A Two-Step Strategy to Enhance Activity of Low Potency Peptides

• Published in: PloS one Vol. 9; no. 11; p. e110502
• Main Authors: Doyle, Jamie R., Harwood, Benjamin N., Krishnaji, Subrahmanian Tarakkad, Krishnamurthy, Vijay M., Lin, Wei-En, Fortin, Jean-Philippe, Kumar, Krishna, Kopin, Alan S.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 12.11.2014; Public Library of Science (PLoS)
• Subjects: Anchoring; Antagonists; Binding sites; Biology and Life Sciences; Cardiology; Chemistry; Cholecystokinin; Fatty acids; G protein-coupled receptors; G proteins; Glycine; Glycine - chemistry; HEK293 Cells; Hormones; Humans; Ligands; Membranes; Modulators; Optimization; Peptides; Peptides - chemistry; Permeability; Pharmaceutical sciences; Pharmacology; Piperidines - chemistry; Plasmids - metabolism; Probes; Protein Structure, Tertiary; Proteins; Prototypes; Receptors; Receptors, G-Protein-Coupled - chemistry; Receptors, Neurokinin-1 - chemistry; Recombinant proteins; Recombinant Proteins - chemistry; Signal Transduction; Signaling; Substance P; Substance P - chemistry; Tetragastrin - chemistry
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0110502

Novel strategies are needed to expedite the generation and optimization of peptide probes targeting G protein-coupled receptors (GPCRs). We have previously shown that membrane tethered ligands (MTLs), recombinant proteins comprised of a membrane anchor, an extracellular linker, and a peptide ligand can be used to identify targeted receptor modulators. Although MTLs provide a useful tool to identify and/or modify functionally active peptides, a major limitation of this strategy is the reliance on recombinant protein expression. We now report the generation and pharmacological characterization of prototype peptide-linker-lipid conjugates, synthetic membrane anchored ligands (SMALs), which are designed as mimics of corresponding MTLs. In this study, we systematically compare the activity of selected peptides as MTLs versus SMALs. As prototypes, we focused on the precursor proteins of mature Substance P (SubP) and Cholecystokinin 4 (CCK4), specifically non-amidated SubP (SubP-COOH) and glycine extended CCK4 (CCK4-Gly-COOH). As low affinity soluble peptides these ligands each presented a challenging test case for assessment of MTL/SMAL technology. For each ligand, MTLs and corresponding SMALs showed agonist activity and comparable subtype selectivity. In addition, our results illustrate that membrane anchoring increases ligand potency. Furthermore, both MTL and SMAL induced signaling can be blocked by specific non-peptide antagonists suggesting that the anchored constructs may be orthosteric agonists. In conclusion, MTLs offer a streamlined approach for identifying low activity peptides which can be readily converted to higher potency SMALs. The ability to recapitulate MTL activity with SMALs extends the utility of anchored peptides as probes of GPCR function.