Peptides or modified peptides as drug moleculesPepducins: lipopeptide allosteric modulators of GPCR signaling
Introduction
G-protein coupled receptors (GPCRs) are cell-surface integral membrane proteins involved in cellular signaling pathways that regulate development, cell migration, homeostasis, endocrine pathways, synaptic transmission, and sensory systems. With over 650 members, the GPCR gene superfamily represents the largest single class of membrane receptors in the human genome [1]. Approximately 400 GPCRs have been established as drug targets or potential drug targets and, as a result, for decades modulators of GPCR-mediated signaling pathways have been a staple in the portfolios of drug companies. Despite many notable successes targeting GPCRs, particularly in the biogenic amine receptor subfamily, optimizing drugs for existing targets and developing drugs for new GPCR targets continues to be a challenge for the pharmaceutical industry. Remarkably only ∼50 GPCRs have been successfully targeted by marketed drugs, yet these drugs generate sales of over $40 billion per year. As many as 350 GPCRs, including ‘orphan’ GPCRs which have no known endogenous ligands, have proven to be intractable to standard drug discovery approaches. Many of these potential GPCR drug targets have been validated in disease models and could represent entirely new therapeutic target classes. An emerging technology that targets GPCRs by utilizing optimized lipopeptides (called pepducins) represents a new approach to exploit this rich family of drug targets.
Section snippets
GPCRs as signaling networks
GPCRs are a well-characterized family of heptahelical transmembrane proteins that interact with a variety of activators or ligands, including light, divalent cations, small molecules, lipids, peptides, and proteins [1]. The interaction between a GPCR and its agonist ligand causes a signal to be transduced across the cell membrane, resulting in changes in downstream cellular effector functions. GPCRs generally regulate normal cellular homeostasis and intercellular communication networks.
Pepducins: discovery and structure
Pepducins were discovered by Kuliopulos and Covic at Tufts New England Medical Center in the late 1990s, and described in a seminal paper in 2002 [4]. These researchers were working to develop tools to study signaling by the PAR-1 (protease-activated receptor-1) receptor, a GPCR important in cardiovascular disease. Kuliopulos and Covic built on previous work from Strader [5], Neubig [6], Lefkowitz [7], and others who had demonstrated that the intracellular loops of GPCRs were crucial sites of
Peptide therapeutics
Peptide drugs are not new to the commercial pharmaceutical landscape. Although they represent one of the oldest drug classes, they have re-emerged as one of the fastest-growing areas of drug discovery and development. There are over 150 peptide drugs on the market or in various stages of development [8]. Several drugs including Cubicin®, Levemir®, Cancidas® and most recently Victoza®, are lipidated peptides similar to pepducins. For example, Victoza® (liraglutide) [9] is longer in length (32
Pepducin identification and characterization
Since the initial description of pepducins in 2002, more than 30 papers have been published on pepducins from 14 labs worldwide. To date, pepducins have been described in the scientific literature for nine GPCRs (see Table 1), eight being Class A GPCRs and one Class C. A recently published comprehensive review of pepducins [10] provides significant detail on in vitro and in vivo characterization and effects in models of disease.
Pepducins in vivo
Pepducin activity has been demonstrated in several in vivo models. For example, subcutaneous (SC) administration of the PAR2 antagonist, pepducin P2pal-18S, significantly reduced γ-carrageenan/kaolin-induced paw edema and inflammation in wild-type but not PAR2-deficient mice [21]. The CXCR4 pepducin agonist, ATI-2341, was shown to dose-dependently mobilize neutrophils from the bone marrow of both mice and cynomolgus monkeys upon subcutaneous administration [12]. In contrast, neither a
Pepducin mechanism of action
Pepducins represent a novel approach to modulate GPCR signaling. Experiments suggest that pepducins enter the cell via the lipid tail by incorporating into the outer leaflet of the lipid bilayer and then traversing the hydrophobic core where they establish an equilibrium between the inner and outer leaflet of the lipid bilayer. The process by which pepducins transverse the plasma membrane and establish equilibrium has been termed ‘insertion and inversion,’ and is not yet completely understood.
Pepducin screening paradigm
Anchor has developed a generic screening paradigm to identify pepducins for any given receptor. Standard bioinformatic techniques are used to identify amino acid sequences encoding the membrane-spanning regions and extra- and intracellular domains. Small libraries of 200–300 pepducins, derived from the primary sequences of intracellular loops and adjacent juxtamembrane regions, are created and differ in length and starting/ending residues. The pepducins are synthesized on solid phase and the
Pepducins as therapeutics: CXCR4 as a case study
Anchor Therapeutics has advanced the understanding of pepducin biology and the development of the potential of pepducins as therapeutics through its study of the chemokine receptor CXCR4. The biology of the CXCR4 receptor and its natural ligand, CXCL12 is well understood [30]. In normal physiology, CXCR4 and CXCL12 are involved in sequestering cells of the hematopoietic lineage in the bone marrow. Genzyme's Mozobil®, a CXCR4 antagonist, has been used to block this interaction, leading to
Conclusions
Pepducins represent a novel allosteric approach to modulate GPCR signaling. Numerous examples of pepducins demonstrating both selective in vitro activity at the target receptor and in vivo efficacy in models of disease have been described in the literature. Pepducins derived from each of the intracellular loop regions have been identified and, most interestingly, a spectrum of pharmacological phenotypes (e.g. agonists, PAMs, and NAMs) has been documented. In addition to their biological
Conflict of interest statement
K.E.C., T.J.M. and S.W.H. are employees of Anchor Therapeutics. T.J.M. is a cofounder of Anchor Therapeutics.
Acknowledgements
The authors would like to Thomas P. Sakmar, Frederick (Rick) Jones, Mary Lou Bell and Janet Smart for their helpful input on the manuscript. We would also like to thank the outstanding scientists and employees of Anchor Therapeutics for their dedication and scientific contributions.
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