Transient receptor potential vanilloid 4 regulates extracellular matrix composition and mediates load-induced intervertebral disc degeneration in a mouse model.

OBJECTIVE: Transient receptor potential vanilloid 4 (TRPV4) is a multi-modally activated cation channel that mediates mechanotransduction pathways by which musculoskeletal tissues respond to mechanical load and regulate tissue health. Using conditional Trpv4 knockout mice, we investigated the role of Trpv4 in regulating intervertebral disc (IVD) health and injury-induced IVD degeneration. METHODS: Col2-Cre;Trpv4fl/f (Trpv4 KO) mice were used to knockout Trpv4 in all type 2 collagen-expressing cells. Effects of gene targeting alone was assessed in lumbar spines, using vertebral bone length measurement, histological, immunohistochemistry and gene expression analyses, and mechanical testing. Disc puncture was performed on caudal IVDs of wild-type (WT) and Trpv4 KO mice at 2.5- and 6.5-months-of-age. Six weeks after puncture (4- and 8-months-of-age at sacrifice), caudal spines were assessed using histological analyses. RESULTS: While loss of Trpv4 did not significantly alter vertebral bone length and tissue histomorphology compared to age-matched WT mice, Trpv4 KO mice showed decreased proteoglycan and PRG4 staining in the annulus fibrosus compared to WT. At the gene level, Trpv4 KO mice showed significantly increased expression of Acan, Bgn, and Prg4 compared to WT. Functionally, loss of Trpv4 was associated with significantly increased neutral zone length in lumbar IVDs. Following puncture, both Trpv4 KO and WT mice showed similar signs of degeneration at the site of injury. Interestingly, loss of Trpv4 prevented mechanically-induced degeneration in IVDs adjacent to sites of injury. CONCLUSION: These studies suggest a role for Trpv4 in regulating extracellular matrix synthesis and mediating the response of IVD tissues to mechanical stress.

Lysophosphatidic Acid Receptor 1 (LPA1) Antagonists as Potential Migrastatics for Triple Negative Breast Cancer.

Metastasis is responsible for about 90 % of cancer deaths. Anti-metastatic drugs, termed as migrastatics, offer a distinctive therapeutic approach to address cancer migration and invasion. However, therapeutic exploitation of metastasis-specific targets remains limited, and the effective prevention and suppression of metastatic cancer continue to be elusive. Lysophosphatidic acid receptor 1 (LPA1) is activated by an endogenous lipid molecule LPA, leading to a diverse array of cellular activities. Previous studies have shown that the LPA/LPA1 axis supports the progression of metastasis for many types of cancer. In this study, we report the synthesis and biological evaluation of fluorine-containing triazole derivatives as potent LPA1 antagonists, offering potential as migrastatic drugs for triple negative breast cancer (TNBC). In particular, compound 12 f, the most potent and highly selective in this series with an IC50 value of 16.0 nM in the cAMP assay and 18.4 nM in the calcium mobilization assay, inhibited cell survival, migration, and invasion in the TNBC cell line. Interestingly, the compound did not induce apoptosis in TNBC cells and demonstrated no cytotoxic effects. These results highlight the potential of LPA1 as a migrastatic target. Consequently, the LPA1 antagonists developed in this study hold promise as potential migrastatic candidates for TNBC.

A survey of stapling methods to increase affinity, activity, and stability of ghrelin analogues.

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor which regulates various important physiological and pathophysiological processes in the body such as energy homeostasis, growth hormone secretion and regulation of appetite. As a result, it has been postulated as a potential therapeutic target for the treatment of cancer cachexia and other metabolic disorders, as well as a potential imaging agent target for cancers and cardiovascular diseases. Ghrelin is the primary high affinity endogenous ligand for GHSR and has limited secondary structure in solution, which makes it proteolytically unstable. This inherent instability in ghrelin can be overcome by incorporating helix-inducing staples that stabilize its structure and improve affinity and activity. We present an analysis of different stapling methods at positions 12 and 16 of ghrelin(1-20) analogues with the goal of increasing proteolytic stability and to retain or improve affinity and activity towards the GHSR. Ghrelin(1-20) analogues were modified with a wide range of chemical staples, including a lactam staple, triazole staple, hydrocarbon staple, Glaser staple, and xylene-thioether staple. Once synthesized, the receptor affinity and α-helicity were measured using competitive binding assays and circular dichroism spectroscopy, respectively. Generally, an increase in alpha-helicity using a flexible staple linker led to improved affinity towards GHSR. Ghrelin(1-20) analogues with a lactam, triazole, and hydrocarbon staple resulted in helical analogues with stronger affinity towards GHSR than unstapled ghrelin(1-20), a compound that lacks helical character. Compounds were also investigated for their agonist activity through ß-arrestin 1 & 2 recruitment BRET assays and for their metabolic stability through serum stability analysis.

S. aureus drives itch and scratch-induced skin damage through a V8 protease-PAR1 axis.

Itch is an unpleasant sensation that evokes a desire to scratch. The skin barrier is constantly exposed to microbes and their products. However, the role of microbes in itch generation is unknown. Here, we show that Staphylococcus aureus, a bacterial pathogen associated with itchy skin diseases, directly activates pruriceptor sensory neurons to drive itch. Epicutaneous S. aureus exposure causes robust itch and scratch-induced damage. By testing multiple isogenic bacterial mutants for virulence factors, we identify the S. aureus serine protease V8 as a critical mediator in evoking spontaneous itch and alloknesis. V8 cleaves proteinase-activated receptor 1 (PAR1) on mouse and human sensory neurons. Targeting PAR1 through genetic deficiency, small interfering RNA (siRNA) knockdown, or pharmacological blockade decreases itch and skin damage caused by V8 and S. aureus exposure. Thus, we identify a mechanism of action for a pruritogenic bacterial factor and demonstrate the potential of inhibiting V8-PAR1 signaling to treat itch.

PAR4 Inhibition Reduces Coronary Artery Atherosclerosis and Myocardial Fibrosis in SR-B1/LDLR Double Knockout Mice.

BACKGROUND: SR-B1 (scavenger receptor class B type 1)/LDLR (low-density lipoprotein receptor) double knockout mice fed a high-fat, high-cholesterol diet containing cholate exhibit coronary artery disease characterized by occlusive coronary artery atherosclerosis, platelet accumulation in coronary arteries, and myocardial fibrosis. Platelets are involved in atherosclerosis development, and PAR (protease-activated receptor) 4 has a prominent role in platelet function in mice. However, the role of PAR4 on coronary artery disease in mice has not been tested. METHODS: We tested the effects of a PAR4 inhibitory pepducin (RAG8) on diet-induced aortic sinus and coronary artery atherosclerosis, platelet accumulation in atherosclerotic coronary arteries, and myocardial fibrosis in SR-B1/LDLR double knockout mice. SR-B1/LDLR double knockout mice were fed a high-fat, high-cholesterol diet containing cholate and injected daily with 20 mg/kg of either the RAG8 pepducin or a control reverse-sequence pepducin (SRQ8) for 20 days. RESULTS: Platelets from the RAG8-treated mice exhibited reduced thrombin and PAR4 agonist peptide-mediated activation compared with those from control SRQ8-treated mice when tested ex vivo. Although aortic sinus atherosclerosis levels did not differ, RAG8-treated mice exhibited reduced coronary artery atherosclerosis, reduced platelet accumulation in atherosclerotic coronary arteries, and reduced myocardial fibrosis. These protective effects were not accompanied by changes in circulating lipids, inflammatory cytokines, or immune cells. However, RAG8-treated mice exhibited reduced VCAM-1 (vascular cell adhesion molecule 1) protein levels in nonatherosclerotic coronary artery cross sections and reduced leukocyte accumulation in atherosclerotic coronary artery cross sections compared with those from SRQ8-treated mice. CONCLUSIONS: The PAR4 inhibitory RAG8 pepducin reduced coronary artery atherosclerosis and myocardial fibrosis in SR-B1/LDLR double knockout mice fed a high-fat, high-cholesterol diet containing cholate. Furthermore, RAG8 reduced VCAM-1 in nonatherosclerotic coronary arteries and reduced leukocyte and platelet accumulation in atherosclerotic coronary arteries. These findings identify PAR4 as an attractive target in reducing coronary artery disease development, and the use of RAG8 may potentially be beneficial in cardiovascular disease.

Discovery of Ghrelin(1-8) Analogues with Improved Stability and Functional Activity for PET Imaging.

The highest affinity ghrelin-based analogue for fluorine-18 positron emission tomography, [Inp1,Dpr3(6-FN),1Nal4,Thr8]ghrelin(1-8) amide (1), has remarkable subnanomolar receptor affinity (IC50 = 0.11 nM) toward the growth hormone secretagogue receptor 1a (GHSR). However, initial in vivo PET imaging and biodistribution of [18F]1 in mice demonstrated an unfavorable pharmacokinetic profile with rapid clearance and accumulation in liver and intestinal tissue, prompting concerns about the metabolic stability of this probe. The aims of the present study were to examine the proteolytic stability of ghrelin analogue 1 in the presence of blood and liver enzymes, structurally modify the peptide to improve stability without impeding the strong binding affinity, and measure the presently unknown functional activity of ghrelin(1-8) analogues. The in vitro stability and metabolite formation of 1 in human serum and liver S9 fraction revealed a metabolic soft spot between amino acids Leu5 and Ser6 in the peptide sequence. A focused library of ghrelin(1-8) analogues was synthesized and evaluated in a structure-activity-stability relationship study to further understand the structural importance of the residues at these positions in the context of stability and receptor affinity. The critical nature of l-stereochemistry at position 5 was identified and substitution of Ser6 with l-2,3-diaminopropionic acid led to a novel ligand with substantially improved in vitro stability while maintaining subnanomolar GHSR affinity. Despite the highly modified nature of these analogues compared to human ghrelin, ghrelin(1-8) analogues were found to recruit all G protein subtypes (Gαq/11/13/i1/oB) known to associate with GHSR as well as ß-arrestins with low micromolar to nanomolar potencies. The study of these analogues demonstrates the ability to balance desirable ligand properties, including affinity, stability, and potency to produce well-rounded candidate molecules for further in vivo evaluation.

Autocrine proteinase-activated receptor signaling in PC3 prostate cancer cells.

Proteinase-activated receptors (PARs) are G protein-coupled receptors (GPCRs) activated by limited n-terminal proteolysis. PARs are highly expressed in many cancer cells, including prostate cancer (PCa), and regulate various aspects of tumor growth and metastasis. Specific activators of PARs in different physiological and pathophysiological contexts remain poorly defined. In this study, we examined the androgen-independent human prostatic cancer cell line PC3 and find the functional expression of PAR1 and PAR2, but not PAR4. Using genetically encoded PAR cleavage biosensors, we showed that PC3 cells secrete proteolytic enzymes that cleave PARs and trigger autocrine signaling. CRISPR/Cas9 targeting of PAR1 and PAR2 combined with microarray analysis revealed genes that are regulated through this autocrine signaling mechanism. We found several genes that are known PCa prognostic factors or biomarker to be differentially expressed in the PAR1-knockout (KO) and PAR2-KO PC3 cells. We further examined PAR1 and PAR2 regulation of PCa cell proliferation and migration and found that absence of PAR1 promotes PC3 cell migration and suppresses cell proliferation, whereas PAR2 deficiency showed opposite effects. Overall, these results demonstrate that autocrine signaling through PARs is an important regulator of PCa cell function.

Crohn's disease proteolytic microbiota enhances inflammation through PAR2 pathway in gnotobiotic mice.

Emerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn's disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2-/-; R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2-/- mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway.

Human osteoarthritis knee joint synovial fluids cleave and activate Proteinase-Activated Receptor (PAR) mediated signaling.

Osteoarthritis (OA) is the most prevalent joint disorder with increasing worldwide incidence. Mechanistic insights into OA pathophysiology are evolving and there are currently no disease-modifying OA drugs. An increase in protease activity is linked to progressive degradation of the cartilage in OA. Proteases also trigger inflammation through a family of G protein-coupled receptors (GPCRs) called the Proteinase-Activated Receptors (PARs). PAR signaling can trigger pro-inflammatory responses and targeting PARs is proposed as a therapeutic approach in OA. Several enzymes can cleave the PAR N-terminus, but the endogenous protease activators of PARs in OA remain unclear. Here we characterized PAR activating enzymes in knee joint synovial fluids from OA patients and healthy donors using genetically encoded PAR biosensor expressing cells. Calcium signaling assays were performed to examine receptor activation. The class and type of enzymes cleaving the PARs was further characterized using protease inhibitors and fluorogenic substrates. We find that PAR1, PAR2 and PAR4 activating enzymes are present in knee joint synovial fluids from healthy controls and OA patients. Compared to healthy controls, PAR1 activating enzymes are elevated in OA synovial fluids while PAR4 activating enzyme levels are decreased. Using enzyme class and type selective inhibitors and fluorogenic substrates we find that multiple PAR activating enzymes are present in OA joint fluids and identify serine proteinases (thrombin and trypsin-like) and matrix metalloproteinases as the major classes of PAR activating enzymes in the OA synovial fluids. Synovial fluid driven increase in calcium signaling was significantly reduced in cells treated with PAR1 and PAR2 antagonists, but not in PAR4 antagonist treated cells. OA associated elevation of PAR1 cleavage suggests that targeting this receptor may be beneficial in the treatment of OA.

Granzyme K contributes to endothelial microvascular damage and leakage during skin inflammation.

BACKGROUND: Granzyme K (GzmK) is a serine protease with minimal presence in healthy tissues while abundant in inflamed tissues. Initially thought to play an exclusive role in immune-mediated cell death, extracellular GzmK can also promote inflammation. OBJECTIVES: To evaluate the role of GzmK in the pathogenesis of atopic dermatitis (AD), the most common inflammatory skin disease. METHODS: A panel of human AD and control samples was analysed to determine if GzmK is elevated. Next, to determine a pathological role for GzmK in AD-like skin inflammation, oxazolone-induced dermatitis was induced in GzmK-/- and wild-type (WT) mice. RESULTS: In human lesional AD samples, there was an increase in the number of GzmK+ cells compared with healthy controls. GzmK-/- mice exhibited reduced overall disease severity characterized by reductions in scaling, erosions and erythema. Surprisingly, the presence of GzmK did not notably increase the overall pro-inflammatory response or epidermal barrier permeability in WT mice; rather, GzmK impaired angiogenesis, increased microvascular damage and microhaemorrhage. Mechanistically, GzmK contributed to vessel damage through cleavage of syndecan-1, a key structural component of the glycocalyx, which coats the luminal surface of vascular endothelia. CONCLUSIONS: GzmK may provide a potential therapeutic target for skin conditions associated with persistent inflammation, vasculitis and pathological angiogenesis.

Protease-activated receptor 2 (PAR2)-targeting peptide derivatives for positron emission tomography (PET) imaging.

The proteolytically-activated G protein-coupled receptor (GPCR) protease-activated receptor 2 (PAR2), is implicated in various cancers and inflammatory diseases. Synthetic ligands and in vitro imaging probes targeting this receptor have been developed with low nanomolar affinity, however, no in vivo imaging probes exist for PAR2. Here, we report the strategic design, synthesis, and biological evaluation of a series of novel 4-fluorobenzoylated PAR2-targeting peptides derived from 2f-LIGRLO-NH2 (2f-LI-) and Isox-Cha-Chg-Xaa-NH2 (Isox-) peptide families, where the 4-fluorobenzoyl moiety acts as the 19F-standard of an 18F-labeled probe for potential use in in vivo imaging. We found that several of the 4-fluorobenzoylated peptides from the 2f-LI-family exhibited PAR2 selectivity with moderate potency (EC50 = 151-252 nM), whereas several from the Isox-family exhibited PAR2 selectivity with high potency (EC50 = 13-42 nM). Our lead candidate, Isox-Cha-Chg-Ala-Arg-Dpr(4FB)-NH2 (EC50 = 13 nM), was successfully synthesized with fluorine-18 with a radiochemical yield of 37%, radiochemical purity of >98%, molar activity of 20 GBq/µmol, and an end of synthesis time of 125 min. Biodistribution studies and preliminary PET imaging of the tracer in mice showed predominantly renal clearance. This 18F-labeled tracer is the first reported PAR2 imaging agent with potential for use in vivo. Future work will explore the use of this tracer in cancer xenografts and inflammation models involving upregulation of PAR2 expression.

Molecular mechanisms regulating Proteinase-Activated Receptors (PARs).

Proteinase-activated receptors (PARs) are a four-member family of G protein-coupled receptors defined by their irreversible proteolytic mechanism of activation. PARs have emerged as important regulators of various physiological responses and are implicated in numerous pathological conditions. Importantly, PAR1 and PAR4 are critical regulators of platelet function, while PAR2 is well established as a driver of inflammatory responses. PAR-targeted drug development efforts are therefore of great interest. In this review, we provide an overview of recent advances in our understanding of molecular mechanisms underlying PAR activation, effector interaction, and signaling. We also provide an overview of the diverse proteolytic enzymes that are now established as PAR regulators and describe the ability of different enzymes to elicit biased signaling through PARs. Finally, we highlight recent advances in the development of PAR-targeted pharmacological agents and discuss recent structure-activity relationship studies.

Biased signaling in platelet G-protein coupled receptors.

Platelets are small megakaryocyte-derived, anucleate, disk-like structures that play an outsized role in human health and disease. Both a decrease in the number of platelets and a variety of platelet function disorders result in petechiae or bleeding that can be life threatening. Conversely, the inappropriate activation of platelets, within diseased blood vessels, remains the leading cause of death and morbidity by affecting heart attacks and stroke. The fine balance of the platelet state in healthy individuals is controlled by a number of receptor-mediated signaling pathways that allow the platelet to rapidly respond and maintain haemostasis. G-protein coupled receptors (GPCRs) are particularly important regulators of platelet function. Here we focus on the major platelet-expressed GPCRs and discuss the roles of downstream signaling pathways (e.g., different G-protein subtypes or ß-arrestin) in regulating the different phases of the platelet activation. Further, we consider the potential for selectively targeting signaling pathways that may contribute to platelet responses in disease through development of biased agonists. Such selective targeting of GPCR-mediated signaling pathways by drugs, often referred to as biased signaling, holds promise in delivering therapeutic interventions that do not present significant side effects, especially in finely balanced physiological systems such as platelet activation in haemostasis.

Role of the Helix-8 and C-Terminal Tail in Regulating Proteinase Activated Receptor 2 Signaling.

The C-terminal tail of G-protein-coupled receptors (GPCR) contain important regulatory sites that enable interaction with intracellular signaling effectors. Here we examine the relative contribution of the C-tail serine/threonine phosphorylation sites (Ser383-385, Ser387-Thr392) and the helix-8 palmitoylation site (Cys361) in signaling regulation downstream of the proteolytically activated GPCR, PAR2. We examined Gαq/11-coupled calcium signaling, ß-arrestin-1/-2 recruitment, and MAPK activation (p44/42 phosphorylation) by wild-type and mutant receptors expressed in a CRISPR/Cas9 PAR2-knockout HEK-293 cell background with both peptide stimulation of the receptor (SLIGRL-NH2) as well as activation with its endogenous trypsin revealed a tethered ligand. We find that alanine substitution of the membrane proximal serine residues (Ser383-385Ala) had no effect on SLIGRL-NH2- or trypsin-stimulated ß-arrestin recruitment. In contrast, alanine substitutions in the Ser387-Thr392 cluster resulted in a large (∼50%) decrease in ß-arrestin-1/-2 recruitment triggered by the activating peptide, SLIGRL-NH2, but was without an effect on trypsin-activated ß-arrestin-1/-2 recruitment. Additionally, we find that alanine substitution of the helix-8 cysteine residue (Cys361Ala) led to a large decrease in both Gαq/11 coupling and ß-arrestin-1/-2 recruitment to PAR2. Furthermore, we show that Gαq/11 inhibition with YM254890, inhibited ERK phosphorylation by PAR2 agonists, while genetic deletion of ß-arrestin-1/-2 by CRISPR/Cas9 enhanced MAPK activation. Knockout of ß-arrestins also enhanced Gαq/11-mediated calcium signaling. In line with these findings, a C-tail serine/threonine mutant that has decreased ß-arrestin recruitment also showed enhanced ERK activation. Thus, our studies point to multiple mechanisms that regulate ß-arrestin interaction with PAR2 and highlight differences in regulation of tethered-ligand- and peptide-mediated activation of this receptor.

Chronic morphine regulates TRPM8 channels via MOR-PKCß signaling.

Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for 5 days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking phospholipase C (PLC) as well as protein kinase C beta (PKCß), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCß-mediated reduction of TRPM8 desensitization. This MOR-PKCß dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCß as important targets for opioid-induced cold hypersensitivity.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and ß-Arrestin.

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

Single Amino Acid Replacement in G-7039 Leads to a 70-fold Increase in Binding toward GHS-R1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) is a class A rhodopsin-like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1-Nal4 ,Lys5 (4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50 =69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G-7039. A 2-fluoropropionic acid (2-FPA) group was added on to Lys5 to determine the potential binding affinity of the [18 F]-2-FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4 ,Lys5 (2-fluoropropionyl)]G-7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS-R1a. Molecular docking revealed a molecular basis for this picomolar affinity.

High Affinity Fluorescent Probe for Proteinase-Activated Receptor 2 (PAR2).

PAR2 is a proteolytically activated G protein-coupled receptor (GPCR) that is implicated in various cancers and inflammatory diseases. Ligands with low nanomolar affinity for PAR2 have been developed, but there is a paucity of research on the development of PAR2-targeting imaging probes. Here, we report the development of seven novel PAR2-targeting compounds. Four of these compounds are highly potent and selective PAR2-targeting peptides (EC50 = 10 to 23 nM) that have a primary amine handle available for facile conjugation to various imaging components. We describe a peptide of the sequence Isox-Cha-Chg-ARK(Sulfo-Cy5)-NH2 as the most potent and highest affinity PAR2-selective fluorescent probe reported to date (EC50 = 16 nM, K D = 38 nM). This compound has a greater than 10-fold increase in potency and binding affinity for PAR2 compared to the leading previously reported probe and is conjugated to a red-shifted fluorophore, enabling in vitro and in vivo studies.

Myopia-Inhibiting Concentrations of Muscarinic Receptor Antagonists Block Activation of Alpha2A-Adrenoceptors In Vitro.

Purpose: Myopia is a refractive disorder that degrades vision. It can be treated with atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, but the mechanism is unknown. Atropine may block α-adrenoceptors at concentrations ≥0.1 mM, and another potent myopia-inhibiting ligand, mamba toxin-3 (MT3), binds equally well to human mAChR M4 and α1A- and α2A-adrenoceptors. We hypothesized that mAChR antagonists could inhibit myopia via α2A-adrenoceptors, rather than mAChR M4. Methods: Human mAChR M4 (M4), chicken mAChR M4 (cM4), or human α2A-adrenergic receptor (hADRA2A) clones were cotransfected with CRE/promoter-luciferase (CRE-Luc; agonist-induced luminescence) and Renilla luciferase (RLuc; normalizing control) into human cells. Inhibition of normalized agonist-induced luminescence by antagonists (ATR: atropine; MT3; HIM: himbacine; PRZ: pirenzepine; TRP: tropicamide; OXY: oxyphenonium; QNB: 3-quinuclidinyl benzilate; DIC: dicyclomine; MEP: mepenzolate) was measured using the Dual-Glo Luciferase Assay System. Results: Relative inhibitory potencies of mAChR antagonists at mAChR M4/cM4, from most to least potent, were QNB > OXY ≥ ATR > MEP > HIM > DIC > PRZ > TRP. MT3 was 56× less potent at cM4 than at M4. Relative potencies of mAChR antagonists at hADRA2A, from most to least potent, were MT3 > HIM > ATR > OXY > PRZ > TRP > QNB > MEP; DIC did not antagonize. Conclusions: Muscarinic antagonists block hADRA2A signaling at concentrations comparable to those used to inhibit chick myopia (≥0.1 mM) in vivo. Relative potencies at hADRA2A, but not M4/cM4, correlate with reported abilities to inhibit chick form-deprivation myopia. mAChR antagonists might inhibit myopia via α2-adrenoceptors, instead of through the mAChR M4/cM4 receptor subtype.