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A two-step strategy to enhance activity of low potency peptides.

Doyle JR, Harwood BN, Krishnaji ST, Krishnamurthy VM, Lin WE, Fortin JP, Kumar K, Kopin AS - PLoS ONE (2014)

Bottom Line: In addition, our results illustrate that membrane anchoring increases ligand potency.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.

View Article: PubMed Central - PubMed

Affiliation: Tufts Medical Center, Molecular Cardiology Research Institute, Molecular Pharmacology Research Center, Boston, Massachusetts, United States of America.

ABSTRACT
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.

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Related in: MedlinePlus

YM022 inhibits CCK2R signaling induced by tethered CCK4-Gly, soluble CCK4-Gly, or lipidated CCK4-Gly.A) YM022 blocks tethered CCK4-Gly mediated CCK2R signaling. HEK293 cells were cotransfected with cDNAs encoding: CCK2R, a 5X-SRE-Luc-pest reporter construct, tCCK4-Gly (as indicated), and a β-galactosidase gene to control for transfection efficiency. Four hours following transfection, cells were with treated with increasing concentrations of YM022 for 20 hours. Luciferase activity was quantified and normalized relative to a parallel preparation of CCK2R expressing cells stimulated for 4 hours with soluble amidated CCK4 (s-CCK4-NH2, 10 µM). B) YM022 blocks s-CCK4-Gly-COOH and l-CCK4-Gly-COOH mediated activation of CCK2R. HEK293 cells were transfected as indicated above. Twenty hours after transfection, cells were with treated with increasing concentrations of YM022 together with either 10 µM of l-CCK4-Gly-COOH or s-CCK4-Gly-COOH. Following an additional four hour stimulation, luciferase activity was quantified and normalized as outlined for panel A. Data represent the mean ± SEM from 3 independent experiments, each performed in triplicate. Abbreviations: tCCK4-Gly, tethered glycine extended CCK4; s-CCK4-Gly-COOH, soluble glycine extended CCK4 with a C-terminal free acid; l-CCK4-Gly-COOH, lipidated glycine extended CCK4 with a C-terminal free acid; CCK2R, cholecystokinin 2 receptor.
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pone-0110502-g006: YM022 inhibits CCK2R signaling induced by tethered CCK4-Gly, soluble CCK4-Gly, or lipidated CCK4-Gly.A) YM022 blocks tethered CCK4-Gly mediated CCK2R signaling. HEK293 cells were cotransfected with cDNAs encoding: CCK2R, a 5X-SRE-Luc-pest reporter construct, tCCK4-Gly (as indicated), and a β-galactosidase gene to control for transfection efficiency. Four hours following transfection, cells were with treated with increasing concentrations of YM022 for 20 hours. Luciferase activity was quantified and normalized relative to a parallel preparation of CCK2R expressing cells stimulated for 4 hours with soluble amidated CCK4 (s-CCK4-NH2, 10 µM). B) YM022 blocks s-CCK4-Gly-COOH and l-CCK4-Gly-COOH mediated activation of CCK2R. HEK293 cells were transfected as indicated above. Twenty hours after transfection, cells were with treated with increasing concentrations of YM022 together with either 10 µM of l-CCK4-Gly-COOH or s-CCK4-Gly-COOH. Following an additional four hour stimulation, luciferase activity was quantified and normalized as outlined for panel A. Data represent the mean ± SEM from 3 independent experiments, each performed in triplicate. Abbreviations: tCCK4-Gly, tethered glycine extended CCK4; s-CCK4-Gly-COOH, soluble glycine extended CCK4 with a C-terminal free acid; l-CCK4-Gly-COOH, lipidated glycine extended CCK4 with a C-terminal free acid; CCK2R, cholecystokinin 2 receptor.

Mentions: In addition to studying how membrane anchoring influences SubP activity, we also evaluated a second low potency peptide, glycine extended CCK4 (CCK4-Gly). Like SubP, the mature CCK4 peptide is also endogenously α amidated at the C-terminus. CCK4-NH2 is a well-established CCK2R ligand [12], [18]. In preliminary experiments, membrane tethered CCK4 (tCCK4), minimally activated CCK2R (data not shown). In contrast to tCCK4, we noted that with addition of a C-terminal glycine residue (tCCK4-Gly), activity of this construct at the CCK2R significantly increased. As illustrated in Figure 5A, tCCK4-Gly activated the CCK2R in a cDNA concentration dependent manner. In contrast, this construct showed no activity on the CCK1R (Figure 5B). To determine if the activity of the corresponding lipidated peptide would again (as with SubP) parallel the signaling observed with the tethered ligand, we next tested signaling induced by lipidated, glycine extended CCK4 (l-CCK4-Gly-COOH). As with tethered glycine extended CCK4, l-CCK4-Gly-COOH activated the CCK2R (Figure 5C) and lacked activity at the CCK1R (Figure 5D). Furthermore, lipidation of CCK4-Gly increased the potency of this ligand when compared with its soluble counterpart (s-CCK4-Gly-COOH) at the CCK2R. To further explore the mechanism underlying agonist mediated signaling, we evaluated the potential of a well-established CCK2R non-peptide antagonist, YM022 [26], [27], to block receptor mediated signaling. As illustrated in Figure 6, YM022 inhibits CCK2 receptor signaling induced by tethered CCK4-Gly-COOH (Figure 6A) as well as soluble and lipidated CCK4-Gly-COOH (Figure 6B). IC50 values are as follows: tCCK4-Gly (IC50 = 0.54 nM), l-CCK4-Gly-COOH (IC50 = 10.2 nM), and s-CCK4-Gly-COOH (IC50 = 0.84 nM).


A two-step strategy to enhance activity of low potency peptides.

Doyle JR, Harwood BN, Krishnaji ST, Krishnamurthy VM, Lin WE, Fortin JP, Kumar K, Kopin AS - PLoS ONE (2014)

YM022 inhibits CCK2R signaling induced by tethered CCK4-Gly, soluble CCK4-Gly, or lipidated CCK4-Gly.A) YM022 blocks tethered CCK4-Gly mediated CCK2R signaling. HEK293 cells were cotransfected with cDNAs encoding: CCK2R, a 5X-SRE-Luc-pest reporter construct, tCCK4-Gly (as indicated), and a β-galactosidase gene to control for transfection efficiency. Four hours following transfection, cells were with treated with increasing concentrations of YM022 for 20 hours. Luciferase activity was quantified and normalized relative to a parallel preparation of CCK2R expressing cells stimulated for 4 hours with soluble amidated CCK4 (s-CCK4-NH2, 10 µM). B) YM022 blocks s-CCK4-Gly-COOH and l-CCK4-Gly-COOH mediated activation of CCK2R. HEK293 cells were transfected as indicated above. Twenty hours after transfection, cells were with treated with increasing concentrations of YM022 together with either 10 µM of l-CCK4-Gly-COOH or s-CCK4-Gly-COOH. Following an additional four hour stimulation, luciferase activity was quantified and normalized as outlined for panel A. Data represent the mean ± SEM from 3 independent experiments, each performed in triplicate. Abbreviations: tCCK4-Gly, tethered glycine extended CCK4; s-CCK4-Gly-COOH, soluble glycine extended CCK4 with a C-terminal free acid; l-CCK4-Gly-COOH, lipidated glycine extended CCK4 with a C-terminal free acid; CCK2R, cholecystokinin 2 receptor.
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pone-0110502-g006: YM022 inhibits CCK2R signaling induced by tethered CCK4-Gly, soluble CCK4-Gly, or lipidated CCK4-Gly.A) YM022 blocks tethered CCK4-Gly mediated CCK2R signaling. HEK293 cells were cotransfected with cDNAs encoding: CCK2R, a 5X-SRE-Luc-pest reporter construct, tCCK4-Gly (as indicated), and a β-galactosidase gene to control for transfection efficiency. Four hours following transfection, cells were with treated with increasing concentrations of YM022 for 20 hours. Luciferase activity was quantified and normalized relative to a parallel preparation of CCK2R expressing cells stimulated for 4 hours with soluble amidated CCK4 (s-CCK4-NH2, 10 µM). B) YM022 blocks s-CCK4-Gly-COOH and l-CCK4-Gly-COOH mediated activation of CCK2R. HEK293 cells were transfected as indicated above. Twenty hours after transfection, cells were with treated with increasing concentrations of YM022 together with either 10 µM of l-CCK4-Gly-COOH or s-CCK4-Gly-COOH. Following an additional four hour stimulation, luciferase activity was quantified and normalized as outlined for panel A. Data represent the mean ± SEM from 3 independent experiments, each performed in triplicate. Abbreviations: tCCK4-Gly, tethered glycine extended CCK4; s-CCK4-Gly-COOH, soluble glycine extended CCK4 with a C-terminal free acid; l-CCK4-Gly-COOH, lipidated glycine extended CCK4 with a C-terminal free acid; CCK2R, cholecystokinin 2 receptor.
Mentions: In addition to studying how membrane anchoring influences SubP activity, we also evaluated a second low potency peptide, glycine extended CCK4 (CCK4-Gly). Like SubP, the mature CCK4 peptide is also endogenously α amidated at the C-terminus. CCK4-NH2 is a well-established CCK2R ligand [12], [18]. In preliminary experiments, membrane tethered CCK4 (tCCK4), minimally activated CCK2R (data not shown). In contrast to tCCK4, we noted that with addition of a C-terminal glycine residue (tCCK4-Gly), activity of this construct at the CCK2R significantly increased. As illustrated in Figure 5A, tCCK4-Gly activated the CCK2R in a cDNA concentration dependent manner. In contrast, this construct showed no activity on the CCK1R (Figure 5B). To determine if the activity of the corresponding lipidated peptide would again (as with SubP) parallel the signaling observed with the tethered ligand, we next tested signaling induced by lipidated, glycine extended CCK4 (l-CCK4-Gly-COOH). As with tethered glycine extended CCK4, l-CCK4-Gly-COOH activated the CCK2R (Figure 5C) and lacked activity at the CCK1R (Figure 5D). Furthermore, lipidation of CCK4-Gly increased the potency of this ligand when compared with its soluble counterpart (s-CCK4-Gly-COOH) at the CCK2R. To further explore the mechanism underlying agonist mediated signaling, we evaluated the potential of a well-established CCK2R non-peptide antagonist, YM022 [26], [27], to block receptor mediated signaling. As illustrated in Figure 6, YM022 inhibits CCK2 receptor signaling induced by tethered CCK4-Gly-COOH (Figure 6A) as well as soluble and lipidated CCK4-Gly-COOH (Figure 6B). IC50 values are as follows: tCCK4-Gly (IC50 = 0.54 nM), l-CCK4-Gly-COOH (IC50 = 10.2 nM), and s-CCK4-Gly-COOH (IC50 = 0.84 nM).

Bottom Line: In addition, our results illustrate that membrane anchoring increases ligand potency.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.

View Article: PubMed Central - PubMed

Affiliation: Tufts Medical Center, Molecular Cardiology Research Institute, Molecular Pharmacology Research Center, Boston, Massachusetts, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus