Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics.

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

Systemic delivery of a Peptide-linked morpholino oligonucleotide neutralizes mutant RNA toxicity in a mouse model of myotonic dystrophy.

Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.

Targeting nuclear RNA for in vivo correction of myotonic dystrophy.

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUG(exp)) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG(exp) RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

BACKGROUND: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. METHODS: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma. RESULTS: Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. CONCLUSIONS: Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

Inhibition of osteoclastogenesis by prolyl hydroxylase inhibitor dimethyloxallyl glycine.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

Platelet matrix metalloprotease-1 mediates thrombogenesis by activating PAR1 at a cryptic ligand site.

Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.

'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage.

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.

Protease-activated receptors in cardiovascular diseases.

Thrombosis associated with the pathophysiological activation of platelets and vascular cells has brought thrombin and its receptors to the forefront of cardiovascular medicine. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiological responses including platelet activation, intimal hyperplasia, inflammation, and maintenance of vascular tone and barrier function. The thrombin receptors PAR1 and PAR4 can be effectively targeted in animals in which acute or prolonged exposure to thrombin leads to thrombosis and/or restenosis. In the present study, we describe the molecular and pharmacological basis of small-molecule inhibitors that target PAR1. In addition, we discuss a new class of cell-penetrating inhibitors, termed pepducins, that provide insight into previously unidentified roles of PAR1 and PAR4 in protease signaling.

Therapeutic targeting of molecules involved in leukocyte-endothelial cell interactions.

Inflammation is traditionally viewed as a physiological reaction to tissue injury. Leukocytes contribute to the inflammatory response by the secretion of cytotoxic and pro-inflammatory compounds, by phagocytotic activity and by targeted attack of foreign antigens. Leukocyte accumulation in tissues is important for the initial response to injury. However, the overzealous accumulation of leukocytes in tissues also contributes to a wide variety of diseases, such as atherosclerosis, chronic inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, vasculitis, systemic inflammatory response syndrome, juvenile diabetes and psoriasis. Many therapeutic interventions target immune cells after they have already migrated to the site of inflammation. This review addresses different therapeutic strategies, used to reduce or prevent leukocyte-endothelial cell interactions and communication, in order to limit the progression of inflammatory diseases.

Blocking the protease-activated receptor 1-4 heterodimer in platelet-mediated thrombosis.

BACKGROUND: Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. METHODS AND RESULTS: Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. CONCLUSIONS: We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism.

The protease-activated receptors are tethered ligand G protein-coupled receptors that are activated by proteolytic cleavage of the extracellular domain of the receptor. The archetypic protease-activated receptor PAR1 strongly activates G(q) signaling pathways, but very little is known regarding the mechanism of signal transference between receptor and internally located G protein. The recent x-ray structure of rhodopsin revealed the presence of a highly conserved amphipathic 8th helix that is likely to be physically interposed between receptor and G protein. We found that the analogous 8th helix region of PAR1 was critical for activation of G(q)-dependent signaling. Engineering an 8th helix alpha-aneurysm with a downwards-directed alanine residue markedly interfered with signal transference to G(q). The 8th helix-anchoring cysteine palmitoylation sites were important for the affinity of ligand-dependent G protein coupling but did not affect the maximal signal. A network of H-bond and ionic interactions was found to connect the N-terminal portion of the 8th helix to the nearby NPXXY motif on transmembrane helix 7 and also to the adjacent intracellular loop-1. Disruption of these pairwise interactions caused additive defects in coupling to G protein, indicating that the transmembrane 7-8th helix-i1 loop may move in a coordinated manner to transfer the signal from PAR1 to G protein. This "7-8-1" interaction network was found to be prevalent in G protein-coupled receptors involved in endothelial signaling and angiogenesis.

Reversing systemic inflammatory response syndrome with chemokine receptor pepducins.

We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides-termed pepducins-that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.