Review
Structure-based drug screening for G-protein-coupled receptors

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G-protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past 4 years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.

Introduction

GPCRs mediate cellular responses to the majority of hormones and neurotransmitters, and are therefore attractive targets for drug discovery. In the decade following the cloning of the first genes and cDNAs for GPCRs 1, 2, there was great hope that these discoveries would rapidly translate into new and more effective therapeutics. Cloning and later mining the human genome sequence led to the identification of new GPCR subtypes [3] and the establishment of cell lines that could be used for high-throughput screening (HTS) of large compound libraries. The identification of polymorphisms for specific GPCRs suggested the potential for individualized medicines. Unfortunately, the promise of new drugs for new GPCR targets or safer and more effective drugs for previously identified targets has largely gone unfulfilled [4].

Several reasons may explain the slow pace of drug discovery in the face of more targets and screening modalities. If the advent of the molecular era gave us unprecedented tools and abundant targets, it also disrupted the integrated, tissue-based pharmacology of the classical era of drug discovery 5, 6; the underlying biology was more complicated than anticipated by the reductionist, molecular view. Many GPCRs signal through multiple pathways, often in a ligand-specific manner. For example, the β2 adrenergic receptor (β2AR) activates specific cellular signaling pathways through Gs, the stimulatory G protein for adenylyl cyclase, and independently through arrestin. Thus, the drug carvedilol is an inverse agonist for β2AR activation of Gs, but a partial agonist for activation of arrestin [7]. Consequently, high-throughput assays that only monitor a single readout, for instance cAMP accumulation or calcium flux, may not reflect the physiologically relevant signaling pathway [8]. Not only do we need to identify the correct GPCR target and signaling pathway, we must find a drug with the appropriate efficacy profile: agonist, partial agonist, neutral antagonist and inverse agonist. Drugs that satisfy these criteria must then pass through a gauntlet of assays to assess toxicology and pharmacokinetics. For this and other reasons, the cost of drug development has escalated whereas revenue from new drugs has slipped [9]. Consequently, some pharmaceutical companies are abandoning small molecule development programs in favor of biologics [10] and the cost of the few new drugs that make it to the market will further escalate the cost of healthcare.

In 2007, we entered the new era of GPCR structural biology. Since the initial crystal structures of the β2AR [11] and the β1AR [12], the number of published GPCRs which have yielded to crystallography has grown to ten and includes the adenosine A2A receptor [13], the D3 dopamine receptor [14], the CXCR4 receptor [15], the histamine H1 receptor, [16], the sphingosine 1 phosphate receptor [17], the M2 and M3 muscarinic receptors 18, 19, and the mu opioid receptor [20], with at least two new structures anticipated in 2012. This is largely attributable to the application of high-throughput methods for lipidic cubic phase (LCP) crystallography [21] and protein engineering with GPCR–T4 lysozyme 11, 22 and thermostabilization [23] methods being generally applicable to structurally diverse GPCRs. Although structural biology is not a panacea for the challenges described above, there is reason to hope that GPCR crystal structures can facilitate drug discovery based on success with soluble protein targets such as kinases and proteases. In this review, we will discuss the application of structure-based screens of large compound libraries to GPCR drug discovery.

Section snippets

Structure-based screens for new ligands

Structure-based design has been pivotal in the development of over ten marketed drugs, including recent successes against renin with aliskiren [24], and against hepatitis C virus protease with telapravir [25], and has contributed to the development of multiple others, since the technique came into widespread use in the 1990s. Although this is far fewer than initially promised by advocates of the technique, it is probably larger than the number of drugs whose origins can be traced directly to

Expanding structural coverage with homology models

As of this writing, we are aware of 12 GPCRs whose structures have been determined, including published and unpublished structures. If this is a great expansion from only 4 years ago, it still represents only 4% of the pharmacologically relevant GPCRs, considering that there are approximately 300 nonolfactory GPCRs in the human genome [3]. Even with the current pace of structure determination, for the foreseeable future there are likely to be many fewer structures than good targets for

State-specific structures

All of the initial GPCR structures captured the receptors in inactive states, and in silico screens against the inactive state structure of the β2AR [32], the A2a adenosine 31, 36, and the D3 dopamine receptor [35] yielded only antagonists and inverse agonists. Whereas retrospective modeling suggested that modest manipulation of the inactive states led to recognition of agonists in docking studies 37, 38, prospective screens – where new molecules were tested – returned no agonists whatsoever.

Concluding remarks

When the field began docking against the new GPCR structures, it was uncertain whether the technique would find potent and novel chemical matter at all. What we have learned over the past 3 years is that GPCRs are unusually well suited to docking screens, returning molecules whose affinities and hit rates are several logs better than we have come to expect in docking against soluble proteins. The campaigns to date have focused on technical features of hit rates, affinities, compound novelty,

Acknowledgments

Supported by GM59957 and GM72970 (PI R. Altman) (to B.K.S.) and NS028471 and GM083118 (to B.K.K.). We thank R. Coleman for sequence identity calculations.

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