Ntroduction

G protein-coupled receptors (GPCRs) span six subtypes and constitute the single largest protein target class of all marketed drugs [ 1, 2] . All GPCRs contain a transmembrane-spanning region comprised of seven a-helices, linked by alternating intracellular and extracellular loops (ICLs and ECLs). X-ray crystallography-derived structures would facilitate the application of structure-based drug-design approaches to these targets. Due to low concentrations, conformational flexibility, and instability in detergent solutions, the

GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions,

Edited by Annette Gilchrist

Copyright © 2010 John Wiley & Sons, Inc.

purification and crystallization of GPCR targets have been challenging. Consequently, the only GPCR X-ray structures of the 7TM region available until 2007 have been those of bovine rhodopsin (a Class A GPCR), which is not ligand-activated and generally has less than 20% sequence identity relative to other Class A GPCR proteins. Thus, the use of a bovine rhodopsin X-ray structure as a template for homology modeling of other GPCR targets, particularly non-Class A GPCRs, may provide questionable results due to uncertainties in the alignment in loop regions (where the homology is generally very low) and expected variations between different proteins in binding-site relaxation (which may require the rearrangement of a-helices).

The first high-resolution X-ray structures of GPCR proteins began to emerge in 2000 (see Table 16.1 and references therein). Important insights into the mechanism of rhodopsin activation were facilitated [3] by studies that followed the publication of the bovine rhodopsin X-ray structures. However, it soon became apparent (e.g., References 4-6) that the construction and use of homology models for ligand-mediated Class A GPCRs would be challenging and likely produce results with limited accuracy. This is in contrast to other target classes such as kinases and proteases, where the use of X-ray structures and homology models based on closely related proteins is often possible. For structure-based drug design, the recently published X-ray structures of ligand-activated GPCR proteins should provide a distinct advantage over previously published X-ray structures of bovine rhodopsin, a light-activated Class A GPCR to which the endogenous chromophore retinal is covalently bound in the "dark" (inactive) state. The now commonly used structure -based drug-design methods, including the construction and use of homology models, may be applied to GPCR targets, especially in the more homologous, common seven-transmembrane structural core. Early questions to be addressed include the following: (1) Would GPCR structures within the same class or across classes demonstrate significant differences? (2) Could reliable models be built for other states of GPCRs, especially because it is believed that GPCR proteins exist in multiple states that depend on the nature and function of the target's ligands [7-13]? Most likely, separate models would have to be generated to analyze agonists, antagonists, and inverse agonists. In addition, as multiple conformations of an active state may exist (possibly leading to ligand-biased trafficking [14]), separate models for different activating ligands may be needed. (3) Should a GPCR target be modeled as a monomer, homodimer, heterodimer, or oligomer? These potential differences could profoundly affect structure -based drug design. (4) Do the available X-ray structures provide enough information to understand the molecular mechanisms of GPCR activation and G protein coupling?

In this chapter, we summarize the impact of published Class A and Class C GPCR X-ray structures that help to address several pertinent questions, and we examine early studies that may provide partial answers. More detailed information on the Class A GPCR X-ray structures is available [15-17].

436 gpcr molecular pharmacology and drug targeting TABLE 16.1 X-Ray Diffraction GPCR Structures Released by the PDB

PDB

Resolution

R-Free

Date

Protein and

Literature

ID

(Â)

Released

Chromophore/Ligand

Reference

A. (Rhod)opsin (a Class A GPCR)

1F88

2.8

0.238

August 4,

Bovine rhodopsin with

[85]

2000

retinal

1HZX

2.8

0.212

July 4,

Bovine rhodopsin with

[86]

2001

retinal

1L9H

2.6

0.225

May 15,

Bovine rhodopsin with

[3]

2002

retinal

1GZM

2.65

0.235

November

Bovine rhodopsin with

[87]

20, 2003

retinal

1U19

2.2

0.222

October

Bovine rhodopsin with

[88]

12, 2004

retinal

2HPY

2.8

0.238

August 22,

Bovine lumirhodopsin

[89]

2006

with retinal

2G87

2.6

0.181

September

Bovine bathorhodopsin

[90]

2, 2006

with retinal

2I35

3.8

0.418

October

Bovine rhodopsin with

[91]

17,2006

retinal

2I36

4.1

0.412

October

Bovine rhodopsin with

[91]

17, 2006

retinala

2I37

4.15

0.382

October

Bovine rhodopsin with

[91]

17, 2006

retinala

2J4Y

3.4

0.330

September

Bovine rhodopsin with

[20]

25, 2007

retinal

2PED

2.95

0.289

October

Bovine rhodopsin with

[92]

30, 2007

9-cis-retinal

2ZIY

3.7

0.330

May 6,

Squid rhodopsin with

[49]

2008

retinal

2Z73

2.5

0.206

May 13,

Squid rhodopsin with

[50]

2008

retinal

3CAP

2.9

0.266

June 24,

Bovine opsin, ligand -

[31]

2008

free rhodopsin

3C9L

2.65

0.216

August 5,

Bovine rhodopsin with

[93]

2008

retinal

3C9M

3.4

0.219

August 5,

Bovine rhodopsin with

[93]

2008

retinal

3DQB

3.2

0.248

September

Bovine opsin, ligand -

[51]

23, 2008

free rhodopsin

B. Other Class A

GPCRs

2RH1

2.4

0.232

October

Human p2AR with

[22]

30, 2007

carazolol

2R4R

3.4

0.270

November

Human p2AR with

[21]

6, 2007

carazolola

2R4S

3.4

0.280

November

Human p2AR with

[21]

6, 2007

carazolola

TABLE 16.1 (Continued)

PDB

Resolution

R-Free

Date

Protein and

Literature

ID

(Â)

Released

Chromophore/Ligand

Reference

3D4S

2.8

0.273

June 17,

Human p2AR with

[27]

2008

timolol

2VT4

2.7

0.268

June 24,

Turkey p1Ar with

[28]

2008

cyanopindolol

3EML

2.6

0.231

October

Human A2a adenosine

[29]

14, 2008

receptor with

ZM241385

C. Class C GPCRs

1EWK

2.2

0.227

December

Rat mGluRl EC

[73]

18, 2000

domain with

glutamate

1EWT

3.7

0.287

December

Rat mGluRl EC

[73]

18, 2000

domain, free form I

1EWV

4.0

0.328

December

Rat mGluRl EC

[73]

18, 2000

domain, free form II

1ISR

4.0

0.259

March 13,

Rat mGluRl EC

[74]

2002

domain with

glutamate/Gd3+

1ISS

3.3

0.314

March 13,

Rat mGluRl EC

[74]

2002

domain with

S-MCPG

2E4U

2.35

0.267

February

Rat mGluR3 EC

[75]

27, 2007

domain with

glutamate

2E4V

2.4

0.268

February

Rat mGluR3 EC

[75]

27, 2007

domain with

DCG-IV

2E4W

2.4

0.267

February

Rat mGluR3 EC

[75]

27, 2007

domain with

1^,3^-ACPD

2E4X

2.75

0.265

February

Rat mGluR3 EC

[75]

27, 2007

domain with

1S,3R-ACPD

2E4Y

3.4

0.284

February

Rat mGluR3 EC

[75]

27, 2007

domain with

2R,4R-APDC

2E4Z

3.3

0.324

February

Rat mGluR7 EC

[75]

27, 2007

domain with 2-MES

aThe resolution in the active site was insufficient to determine the chromophore ' s or ligand' s coordinates.

aThe resolution in the active site was insufficient to determine the chromophore ' s or ligand' s coordinates.

0 0

Post a comment