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Apr 15, 2011 (Vol. 31, No. 8)

Refining GPCR Discovery Approaches

Emerging Platforms Afford Timelines More Comparable to Those for Soluble Targets

  • Click Image To Enlarge +
    Osteoblasts expressing a fusion protein of green fluorescent protein and ß-arrestin and the PTH receptor were incubated with the natural hormone PTH (left), the low molecular weight agonist (middle), or vehicle control (right). [MSD/Merck]

    GPCRs constitute the largest class of known targets in commercial drug discovery today—collectively the targets of at least 30% of currently marketed drugs. Yet, as heptahelical transmembrane proteins involved in divergent and manifold signaling pathways, they can confound conventional drug discovery techniques.

    The recent “Congress on G Protein-Coupled Receptors in Drug Discovery” meeting highlighted key approaches from companies pursuing developments in biased agonism, structure elucidation for ligand prediction, allosteric modulation, and advancement of GPCRs from preclinical to clinical studies, particularly with small molecule drug candidates.

    Guido Zaman, Ph.D., who heads assay development and pharmacology support for GPCR and enzyme projects at MSD/Merck, presented data on low molecular weight β–arrestin biased ligands of a class B GPCR, the parathyroid hormone receptor PTH-R1.

    “Identification of small molecule drugs for GPCRs is not trivial,” Dr. Zaman said, citing a general paucity of structural information on most GPCRs. Further, class B GPCRs, whose native activating ligands are proteins and large peptides, can prove particularly recalcitrant to pharmacological intervention by small molecules.

    Dr. Zaman’s lab and others are focused on bypassing these challenges by means of biased ligand binding, specifically through manipulation of β-arrestin. Because β-arrestin attenuates G protein coupling to GPCRs either by binding-site occlusion or by promoting cellular internalization of the GPCR, it is a key intervention point in GPCR-related drug discovery.

    Binding of either β-arrestin or G protein will be associated with discrete downstream signaling cascades; depending on the particular disease pathway or side-effect profile, either could be desired. As Dr. Zaman noted, in the case of PTH, some models in the literature have shown that an anabolic (tissue-building) response is mediated by β-arrestin, and an osteoclast (bone-degrading) pathway is mediated through G proteins.

    Precise and informative assays are thus critical to understanding these complex interactions such that they may be harnessed for effective pharmaceutical development. Dr. Zaman explained that MSD has refined a two-part assay system that confirms biased β-arrestin signaling recruitment: a live-cell, high-content green fluorescent protein imaging assay and a beta-galactosidase enzyme fragment complementation assay. The former permits study of the receptor in nonmodified form; the latter allows cost-effective, high-throughput measurements on standard chemiluminescence readers, he remarked.

    MSD has developed an in silico modeling approach that Dr. Zaman said is predictive of small molecule interactions with GPCRs, for in silico selection of compound libraries. Such developments may contribute to a move away from ultrahigh-throughput screening while allowing for increased specificity, always a key concern with any drug candidate but of particular importance with GPCRs given their involvement with numerous signaling pathways.

    MSD is using these approaches to develop low molecular weight compounds whose effects mimic those of natural hormones. Its most advanced project in this area, focused on gonadotropins, is currently in the clinic.

  • Click Image To Enlarge +
    Trevena uses its ABLE platform to discover and develop differentiated GPCR-targeted biased ligand therapeutics.

    Trevena is focused on GPCR therapeutics, specifically identification of ligands that bias either β-arrestin or G protein signaling. The company applies its Advanced Biased Ligand Explorer (ABLE™) platform, comprising assay tools and techniques for identifying and pharmacologically characterizing signal-selective ligands.

    “Drugs that are marketed today typically turn the receptor on or off, like a light switch,” said Michael Lark, Ph.D., CSO and svp of research. “In actuality, the biology is much more complicated than that. The µ-opioid receptor, for instance, has a side-effect profile associated with β-arrestin that includes respiratory suppression and gastrointestinal side effects; we’re working to identify a ligand that selectively favors the G protein pathway, which induces analgesia without classical opioid side effects.” That project is currently at the lead-optimization stage.

    In February, Trevena announced a Phase IIa trial with its lead candidate, TRV120027, a β-arrestin-biased angiotensin II type 1 receptor (AT1R) ligand to treat acute heart failure. Using its platform to characterize the compound, the company noted that the biased ligand favors increased cardiac performance due to its β-arrestin activation, while also lowering blood pressure due to its inhibition of G protein activation. Further, the compound also improves renal perfusion while preserving renal function.

    “When we formulate our hypotheses, we’re always thinking about what benefit we can harness by biasing the signaling one way or the other,” Dr. Lark explained.

    Once initial hits are identified, assays to clearly functionally characterize their activity at a cellular level are paramount. As Dr. Lark noted, appropriate pharmaceutical activity must obviously be validated in the cell and whole animal: “Does the compound change cellular signaling in the way we predicted? How does it change receptor trafficking and downstream signaling? What biological consequences are associated with this biased ligand?”

    All this can provide critical mechanism-of-action information early on in the pipeline efforts. And GPCR drug companies are finding success with approaches based on strong biology and robust data interpretation. Trevena, for instance, was able to screen 11 receptors (from a Ligand Pharmaceuticals five-million-compound library) within an 18-month time frame, and Trevena’s lead program moved from discovery through candidate selection in the same amount of time.


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