127.39 as de-scribed above (see Table S6.8).

135


6 6

Liga

ndAl

pren

olol

3NYA

_Aβ2

89%

25%

Buci

ndol

ol4A

MI_A

β147

%31

%Bu

cind

olol

4AMI

_Bβ1

47%

38%

Cara

zolo

l2R

H1_A

β284

%6%

Cara

zolo

l2Y

CW_A

β189

%13

%Ca

razo

lol

2YCW

_Bβ1

89%

0%Ca

razo

lol

4GBR

_Aβ2

63%

0%Ca

rved

ilol

4AMJ

_Aβ1

42%

13%

Carv

edilo

l4A

MJ_B

β126

%6%

Cyan

opin

dolo

l2V

T4_A

-Dβ1

89%

44%

Cyan

opin

dolo

l2Y

CX_A

β174

%0%

Cyan

opin

dolo

l2Y

CX_B

β174

%13

%Cy

anop

indo

lol

2YCY

_Aβ1

95%

25%

Cyan

opin

dolo

l2Y

CY_B

β184

%13

%Cy

anop

indo

lol

4BVN

_Aβ1

89%

50%

ICI51

18,5

513N

Y8_A

β279

%25

%Io

docy

anop

indo

lol2Y

CZ_A

-Bβ1

95%

31%

Tim

olol

3D4S

_Aβ2

84%

31%

VS5h

it5(K

olb)

3NY9

_Aβ2

74%

6%Ar

ylpi

pera

zine

519

3ZPQ

_Aβ1

11%

0%Ar

ylpi

pera

zine

519

3ZPQ

_Bβ1

21%

0%Ar

ylpi

pera

zine

519

3ZPQ

_doc

kβ1

58%

6%Ar

ylpi

pera

zine

520

3ZPR

_Aβ1

68%

0%Ar

ylpi

pera

zine

520

3ZPR

_Bβ1

42%

0%BI

G167

107

3P0G

_Aβ2

16%

63%

BIG1

6710

73S

N6_R

β258

%56

%BI

G167

107

4LDE

_Aβ2

47%

69%

Carm

oter

ol2Y

02_A

β116

%75

%Ca

rmot

erol

2Y02

_Bβ1

16%

81%

Dobu

tam

ine

2Y00

_Aβ1

0%38

%Do

buta

min

e2Y

00_B

β111

%38

%Do

buta

min

e2Y

01_A

β10%

56%

Dobu

tam

ine

2Y01

_Bβ1

5%50

%Ep

inep

hrin

e4L

DO_A

β20%

50%

FAU

C50

3PDS

_Aβ2

0%44

%Hy

drox

yben

zylIS

O4L

DL_A

β211

%63

%Is

opro

tere

nol

2Y03

_A-B

β126

%75

%Sa

lbut

amol

2Y04

_Aβ1

42%

88%

Salb

utam

ol2Y

04_B

β163

%81

%63

%N

orep

inep

hrin

ea2Y

03_A

β10%

63%

Hydr

opho

bic5

cont

act

Arom

atic

5face

GtoG

face

Arom

atic

5edg

eGto

Gface

Hbon

d5(r

esid

ue5d

onor

)Hb

ond5

(res

idue

5acc

epto

r)Io

nic5

(res

idue

5pos

itive

)Io

nic5

(res

idue

5neg

ativ

e)

W10

73.

285.

466.

48

GL/

HV/

IW

99W

6.51

45.5

145

.50

6.52

45.5

245

.54

5.38

5.39

5.42

5.43

7.40

7.43

6.55

7.35

555555

555Re

sidu

e5C

hain

555555

555555

555555

5552.61

2.64

2.65

7.36

7.39

T

3.32

3.33

3.36

3.37

3.29

D

TM3

ECL2

TM5

FT

YA

SS

DV

VT

CS

Receptor

TM2

ECL1

ANT/iAGO5IFP5≥50.6

f/pAGO5IFP5≥50.6

TM7

V/I

NW

YF/

Y

TM6

WF

FN

Figu

re 6

.2 A

n ov

ervi

ew o

f the

uni

que

inte

ract

ion

finge

rprin

ts o

f all

co-c

ryst

alliz

ed li

gand

s. Th

e co

lors

indi

cate

the

pres

ence

of a

n in

tera

ctio

n (a

s see

n fr

om th

e re

sidue

) ac

cord

ing

to th

e co

lors

des

crib

ed a

t the

bot

tom

of t

he fi

gure

. Ide

ntic

al IF

Ps fo

r mul

tiple

mon

omer

s w

ithin

a P

DB-

entr

y ar

e gr

oupe

d (e

.g. 2

VT4_

chai

nA-D

). Th

e la

st

two

colu

mns

des

crib

e th

e am

ount

of t

imes

(as a

per

cent

age

of th

e to

tal c

ompa

rison

s) a

n IF

P co

mpa

rison

resu

lts in

a sc

ore ≥

0.6

whe

n co

mpa

red

with

the

ANT/

iAG

O

refe

renc

e IF

Ps (a

blu

e ba

ckgr

ound

indi

cate

s a h

igh

perc

enta

ge) a

nd th

e f/

pAG

O re

fere

nce

IFPs

(a re

d ba

ckgr

ound

indi

cate

s a h

igh

perc

enta

ge).

a) Th

e IF

P of

the

high

est

scor

ing

dock

ing

pose

of n

orep

inep

hrin

e in

2Y0

3-ch

ainA

(see

Fig

ure

6.6)

. Nam

es a

nd ic

ons:

f/pA

GO

(red

), AN

T/iA

GO

(blu

e), o

r unv

alid

ated

AN

T392

(cya

n) w

ith n

o or

un

know

n sig

nalin

g pr

efer

ence

(circ

le),

or β

-arr

estin

bia

sed

ligan

ds (t

riang

le) (

see

Tabl

e 6.

1).

136


6 6

All interaction profiles (IFPs) of co-crystallized ligands (8 f/pAGO and 11 ANT/iAGO) in 33 β1R and 15 β2R binding sites were compared and their Tanimoto similarity scores were determined (Table S6.4). Figure 6.2c reports for each individual IFP the percentage of similar IFPs (Tanimoto similarity score ≥ 0.645) derived from (other) ANT/iAGO and f/pAGO co-crystal structures com-plexes. This analysis shows that the IFPs of ANT/iAGO ligands are more similar to each other than to f/pAGO IFPs (75% versus 19% similar pairs respectively). Vice versa, the pairwise similarity between f/pAGO IFPs is higher (62%) than similarity to ANT/iAGO (62% versus 21% respectively, Figure 6.2). Moreover, the arylpiperazine ligands cluster together with the ANT/iAGO IFPs (on average 40% similar pairs compared to 1% similarity with f/pAGO IFPs) suggesting that these compounds are antagonists, as previously proposed.392 These analyses show that the functional effect of a ligand is encoded in the ligand-protein interaction patterns, and suggest that these IFPs can be used to discriminate ANT/iAGO from f/pAGO ligands in structure-based virtual screening studies.

Figure 6.3 Overall enrichment at 1% false positive rate (FP-rate) for the retrieval of 25 partial/full agonists and 25 inverse agonists/antagonists over a set of 980 decoy molecules using IFP scoring (a). Full ROC curves visualizing the retrieval rate (TP-rate) of f/pAGO (red) and ANT/iAGO (blue) in the best ANT/iAGO structure (b) and best f/pAGO structure (c, legend shown in b). The structures are indicated by their PDB code followed by an underscore and the chain identifier (except when there was only one chain or all chains of the structure had similar performance). The 2D structures represent the co-crystallized ligand for selected structures. The ANT/iAGO-axis is scaled from 0 to 60 and the f/pAGO axis from 0 to 100. *1) 3ZPQ_A, 3ZPQ_dock, 2Y00_B. *2) 4AMJ_B, 3ZPQ_B. Icons: fpAGO (red), ANT/iAGO (blue), or unvalidated ANT392 (cyan) with no or unknown signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

6.2.2 Selective retrieval of f/pAGO over ANT/iAGO in structure-based virtual screen-ing

In order to quantify to what extent the differences between the individual structures and the reference IFPs have an influence on the outcome and selectivity of a VS, we docked a set of 1030

137


6 6

compounds comprising 25 f/pAGO, 25 ANT/iAGO and 980 physicochemically similar decoys162 in all β1R and β2R structures. The docking studies were performed using PLANTS345 using the ChemPLP scoring function and the resulting binding modes were post-processed using IFP45 and ranked according to their PLANTS and IFP score. In 85% of all f/pAGO and ANT/iAGO structures IFP scoring yielded equal or higher enrichment factors (EF1%) than ChemPLP-scoring (Figure 6.4, Figure S6.6). Using IFP to rank the docking poses of ligands of the same functional activity class gives a virtual screening enrichment that is on average two times as high as the enrichment obtained by ChemPLP-scoring. Moreover, in 90% of the structures IFP scoring yielded an equal or higher selectivity (higher retrieval of f/pAGO than ANT/iAGO over decoys for f/pAGO structures and vice versa) than when using ChemPLP scoring (Figure 6.4, Figure S6.6). The resulting EF1% values of f/pAGO and ANT/iAGO based on IFP similarity scoring (using the co-crystallized ligand reference IFP) are shown in Figure 6.4a. For 36 of the 43 protein-ligand complexes, the EF1% of ligands with the same functional effect as the co-crystallized ligand was higher than the enrichment of ligands with a different functional effect. Virtual screening runs against five (putative) ANT/iAGO bound receptor struc-tures, namely the arylpiperazine-19-bound β1R complexes (3ZPQ), the bucindolol-β2R complexes (4AMI), and one of the carvedilol-β2R complexes (4AMJ), resulted in a higher enrichment for p/fAGO ligands than for ANT/iAGO ligands. For the arylpiperazine-20-bound β1R structures392 (PDB-code 3ZPR) no enrichment was obtained for any of the known ligands (EF1% = 0). For 11 unique crystal structures (of the 14 structures with multiple monomers) a significant dif-ference in the EF1% is observed (ΔEF1% > 5) between the individual monomers, demonstrating the impact of subtle differences in the reference IFP and/or binding site structure in different monomers of the same crystal structure. For example, the EF1% for f/pAGO over decoys for chain A of structure 2Y02 is 84 compared to 52 for chain B, while the reference IFPs of both chains is identical. This can potentially be ascribed to the larger pocket volume of chain A (490 versus 389 Å3 for chain B, which is mainly due to the rotation of F3257.35), therefore the pocket of chain A is better able to accommodate the larger ligands. In contrast, the small pocket of the ligand-free β1R structure393 (4GPO) is not able to effectively accommodate ligands as D1213.32, F3076.52, and N3297.39 are moved inwards (Figure S6.6), thereby preventing the prediction of correct binding modes using rigid-protein docking simulations. Similarly, the small pocket of the active-state, adrenaline-bound β2R structure (4LDO103) is not able to accommodate the relatively bigger TM5/6 binding moieties of most ANT/iAGO and is therefore more selective for f/pAGO.Figure 6.3 shows that overall the ANT/iAGO ligand-protein complexes are more selective (av-erage EF1% of 27 and 4 for ANT/iAGO and f/pAGO respectively) than the f/pAGO complexes (average EF1% of 13 and 46 for ANT/iAGO and f/pAGO respectively). However, virtual screening against f/pAGO bound structures in many cases gives a high enrichment for both ANT/iAGO and (an even higher enrichment for) p/fAGO ligands. For example, virtual screening against chain A of the salbutamol-bound β1R structure (PDB-code 2Y04), results in a remarkably high enrich-ment factor for both f/pAGO and ANT/iAGO (EF1% of 72 and 32 respectively). To evaluate the individual contribution of the reference IFPs and the protein structure we re-scored all docked compounds in all structures with all reference IFPs (i.e. not only with the IFP that was derived from the corresponding receptor-ligand complex). This furthermore allowed us to rescore the docked compounds with IFP in the structures without a ligand (PDB-codes: 2R4R, 2R4S, 3KJ6, and 4GPO). The resulting scoring-matrices containing the EF1% of f/pAGO and ANT/iAGO are shown in Figure 6.4a and 6.4b respectively.

138


6 6

03

58

1013

1518

2023

2528

3033

3538

4043

4548

5053

5558

6063

6568

7073

7578

8083

8588

9093

9598

100

EF 1

% (f

/pA

GO

)

PLANTS

2RH1_A

2VT4_A-D

2YCW_A

2YCW_B

2YCX_A

2YCX_B

2YCY_A

2YCY_B

2YCZ_A-B

3D4S_A

3NY8_A

3NY9_A

3NYA_A

4AMI_A

4AMI_B

4AMJ_A

4AMJ_B

4BVN_A

4GBR_A

3ZPQ_A

3ZPQ_B

3ZPQ_dock

3ZPR_A

3ZPR_B

2Y00_A

2Y00_B

2Y01_A

2Y01_B

2Y02_A

2Y02_B

2Y03_A-B

2Y04_A

2Y04_B

3P0G_A

3PDS_A

3SN6_R

4LDE_A

4LDL_A

4LDO_A

Norepiphrine

2RH1

_Aβ2

80

80

00

88

08

40

012

2016

00

120

816

360

016

2012

1232

1656

4044

2028

4044

4452

482V

T4_A

β18

00

40

04

40

00

120

08

200

00

012

1628

00

128

812

2424

4452

4824

80

1216

5256

2VT4

_Bβ1

80

04

00

00

00

00

40

2424

88

40

164

240

016

2828

2424

3636

4036

2836

440

4432

442 V

T4_C

β112

00

40

00

00

04

00

424

2412

120

08

428

00

1216

1624

3632

4444

3632

248

4836

5244

2VT4

_Dβ1

80

00

04

88

00

012

00

1628

04

80

812

160

00

00

812

1648

4852

124

44

1656

602Y

CW_A

β120

00

00

44

80

84

120

424

124

420

04

1220

00

1216

1216

3216

6464

6036

1624

4436

3272

2YCW

_Bβ1

160

80

00

412

08

412

012

208

88

200

824

320

012

1620

1232

3272

5660

2416

816

4044

482Y

CX_A

β120

012

00

08

120

160

44

012

160

020

04

836

00

88

128

160

4444

368

164

1616

4048

2YCX

_Bβ1

280

80

00

88

08

48

04

812

44

160

412

440

012

1616

2020

844

4036

1612

828

2832

442 Y

CY_A

β116

00

00

00

00

00

00

012

2012

120

04

1216

00

424

164

2020

5244

4040

164

2828

4444

2YCY

_Bβ1

80

00

00

00

00

00

00

2816

00

00

84

40

08

812

1220

2044

4036

3616

828

3640

402Y

CZ_A

β18

00

00

00

00

00

00

016

160

80

08

412

00

80

412

128

4036

4020

40

1632

2044

2YCZ

_Bβ1

120

00

00

00

00

00

00

48

00

00

04

160

012

128

48

848

3636

2416

432

2020

443D

4S_A

β216

00

00

00

00

00

00

412

44

48

04

820

00

812

1216

4036

4040

4032

2412

4844

4036

3NY8

_Aβ2

820

00

124

00

00

00

00

2432

00

80

2020

320

012

128

436

1236

2432

168

848

5244

403 N

Y9_A

β212

08

04

00

80

80

00

420

160

08

08

820

00

812

88

3212

4036

3220

424

4848

3244

3NYA

_Aβ2

360

00

00

00

04

00

00

2816

44

80

44

240

012

1616

2420

2440

4036

248

1644

4440

404A

MI_A

β116

812

44

48

160

208

80

824

284

816

08

1220

00

1616

1620

3636

3644

3620

248

3240

2836

4AMI

_Bβ1

244

160

08

420

012

48

08

2828

04

80

1216

324

012

1216

2452

3656

5248

3228

832

2848

484A

MJ_A

β112

00

00

00

40

00

44

020

168

48

08

2436

00

2016

2028

4028

5632

4428

168

2428

5240

4AMJ

_Bβ1

120

00

00

44

00

40

00

44

128

40

1624

160

016

2416

1636

2464

4452

2820

420

3256

644 B

VN_A

β18

00

00

04

40

04

00

028

124

48

016

1216

00

1612

1216

3620

4840

4020

288

3628

5244

4GBR

_Aβ2

80

00

00

00

80

00

00

2028

44

40

88

160

04

44

04

1240

1624

1220

012

3224

403Z

PQ_A

β18

00

00

08

80

00

120

40

164

48

016

828

00

1620

1620

3220

5244

4024

288

2424

4840

3ZPQ

_Bβ1

124

00

00

88

04

08

04

124

04

40

168

160

016

1616

1644

3244

4836

1632

824

3256

443Z

PR_A

β112

00

04

08

40

00

120

816

244

40

08

1616

00

816

1616

412

5232

3612

160

020

4440

3ZPR

_Bβ1

120

80

00

128

08

84

04

84

40

00

128

80

020

1616

1212

840

4032

1620

08

2440

362 Y

00_A

β132

820

44

812

288

128

48

1212

2016

1612

012

824

00

1616

1616

7252

6464

6440

4412

6836

6460

2Y00

_Bβ1

288

248

44

820

412

412

04

1620

84

120

812

200

016

1616

2468

5652

5248

3648

2072

3260

562Y

01_A

β112

824

120

128

288

44

120

820

2012

416

48

1616

00

1216

1224

4852

5256

6024

3212

6424

4448

2Y01

_Bβ1

1212

164

04

012

44

812

00

2016

1212

124

1212

204

016

1212

2472

4436

5232

2840

2468

2040

362Y

02_A

β120

1216

44

48

2812

128

08

2012

1612

824

48

420

00

1620

1628

8456

6860

4840

4444

8060

6472

2Y02

_Bβ1

1620

1612

412

424

2412

124

412

2020

1624

164

48

284

012

2012

2080

5264

6868

4044

5284

5248

722 Y

03_A

β112

420

00

04

164

88

44

812

812

1216

08

412

00

1212

2020

6864

6468

6444

4016

8456

5268

2Y03

_Bβ1

3212

2412

48

412

124

200

012

168

1616

164

84

40

024

1620

2476

5256

4844

3640

2872

4068

562Y

04_A

β112

412

00

820

1612

120

40

416

1620

1220

04

1216

00

1620

1624

8052

6472

7236

4420

4440

8080

2Y04

_Bβ1

124

124

08

816

816

80

00

3216

124

204

816

440

016

1612

1636

3260

5644

2828

3240

3248

443P

0G_A

β244

012

00

48

164

1612

80

128

84

828

00

08

00

016

120

4440

4440

4036

3244

6440

4448

3PDS

_Aβ2

280

44

08

416

120

48

00

124

128

120

44

40

012

84

1248

3640

4840

2832

3660

4056

523S

N6_R

β248

1224

48

48

4012

248

124

832

284

2824

04

020

00

1220

1612

6032

6036

3644

4464

6432

6460

4 LDE

_Aβ2

164

40

04

416

40

124

40

2416

08

160

812

40

012

128

1676

4836

3636

4036

4872

3652

524L

DL_A

β216

40

80

84

120

012

40

012

324

08

08

48

00

08

88

6032

5240

4820

3648

5232

4452

4LDO

_Aβ2

284

40

04

48

40

84

00

128

88

40

44

40

012

208

2036

3636

5232

2428

2880

2448

404G

PO_A

β116

00

00

40

04

40

00

00

04

40

00

416

00

2012

2424

2832

820

820

368

324

1628

4GPO

_Bβ1

200

80

00

00

00

40

00

00

40

00

44

120

024

2028

2420

2024

2416

424

828

020

162 R

4R_A

β212

2032

4428

2416

3232

3228

128

1216

164

432

020

2044

164

40

48

2020

2020

2012

436

1212

2824

2R4S

_Aβ2

1212

3636

1624

2436

2436

208

04

2020

2020

168

2416

4432

80

120

1216

1616

164

128

4412

1620

123K

J6_A

β28

1620

2024

160

2012

2016

168

1216

164

48

416

2420

2416

44

84

48

1212

124

80

48

1220

IFP

Structure

Figure 6.4a Enrichment factors at a 1% false positive rate using all reference IFPs (columns) on all β-adren-ergic monomers for the retrieval of for f/pAGO over physicochemically similar decoys. The white to red and white to blue gradients as background color mark a low to high enrichment for f/pAGO (A) and ANT/iAGO respectively. Icons: fpAGO (red), ANT/iAGO (blue), or unvalidated ANT392 (cyan) with no or unknown signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

139


6 6

PLANTS

2RH1_A

2VT4_A-D

2YCW_A

2YCW_B

2YCX_A

2YCX_B

2YCY_A

2YCY_B

2YCZ_A-B

3D4S_A

3NY8_A

3NY9_A

3NYA_A

4AMI_A

4AMI_B

4AMJ_A

4AMJ_B

4BVN_A

4GBR_A

3ZPQ_A

3ZPQ_B

3ZPQ_dock

3ZPR_A

3ZPR_B

2Y00_A

2Y00_B

2Y01_A

2Y01_B

2Y02_A

2Y02_B

2Y03_A-B

2Y04_A

2Y04_B

3P0G_A

3PDS_A

3SN6_R

4LDE_A

4LDL_A

4LDO_A

Norepiphrine

2RH1

_Aβ2

812

4840

836

3652

4448

3624

1244

128

48

520

164

204

84

84

420

824

2824

88

4424

816

02V

T4_A

β112

1248

368

3220

4436

4836

3628

3212

208

1640

04

020

40

48

44

2016

2036

288

1232

204

2812

2VT4

_Bβ1

124

4824

836

3248

4848

4040

1644

2028

88

440

120

160

44

44

412

1224

3636

128

2832

88

82V

T4_C

β112

2044

3216

2824

4440

4436

3232

3612

168

844

08

012

00

88

412

168

1636

404

420

3212

208

2VT4

_Dβ1

816

3628

1628

2432

4436

4028

1632

128

812

444

40

168

04

44

424

1220

3628

1612

2820

824

202Y

CW_A

β116

2428

3620

4024

4440

3628

3220

3616

1212

1652

412

012

04

412

44

2012

2432

2416

1224

2012

012

2YCW

_Bβ1

1216

4432

2040

2832

4444

4040

2040

1212

88

444

120

120

44

412

416

824

2424

1212

2820

1212

82 Y

CX_A

β116

1244

2420

2832

4444

4444

448

248

88

440

48

016

04

44

44

204

2424

244

416

248

2016

2YCX

_Bβ1

1212

4012

1616

2032

4440

3636

1220

412

84

364

40

160

04

48

816

816

2420

164

2416

1212

02Y

CY_A

β18

1252

4024

2828

4852

5636

4016

4016

44

452

44

012

00

412

44

128

432

2016

832

2812

128

2YCY

_Bβ1

436

4036

3224

3240

4056

2820

040

1212

88

440

80

200

44

44

416

816

3624

1612

2020

168

122Y

CZ_A

β18

1640

2812

2836

4036

3636

408

408

128

844

44

028

00

44

44

84

1632

368

412

1612

1212

2YCZ

_Bβ1

84

3620

824

2840

4436

2816

1240

816

44

360

40

120

04

40

412

420

3632

84

1624

820

163D

4S_A

β220

1260

2416

2832

4448

5236

3232

488

124

448

48

04

00

48

44

2012

424

244

844

284

164

3NY8

_Aβ2

836

3632

3232

2036

3236

3224

1232

812

48

324

84

200

08

44

412

412

424

44

3228

88

203N

Y9_A

β216

1644

2020

3632

3228

4428

2020

284

84

444

012

416

04

44

48

88

2024

204

440

244

168

3NYA

_Aβ2

2012

3612

816

2040

2836

3232

2028

08

88

320

80

40

04

44

44

420

2020

40

2832

128

204A

MI_A

β124

2432

2824

2816

3236

3224

2016

3212

1212

428

48

416

416

412

84

84

1224

164

828

128

812

4AMI

_Bβ1

1632

2428

2820

2428

2832

3232

1620

1216

84

284

124

2012

124

84

416

88

2020

48

2420

84

124A

MJ_A

β124

1648

3220

2420

3636

4432

2420

368

2012

840

44

012

00

88

84

44

2020

408

1220

164

208

4AMJ

_Bβ1

2824

4428

1632

2036

4444

3636

1644

88

84

524

204

120

84

48

48

1236

4844

1212

1612

1212

84B

VN_A

β112

432

2412

1628

3624

3236

2420

248

168

436

08

48

00

44

44

124

3232

324

1236

324

824

4GBR

_Aβ2

816

3616

1236

2028

2036

3212

832

816

84

2016

120

120

04

44

08

016

2024

80

128

80

43Z

PQ_A

β112

2028

3228

4020

3236

2840

1616

244

1212

424

84

48

04

412

1216

1216

2028

3612

012

248

84

3ZPQ

_Bβ1

1628

3632

2432

2036

3236

4824

2020

1616

88

204

84

40

04

124

1220

824

1220

88

2020

816

243Z

PR_A

β18

1632

3220

4020

2824

3228

2420

2812

124

432

88

44

00

48

44

80

820

244

84

44

44

3ZPR

_Bβ1

812

3628

1236

1620

2832

4024

812

412

84

164

124

80

04

44

48

012

1616

40

04

88

02Y

00_A

β112

2816

2824

1612

2420

1616

84

1612

1612

1212

08

412

04

812

48

168

824

2012

420

208

128

2Y00

_Bβ1

1612

2424

1212

424

2820

168

1220

120

124

204

40

00

44

44

420

1212

2812

812

2416

84

42Y

01_A

β18

1616

2412

1212

2424

2820

1216

248

412

824

44

04

416

48

44

128

1216

2012

424

168

84

2Y01

_Bβ1

1212

168

44

04

812

84

128

1620

48

40

00

40

44

84

412

88

2012

84

88

44

42Y

02_A

β18

3216

2820

164

1224

124

812

812

1212

420

164

04

04

48

48

2412

1620

1612

1228

328

48

2Y02

_Bβ1

1224

2828

1624

820

2420

2016

820

88

1212

124

00

84

84

44

412

412

2020

44

2020

44

82Y

03_A

β112

2024

2428

168

2428

2020

88

164

412

1224

80

04

44

44

44

1616

2024

248

812

2812

48

2Y03

_Bβ1

1228

2832

1616

816

2020

2012

1216

44

88

244

48

80

04

84

424

128

1216

84

2020

820

122Y

04_A

β18

1224

288

3220

2836

2424

3212

2020

164

428

84

04

44

48

44

128

2032

368

824

208

1216

2Y04

_Bβ1

1216

2424

1224

1624

3220

2420

1220

88

124

284

00

88

124

48

44

1216

3232

124

2012

80

43P

0G_A

β212

48

04

00

40

124

44

120

04

412

00

124

00

04

44

124

44

44

416

168

84

3PDS

_Aβ2

80

120

00

1212

1212

1616

812

84

44

160

00

00

04

40

412

816

1624

1212

2820

88

123S

N6_R

β212

820

40

124

1224

2012

1616

48

012

1620

08

08

44

44

44

44

416

88

412

84

48

4LDE

_Aβ2

124

2420

88

1228

2412

2428

1212

44

88

240

80

80

00

44

416

40

2016

84

2820

412

44L

DL_A

β212

824

208

124

2420

2012

84

88

120

1228

04

88

48

08

04

164

44

84

416

124

04

4LDO

_Aβ2

120

164

08

812

812

812

128

88

48

40

44

80

04

44

44

84

124

84

1616

412

04G

PO_A

β10

00

00

04

40

00

00

04

04

40

00

00

00

00

00

00

00

04

04

40

00

4GPO

_Bβ1

40

40

00

44

44

00

04

00

00

00

00

00

04

04

40

04

44

04

00

00

02R

4R_A

β28

2024

2824

2012

2420

2420

128

816

164

412

00

016

816

00

00

1616

168

124

44

416

04

2R4S

_Aβ2

1212

2832

2824

1228

3228

2812

1616

2424

44

200

124

812

164

84

432

3228

84

44

284

324

03K

J6_A

β212

424

2420

44

2412

2424

208

2416

164

416

08

208

04

88

1212

88

1624

244

1212

1212

44

03

58

1013

1518

2023

2528

3033

3538

4043

4548

5053

5558

6063

6568

7073

7578

8083

8588

9093

9598

100

EF 1

% (A

NT/

iAG

O)

IFP

Structure

Figure 6.4b Enrichment factors at a 1% false positive rate using all reference IFPs (columns) on all β-adren-ergic monomers for the retrieval of for ANT/iAGO over physicochemically similar decoys. The white to red and white to blue gradients as background color mark a low to high enrichment for f/pAGO (A) and ANT/iAGO respectively. Icons: fpAGO (red), ANT/iAGO (blue), or unvalidated ANT392 (cyan) with no or unknown signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

140


6 6

In Figure 6.4a we see that also ANT/iAGO structures can be used to efficiently retrieve f/pAGO ligands when using the right reference IFP. This is surprising, as this contrasts the results of the previous VS studies in which the rotamers of the TM5 serines and/or whole TM5 had to be rotated for the efficient retrieval of f/pAGO ligands.162, 276, 404, 412 Interestingly, the isoproterenol IFP (PDB-code 2Y03) allows efficient retrieval of f/pAGO ligands in all ANT/iAGO structures with an average EF1% of 47. Ranking docking poses in f/pAGO structures with an ANT/iAGO reference IFP on the other hand does not yield high enrichments of ANT/iAGO ligands (with an average ANT/iAGO EF1% of 13). The highest average EF1% (21) was obtained by the cyanopindolol IFP (2YCY_B) in the f/pAGO structures. An unanticipated finding was that when pairing specific f/pAGO IFPs with ANT/iAGO structures also high EF1% values were obtained for the selective retrieval of ANT/iAGO. When using the IFPs of salbutamol (pAGO, PDB-code 2Y04) an average ANT/iAGO EF1% of 27 was obtained (Figure 6.4a/b). In short, these statistics emphasize that not only the choice of structure but also the choice of reference IFP has a high impact on the resulting enrichment factors and, more inter-estingly, on the ligand selectivity of the results.

6.2.3 Selective structure-based VS for ligands with the desired functional effect

Based on the highest EF1%s (Figure 6.3) we selected one f/pAGO and one ANT/iAGO ligand-pro-tein complex for a more detailed investigation. From all ANT/iAGO-bound complexes the cy-anopindolol-bound (iAGO) β1R structure 2VT4 (both chains A and B) obtained the highest ANT/iAGO EF1% value (48) and a f/pAGO EF1% value of 0. As both chains gave similar virtual screening enrichments (Figure 6.4b), we randomly selected chain A of 2VT4 (Figure 6.5b) for further in-vestigation. From all f/pAGO complexes chain A of the carmoterol (fAGO) bound structure β1R structure 2Y02 (Figure 6.5a) obtained the highest f/pAGO EF1% value (84) and an ANT/iAGO EF1% value of 24. In figure 6.3b and 6.3c the individual ROC-plots are displayed for the retrieval of ANT/iAGO and f/pAGO over decoys by IFP-similarity scoring of docking poses generated in the 2VT4 and 2Y02 complexes using the reference IFPs of the corresponding co-crystallized ligands. Figures 6.3b/c show the high early and overall enrichment for ligands with the same functional effect of the co-crystallized ligand and much lower early and overall enrichment for ligands with a different functional effect.By rescoring all the docking poses of the compounds that were docked in all 48 structures with the reference IFPs from the two selected structures the impact of the reference IFP selection was investigated (Figure 6.5d/e). For the selected f/pAGO IFP (2Y02_A), 93% of all ligand-pro-tein complexes (excluding the 2Y02 structures) yielded an equal or higher f/pAGO EF1% than when using their own IFP, in 64% of all cases a lower ANT/iAGO EF1% and for almost all ligand-pro-tein complexes a higher f/pAGO EF1% than ANT/iAGO EF1% (Figure 6.5d, 89% of all cases). When using the selected ANT/iAGO IFP (2VT4_A) instead of the IFP of the co-crystallized ligand to rescore all docked compounds in the ligand-protein complexes a higher ANT/iAGO EF1% (80% of all cases), a lower f/pAGO EF1% (51% of all cases) and a higher ANT/iAGO EF1% than f/pAGO EF1% (82% of all cases) was obtained (Figure 6.5e). In two cases the use of the selected ANT/iAGO IFP resulted in an even higher ANT/iAGO EF1% than with its own structure, namely in combination with the structure of cyanopindolol-bound β1R (EF1% 52, 2YCY_A) and timolol-bound β1R (EF1% 60, 3D4S). This is an increase of 4 and 24 in ANT/iAGO EF1% for the 2YCY_A and 3D4S structures respectively compared to the enrichment obtained with their native IFP. This was not observed for the selected f/pAGO IFP as the combination with its own structure (2Y02_A) resulted in

141


6 6

the highest f/pAGO EF1% (84). The results for one of the Fab-complexes (2R4S) were somewhat surprising as the f/pAGO EF1% in combination with the selected ANT/iAGO IFP were higher than with the selected f/pAGO IFP (36 and 16 respectively). Combining this with the fact that this structure is missing 23 of the binding pocket residues (vide supra), it was even more surprising that these enrichment factors can be obtained when docking in a structure with only half of the binding pocket. This does, however, match the success of previously published VS studies using homology models without ECL2160 and low-resolution homology models.122 Overall it is clear that the selection of the reference IFP is essential and can influence the enrichment as well as selec-tivity of the outcome of a VS study regardless of which structure was used.In order to separately quantify the impact of the structure on the enrichment values we studied the effect of rescoring all docking poses in the selected two structures with all 39 reference IFPs (Figure 6.5f/g). The results of rescoring the docking poses in the 2 selected structures with all reference IFPs show similar trends as rescoring with the reference IFP of the selected complexes (Figure 5d/e). For the selected f/pAGO structure (2Y02_A, Figure 6.5f) we see a shift in the results towards the f/pAGO and the opposite for when using the selected structure (2VT4_A, Figure 6.5g). The observed effect is, however, less pronounced. When we compare the obtained enrichment factors they are on average 38% lower for the selected structures than when using their IFP to rescore the docking poses. Moreover, with the right IFP the selectivity of a crystal structure the can be shifted towards ligands with another functional effect than the co-crystallized ligand (Figure 6.5d/e), the oppo-site (shifting the selectivity by using the right structure) is less frequent and less prominently observed. The selection of the crystal structure does, however, clearly influence the extent to which the IFP to able to shift this selectivity and also the final enrichment values. Combined these results indicate that the correct IFP is more crucial for obtaining selectivity as well as high enrichments than the structure, but they also indicate that obtaining the best results are obtained by finding the right combination of IFP and structure.

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6 6

0 10 20 30 40 50 60

020

4060

8010

0

EF1% ANT/iAGO

EF1%

f/pA

GO

●●

●●●●

●

●●●

●

●

●

●●

●

●

● ●

●

●

●●

●

●●●

p/fAGOANT/iAGOANT (Not validated)

0 10 20 30 40 50 60

020

4060

8010

0

EF1% ANT/iAGO

EF1%

f/pA

GO

●

●●●●●

●●

●

●●●● ●●

●

● ●● ●●

●

●●

●●

● ●●

●

●●

●

●●●●●

p/fAGOANT/iAGOANT (Not validated)Fab−complexLigand−free

0 10 20 30 40 50 60

020

4060

8010

0

EF1% ANT/iAGO

EF1%

f/pA

GO

●

●●

●

●

●●

●●● ●

●●

●●

●●

●

●

●

●

●

●

●●

●

●

●

●

● ●●

●

●●●●●


0 10 20 30 40 50 60

020

4060

8010

0

EF1% ANT/iAGO

EF1%

f/pA

GO

● ●●

● ●● ●

● ●●

●

● ● ●●

●●

●

●●

●

●●

●

●

●●●

p/fAGOANT/iAGOANT (Not validated)

e d

c

N6.55%

D3.32%

N7.39%

S5.46%

S5.42%S5.43%

N6.55%

D3.32%

N7.39%

S5.46%

S5.42%S5.43%

Agonist: 2Y02 (chain A) Antagonist: 2VT4 (chain A) a

2VT4 IFP in all structures 2Y02_A IFP in all structures

All IFPs in 2VT4_A structure All IFPs in 2Y02_A structure

4GPO_A

4GPO_A

4LDE

2YCZ

Ligand StructureCarmoterol 2Y02_chainAProcaterol 2Y02_chainA

Cyanopindolol 2VT4_chainAAlprenolol 2VT4_chainA

N7.39D3.32 S5.42 S5.43 S5.46 N6.55

b

g f

Figure 6.5 Analysis of VS runs using the IFPs (d-e) and structures (f-g) of the most f/p AGO selective (2Y02_A) and ANT/iAGO selective (2VT4_A) protein-ligand complexes (see Figure 3A) show that both the reference IFP and protein structure determine enrichment and functional selectivity of the VS study. The

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binding modes of the selected f/pAGO (a, 2Y02_A) and selected ANT/iAGO (b, 2VT4_A) protein-ligand complex, a docked ligand with the same efficacy class (green carbon atoms) and their corresponding IFPs (c) for the displayed residues. EF1% results for agonists and antagonist versus the decoys when using the reference IFP of the selected f/pAGO (d) and selected ANT/iAGO (e) for scoring docked compounds in all 48 protein structures. EF1% results for f/pAGO and ANT/iAGO versus the decoys when using the 38 unique reference IFPs of all ligand-protein complexes for rescoring the docked compounds in the 2Y02_A (f) and 2VT4_A (g) structure. Icons: fpAGO (red), ANT/iAGO (blue), or unvalidated ANT392 (cyan) with no or un-known signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

6.2.4 Selective structure-based virtual screening for partial/full agonists with a com-putationally predicted norepinephrine interaction fingerprint

From the f/pAGO and ANT/iAGO retrieval rates in all structures (Figure 6.4a/b), we can see that in particular the IFP of epinephrine (PDB-codes 4LDO) is able to selectively retrieve f/pAGO ligands in all structures (with an average f/pAGO and ANT/iAGO EF1% of 43.6 and 9.4 respectively). This small endogenous agonist makes all key interactions with D1133.32, N3127.39 and S2035.42 in β2R (Figure 6.2). Based on this observation we hypothesized that the IFP of an even smaller agonist, that could also make all these interactions, should also be highly effective for the retrieval of f/pAGO over ANT/iAGO in all structures. As the crystal structure of norepinephrine bound to β1R or β2R has not yet been resolved we docked norepinephrine and used the docking pose (Figure 6.6b) with the highest IFP-score (compared to the IFP of isoproterenol) to obtain the reference IFP for norepinephrine in β1R (Figure 6.2).Indeed, when using this computationally predicted IFP to rescore all docked compounds in all structures we were able to retrieve f/pAGO ligands with a high efficiency (the average f/pAGO and ANT/iAGO EF1% were 46 and 8 respectively, Figure 6.4a and 6.6). On average the obtained f/pAGO EF1% was almost twice as high compared to the 2Y02_A IFP (the IFP from the highest-scoring f/pAGO complex). It should be noted that this is mostly due to higher f/pAGO EF1% in the ANT/iAGO structures. At the same time a 20% lower ANT/iAGO EF1% was obtained compared to the 2Y02_A IFP. Moreover, the ANT/iAGO structure that obtained the highest f/pAGO EF1% with this IFP was a cyanopindolol-bound β1R structure (2YCW_A, Figure 6.6a), which coincides with the slight contraction of the pocket and the low f/pAGO RMSD as previously de-termined (Figure 6.1c). Interestingly, also in combination with the selected ANT/iAGO structure (cyanopindolol-bound 2VT4_A) the norepinephrine IFP was able to obtain a high and selective retrieval rate for f/pAGO (EF1% of 56 and 12 for f/pAGO and ANT/iAGO respectively, Figure 6.6c). The norepinephrine IFP combined with the 2Y02_A structure resulted in a slightly reduced f/pAGO EF1%, but at the same time reduced the ANT/iAGO EF1% thereby making the results much more selective (Figure 6.6d).By using the IFP from a small compound that makes all key interactions these key interactions are highly emphasized, yielding more selective results. Whenever a docking pose of a compound misses one or more of the key interactions present in the reference IFP this immediately results in a relatively low IFP score.

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0 10 20 30 40 50 60

020

4060

8010

0

EF1% ANT/iAGO

EF1%

f/pA

GO

●

●

●●

●

●

● ●● ●

●● ●

●●

● ●●●

●●

●

●

●

●

●

●

●

●

●

●

●

●

●

●●●●●


0.1

0.5

1.0

5.0

10.0

50.0

100.0

020

4060

80100

2Y02_A

f/pAGOANT/iAGORandom

0.1

0.5

1.0

5.0

10.0

50.0

100.0

020

4060

80100

2VT4_A

f/pAGOANT/iAGORandom

b!

c!

1.0

1.2

1.4

1.6

1.8

2.0

020

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8010

0

FP−rate (%)

TP−r

ate

(%)

1.0

1.2

1.4

1.6

1.8

2.0

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8010

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FP−rate (%)

TP−r

ate

(%)

a!

d!

1.0

1.2

1.4

1.6

1.8

2.0

020

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8010

0

FP−rate (%)

TP−r

ate

(%)

1.0

1.2

1.4

1.6

1.8

2.0

020

4060

8010

0

FP−rate (%)

TP−r

ate

(%)

N6.55%

D3.32%

N7.39%

S5.46%

S5.42%

S5.43%

2Y03_A

4LDE 4LDL

4LDO 4GPO_A

3KJ6_A

Figure 6.6 EF1% results for agonists and antagonists (a) when using the reference IFP of docked norepi-nephrine (Figure 6.2) in 2Y03_A (b). The individual ROC-plots for the retrieval of agonists (red curve) and antagonists (blue curve) in the selected ANT/iAGO structure 2VT4_A (c) and f/pAGO structure 2Y02_A (d). Icons: fpAGO (red), ANT/iAGO (blue), or unvalidated ANT392 (cyan) with no or unknown signaling preference (circle), or β-arrestin biased ligands (triangle) (see Table 6.1).

6.3 Biased agonists

G-protein-coupled receptors can signal via G-proteins as well as β-arrestins.441 So-called biased ligands can induce a preference of the receptor for one pathway over the other.407 An in-depth literature search learned that for 7 of the 19 co-crystallized ligands there is a record of biased signaling (Table 6.1). Amongst them are bucindolol and carvedilol, two β-blockers that promote signaling via the β-arrestin pathway.105 One should note that the signaling bias of compounds is not always consistently observed and reported (e.g. for cyanopindolol205, 430, 434, 435, carvedilol105, 430, 432, 435, dobutamine430, 436) as the effect seems to be dependent on assay type and condi-tions436, receptor-activation state437, and receptor subtype430 (β1 versus β2). For another 7 of the 19 co-crystallized compounds we consistently found one or more publications reporting that ligand signaling is not biased (Table 6.1). It should be noted that the 7 biased ligands are all, with the exception of alprenolol, relatively large compounds (Figure 6.1, Table 6.1) that stretch from the major pocket (between TM3, 4, 5, and 6) into the minor pocket389 (between TM1, 2, 3, and 7).To identify potential key interactions for β-arrestin biased ligand, we grouped all IFPs (Figure 6.2) of both the reported 7 biased and the reported 7 unbiased ligands. Subsequently, we calculated the difference in abundance for each interaction with each pocket residue (Table S6.8). This highlighted four interactions that had a high abundance (≥50%) and were unique for the biased ligands. Three of them are hydrophobic contacts, namely with L101/H932.64, D200/19245.51, and V326/I3097.36 and the fourth is an aromatic stacking interaction (face-to-face or face-to-edge) with W117/1093.28. Interestingly, this last interaction was present for all biased ligands, except for alprenolol. This could indicate that the signaling bias for these ligands is (partially) due to the aromatic stacking with this tryptophan residue. Moreover, isoproterenol is a full agonist with no signaling bias (it is even used as reference ligand442), but when a hydroxybenzyl moiety is at-tached the ligand (hydroxybenzylisoproterenol, 4LDL) makes an aromatic interaction with W3.28

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and induces a β-arrestin signaling bias.395 This is also in line with mutation studies of the homol-ogous W3.28 in the muscarinic M2 receptor (also a member of the aminergic-GPCR family72) that changed the signaling preference of the receptor when stimulated by specific ligands.443 Also the contacts with L/H2.64, D45.51, a

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