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Allosteric regulation is critical for the functioning of G protein-coupled receptors (GPCRs) and their signaling pathways. Endogenous allosteric regulators of GPCRs are simple ions, various biomolecules, and protein components of GPCR signaling (G proteins and ß-arrestins). The stability and functional activity of GPCR complexes is also due to multicenter allosteric interactions between protomers. The complexity of allosteric effects caused by numerous regulators differing in structure, availability, and mechanisms of action predetermines the multiplicity and different topology of allosteric sites in GPCRs. These sites can be localized in extracellular loops; inside the transmembrane tunnel and in its upper and lower vestibules; in cytoplasmic loops; and on the outer, membrane-contacting surface of the transmembrane domain. They are involved in the regulation of basal and orthosteric agonist-stimulated receptor activity, biased agonism, GPCR-complex formation, and endocytosis. They are targets for a large number of synthetic allosteric regulators and modulators, including those constructed using molecular docking. The review is devoted to the principles and mechanisms of GPCRs allosteric regulation, the multiplicity of allosteric sites and their topology, and the endogenous and synthetic allosteric regulators, including autoantibodies and pepducins. The allosteric regulation of chemokine receptors, proteinase-activated receptors, thyroid-stimulating and luteinizing hormone receptors, and beta-adrenergic receptors are described in more detail.

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PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.

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WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, ß-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, ß-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

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Pepducins are lipidated peptides that target the intracellular loops of G protein-coupled receptors (GPCRs) in order to modulate transmembrane signaling to internally located effectors. With a wide array of potential activities ranging from partial, biased, or full agonism to antagonism, pepducins represent a versatile class of compounds that can be used to potentially treat diverse human diseases or be employed as novel tools to probe complex mechanisms of receptor activation and signaling in cells and in animals. Here, we describe a number of different pepducins including an advanced compound, PZ-128, that has successfully progressed through phase 2 clinical trials in cardiac patients demonstrating safety and efficacy in suppressing myonecrosis and arterial thrombosis.

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Allosteric modulators (AMs) of G-protein coupled receptors (GPCRs) are desirable drug targets because they can produce fewer on-target side effects, improved selectivity, and better biological specificity (e.g., biased signaling or probe dependence) than orthosteric drugs. An underappreciated source for identifying AM leads are peptides and proteins-many of which were evolutionarily selected as AMs-derived from endogenous protein-protein interactions (e.g., transducer/accessory proteins), intramolecular receptor contacts (e.g., pepducins or extracellular domains), endogenous peptides, and exogenous libraries (e.g., nanobodies or conotoxins). Peptides offer distinct advantages over small molecules, including high affinity, good tolerability, and good bioactivity, and specific disadvantages, including relatively poor metabolic stability and bioavailability. Peptidomimetics are molecules that combine the advantages of both peptides and small molecules by mimicking the peptide's chemical features responsible for bioactivity while improving its druggability. This review 1) discusses sources and strategies to identify peptide/peptidomimetic AMs, 2) overviews strategies to convert a peptide lead into more drug-like "peptidomimetic," and 3) critically analyzes the advantages, disadvantages, and future directions of peptidomimetic AMs. While small molecules will and should play a vital role in AM drug discovery, peptidomimetics can complement and even exceed the advantages of small molecules, depending on the target, site, lead, and associated factors.

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Formyl peptide receptors (FPRs) are G protein-coupled receptors mainly expressed in inflammatory myeloid cells. Previous reports demonstrated that human neutrophils express only FPR1 and FPR2 but not FPR3. Here, we found that FPR3 is expressed in sepsis patient derived neutrophils and Fpr3 is expressed in the mouse neutrophils. To test the role of Fpr3 in neutrophil activity, we synthesized Fpr3 pepducins and successfully developed an agonistic pepducin that stimulates Fpr3, eliciting calcium increase and chemotactic migration of neutrophils. We also found that administration of an Fpr3 pepducin in an experimental mouse sepsis model significantly increased the survival rate. The pepducin markedly inhibited lung injury, splenocyte apoptosis, and inflammatory cytokine production. Bacterial counts were significantly decreased by the pepducin in septic mice. Based on these results, we suggest that FPR3 can be regarded as a new target to control sepsis, and the newly generated Fpr3-based pepducin can be used for the development of anti-septic agents.

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BACKGROUND: The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape. METHODS: Three pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration. RESULTS: Assays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gαs and Gαq, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gαs stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain. CONCLUSION: We have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR. GENERAL SIGNIFICANCE: PAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.

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A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.

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Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces further cardiomyocyte death. ß-arrestin (ßarr)-biased ß-adrenergic receptor (ßAR) activation promotes survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death at the time of reperfusion to better preserve function. However, a lack of efficacious ßarr-biased orthosteric small molecules has prevented investigation into whether this pathway relays protection against ischemic injury in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from the first intracellular loop of ß2AR, allosterically engaged pro-survival signaling cascades in a ßarr-dependent manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against ischemia/reperfusion (I/R)-induced injury in vivo. Methods: Wild-type (WT) C57BL/6, ß2AR knockout (KO), ßarr1KO and ßarr2KO mice received intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min) followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM) were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to investigate the associated mechanism(s). Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and improved cardiac function in a ß2AR- and ßarr-dependent manner, which led to improved contractile function early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of ICL1-9 effects on ßarr-dependent ß2AR signaling. Conclusion: Pepducin-based allosteric modulation of ßarr-dependent ß2AR signaling represents a novel therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling benefits.

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The G-protein coupled receptors (GPCRs) belong to a large family of diverse receptors that are well recognized as pharmacological targets. However, very few of these receptors have been pursued as oncology drug targets. The Protease-activated receptor 1 (PAR1), which is a G-protein coupled receptor, has been shown to act as an oncogene and is an emerging anti-cancer drug target. In this paper, we provide an overview of PAR1's biased signaling role in metastatic cancers of the breast, lungs, and ovaries and describe the development of PAR1 inhibitors that are currently in clinical use to treat acute coronary syndromes. PAR1 inhibitor PZ-128 is in a Phase II clinical trial and is being developed to prevent ischemic and thrombotic complication of patients undergoing cardiac catheterization. PZ-128 belongs to a new class of cell-penetrating, membrane-tethered peptides named pepducins that are based on the intracellular loops of receptors targeting the receptor G-protein interface. Application of PZ-128 as an anti-metastatic and anti-angiogenic therapeutic agent in breast, lung, and ovarian cancer is being reviewed.

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G-protein coupled receptor (GPCR) biased agonism or functional selectivity has become an essential concept in GPCR research over the last years. Receptor-specific biased agonists selectively trigger one signaling pathway over another and induce a restricted/directed functional response. In this study, we aimed to characterize the concept of biased agonism for FPR2, a member of the formyl peptide receptor (FPR) subfamily of GPCRs. We show that the earlier described FPR2-activating pepducin F2Pal10 is a biased FPR2 agonist. The effects of F2Pal10 on neutrophil function differed in several aspects compared to those mediated by WKYMVM, a conventional FPR2-specific peptide agonist. Upon interaction with FPR2 expressed by neutrophils both F2Pal10 and WKYMVM activated the PLC-PIP2-Ca2+ signaling pathway and the superoxide-generating NADPH-oxidase, but only WKYMVM activated the receptor to recruit ß-arrestin. The functional consequences linked to a lack of ß-arrestin recruitment were further explored, and we demonstrate that FPR2 desensitization occurred independent of ß-arrestin. Despite this, reactivation of desensitized receptors achieved through a disruption of the cytoskeleton and through a novel FPR2 cross-talk mechanism with P2Y2R (the ATP receptor) and PAFR (the receptor for PAF) differed between F2Pal10-desensitized and WKYMVM-desensitized neutrophils. Further, the inability to recruit ß-arrestin was found to be associated with a reduced rate of receptor internalization and impaired chemotaxis in neutrophils. In summary, we provide experimental evidence of biased agonism for FPR2 and our data disclose critical roles of ß-arrestin in neutrophil chemotaxis and reactivation of desensitized receptors.

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Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

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ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

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Protease-activated receptor 2 (PAR2) is a member of protease-activated receptors (PARs). PAR2 distributed in tissues and cells (such as skin, airway epithelial cell, pancreas, etc.) has a broad biological effects, and is involved in pathogenesis of many diseases, such as mechanical pain, asthma, pain of pancreatic cancer, inflammation, pruritus, etc. Intervention of PAR2 will help us to identify the role of PAR2 in the mechanisms of diseases and in the development of new drugs. This article concentrates on the research progress of agonist, antagonist, and pepducin on PAR2.

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Pepducins constitute a unique class of G-protein coupled receptor (GPCR) modulating lipopeptides. Pepducins with inhibitory effects on neutrophils could potentially be developed into anti-inflammatory pharmaceuticals. A pepducin with a peptide sequence identical to the third intracellular loop of FPR1 was found to inhibit neutrophil functions including granule mobilization and superoxide production. This FPR1-derived pepducin selectively inhibited signaling and cellular responses through FPR2, but not FPR1 as expected. Binding to the neutrophil surface of a conventional FPR2 agonist is also inhibited. The fatty acid is essential for inhibition and pepducins with shorter peptides lose in potency. In summary, a pepducin designed to target FPR1 was found to hijack FPR2 and potently inhibit neutrophil functions.

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The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

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The ß2-adrenergic receptor (ß2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. ß-Agonists used to combat hypercontractility in airway smooth muscle stimulate ß2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the ß2AR by long acting ß-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. ß2AR desensitization to ß-agonists is primarily mediated by G protein-coupled receptor kinases and ß-arrestins that attenuate receptor-Gs coupling and promote ß2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and ß-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the ß2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several ß-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the ß2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or ß-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the ß2AR and provides valuable tools for the study of ß2AR function.

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This Letter describes methodology to enable the identification of tool or therapeutic lipopeptides which modulate the function of membrane bound proteins. The choice of lipopeptides as a chemotype is the amalgamation of multiple medicinal chemistry considerations including duration of action, low systemic exposure and access to intracellular components. The 'lipopeptide shuffle' has been applied here to the APJ receptor and has rapidly resulted in the discovery of a 33 nM APJ agonist hit from an initial 369 member lipopeptide synthetic array.

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Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein-coupled receptors, named S1P(1-5). Among them, S1P(3) has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX-725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P(3). Here, aiming to identify novel S1P(3) antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C- and N-terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala-scan derived compounds (2-10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose-dependent manner, both pepducin 1-induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities.

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