Combinatorial Chemistry & High Throughput Screening - Volume 11, Issue 5, 2008

Continuing for many decades, and even into this post-genomic era, the G protein-coupled receptors (GPCRs) remain attractive targets for the discovery of small molecule therapeutics and still constitute the largest single fraction of the “druggable proteome”, with GPCR-targeted drugs having annual sales in the tens of billions of dollars worldwide [1,2]. Further exploitation of this rich treasure trove of targets, however, demands adding novel, creative approaches of identifying GPCR signaling modulators to the continued application of traditional high-throughput screening and medicinal chemistry efforts. In two special issues of CCHTS, we have compiled accounts from a cross-section of strategies in the hopes of illuminating the breadth of available and emerging approaches to identifying small molecules that modulate GPCR signal transduction in valuable ways. In this issue (CCHTS Vol. 11, No. 5), Kenakin considers the emerging realm of allosteric modulators of GPCR signaling and the attendent modifications to screening strategies required to maximize their discovery in high-throughput screens of biological function. The number of such HTS-amenable biological readouts of GPCR action are ever-expanding; Crouch and Osmond discuss the use of labelled antibody monitoring of ERK phosphorylation status in this context, whereas Fang and coworkers consider the emerging realm of label-free, intact cell readouts. Johnston and colleagues review their work in identifying novel phage display peptides with nucleotide-state-selective affinity for heterotrimeric G-protein alpha subunits, as well as applying them in developing non-radioactive assays of G-protein activation by GPCR signaling. Smrcka and coworkers review their work in identifying novel phage display peptides with affinity for heterotrimeric G-protein beta/gamma subunits and their application to in silico compound screening for Gbeta/gamma inhibitors. Finally, Kimple et al. round out this collection of articles with the most speculative assay development concept, involving a target considerably distant from the orthosteric binding site of GPCRs - namely, the interaction of heterotrimeric G-protein alpha subunits with the GoLoco motifs of G-protein signaling regulators LGN and RGS12. In the next issue (CCHTS Vol. 11, No. 6), Houston and colleagues describe a tour-de-force application of iterative chemical synthesis and biological testing towards the rational design of ligand tools (antagonists, agonists, radioligands) for a single GPCR -- the purinergic P2Y1 receptor that represents an important therapeutic target in platelet aggregation. Jensen and Roth describe a converse, post-genomics approach of screening single molecules against a multitude of receptors, and the consequent surprises and biological insights that can result from such screening. As a unique means to identify new GPCR-binding compounds in an unbiased fashion, Whitehurst and Annis describe the use of affinity selection-mass spectrometry (AS-MS) as applied to HTS of GPCRs. This newly-emergent technique relies on obtaining quantities of purified (and functional) receptor; the multitude of strategies for this technical hurdle are enumerated in encyclopedic detail in the following paper by Chiu and colleagues. In addition to enabling AS-MS and structural biology pursuits, purified GPCR preparations should also greatly facilitate antibody generation; the use of state-selective anti-GPCR antibodies in HTS and drug development is considered in the paper by Gupta and co-workers. We thank all the authors who participated in this project for their creative inputs. We hope that, in the aggregate, these articles spark renewed excitement towards GPCR signaling as a drug discovery platform and also highlight some of the novel and innovative approaches yet to be fully explored in this tried-and-true field. REFERENCES [1] Overington, J.P.; Al-Lazikani, B.; Hopkins, A.L. Nat. Rev. Drug Discov., 2006, 5, 993-6. [2] Jacoby, E.; Bouhelal, R.; Gerspacher, M.; Seuwen, K. ChemMedChem, 2006, 1, 761-82.

In high throughput screening systems, a single concentration of a new compound is tested in a biological system to detect direct effects (agonists) or effects on other ligands (antagonists). In this latter case, the chemical context of the assay is defined by a balance of maximal sensitivity (limited agonist concentration) and maximal window to observe effect (sizable agonist concentration to induce measurable effect). For allosteric modulators, there are other factors that should be considered in high throughput screening environments. Specifically, the saturable aspect of allosteric effect can dissociate the observed ordinate change in response (% inhibition) and potency of effect (concentration at which a given ordinate % effect is obtained). Also, the specter of probe dependence can be important in systems where the physiologically relevant agonist cannot be used for screening (i.e. HIV-1 entry). Finally, the interactive nature of allosteric systems can cause complex relationships between the chemical context of an assay and potency of allosteric modulator. For example, in cases where the efficacy of an agonist is reduced but the affinity is increased by a modulator, it may be more beneficial to have higher concentrations of agonist in the screening assay to optimize sensitivity to modulators. This must be balanced for allosteric potentiators with the need to retain a window to observe increased agonist effect.

G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Due to their broad involvement in cellular responses, GPCRs affect many important body functions both in health and disease. Compared to other receptor families, the GPCRs have been a rich source of extracellularly-acting pharmaceuticals, due largely to the fact that many GPCR ligands are small molecules when compared with ligands for other receptors, such as the tyrosine kinase receptor family. This has allowed the development of small molecule modulators of receptor function that act on specific GPCRs, such as those involved in cardiovascular regulation. However, at several levels, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs, such as the Gi-coupled GPCRs, are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca2+ and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs is likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high throughput methodologies. This article presents recent advances and associated applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery.

G protein-coupled receptors (GPCRs) have been proven to be the largest family of druggable targets in the human genome. Given the importance of GPCRs as drug targets and the de-orphanization of novel targets, GPCRs are likely to remain the frequent targets of many drug discovery programs. With recent advances in instrumentation and understanding of cellular mechanisms for the signals measured, biosensor-centered label-free cell assay technologies become a very active area for GPCR screening. This article reviews the principles and potential of current label-free cell assay technologies in GPCR drug discovery.

Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive state in which GDP-bound Gα is complexed with the Gβγ dimer, and an active state in which GTP-bound Gα is freed of its Gβγ binding partner. Structural studies have illustrated the basis for the distinct conformations of these states which are regulated by alterations in three precise ‘switch regions’ of the Gα subunit. Discrete differences in conformation between GDP- and GTP-bound Gα underlie its nucleotide-dependent protein-protein interactions (e.g., with Gβγ/receptor and effectors, respectively) that are critical for maintaining their proper nucleotide cycling and signaling properties. Recently, several screening approaches have been used to identify peptide sequences capable of interacting with Gα (and free Gβγ) in nucleotide-dependent fashions. These peptides have demonstrated applications in direct modulation of the nucleotide cycle, assessing the structural basis for aspects of Gα and Gβγ signaling, and serving as biosensor tools in assays for Gα activation including high throughput drug screening. In this review, we highlight some of the methods used for such discoveries and discuss the insights that can be gleaned from application of these identified peptides.

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein β γ subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gβ γ signaling can be detrimental and blocking Gβ γ signaling has salutary effects in a number of pathological models. Gβ γ regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gβ γ functions requires development of small molecule agents that disrupt Gβ γ protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gβ γ could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a “hot spot” on Gβ γ is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gβ γ functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gβ γ binding molecules. However, further refinement of the approach could significantly improve the yield of Gβ γ binding molecules from this screen that could result in multiple candidate leads for future drug development.

The GoLoco motif is a short Gα-binding polypeptide sequence. It is often found in proteins that regulate cellsurface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Gαi1. The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 μL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fullyautomated run, the Sigma-Aldrich LOPAC1280 collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.