Total Synthesis of a PSGL-1 Glycopeptide Analogue for Targeted Inhibition of P-Selectin.

The high affinity interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin is mediated by a multimotif glycosulfopeptide (GSP) recognition domain consisting of clustered tyrosine sulfates and a Core 2 O-glycan terminated with sialyl LewisX (C2-O-sLeX). These distinct GSP motifs are much more common than previously appreciated within a wide variety of functionally important domains involved in protein-protein interactions. However, despite the potential of GSPs to serve as tools for fundamental studies and prospects for drug discovery, their utility has been limited by the absence of chemical schemes for synthesis on scale. Herein, we report the total synthesis of GSnP-6, an analogue of the N-terminal domain of PSGL-1, and potent inhibitor of P-selectin. An efficient, scalable, hydrogenolysis-free synthesis of C2-O-sLeX-Thr-COOH was identified by both convergent and orthogonal one-pot assembly, which afforded this crucial building block, ready for direct use in solid phase peptide synthesis (SPPS). C2-O-sLeX-Thr-COOH was synthesized in 10 steps with an overall yield of 23% from the 4-O,5-N oxazolidinone thiosialoside donor. This synthesis represents an 80-fold improvement in reaction yield as compared to prior reports, achieving the first gram scale synthesis of SPPS ready C2-O-sLeX-Thr-COOH and enabling the scalable synthesis of GSnP-6 for preclinical evaluation. Significantly, we established that GSnP-6 displays dose-dependent inhibition of venous thrombosis in vivo and inhibits vaso-occlusive events in a human sickle cell disease equivalent microvasculature-on-a-chip system. The insights gained in formulating this design strategy can be broadly applied to the synthesis of a wide variety of biologically important oligosaccharides and O-glycan bearing glycopeptides.

A PSGL-1 glycomimetic reduces thrombus burden without affecting hemostasis.

Events mediated by the P-selectin/PSGL-1 pathway play a critical role in the initiation and propagation of venous thrombosis by facilitating the accumulation of leukocytes and platelets within the growing thrombus. Activated platelets and endothelium express P-selectin, which binds P-selectin glycoprotein ligand-1 (PSGL-1) that is expressed on the surface of all leukocytes. We developed a pegylated glycomimetic of the N terminus of PSGL-1, PEG40-GSnP-6 (P-G6), which proved to be a highly potent P-selectin inhibitor with a favorable pharmacokinetic profile for clinical translation. P-G6 inhibits human and mouse platelet-monocyte and platelet-neutrophil aggregation in vitro and blocks microcirculatory platelet-leukocyte interactions in vivo. Administration of P-G6 reduces thrombus formation in a nonocclusive model of deep vein thrombosis with a commensurate reduction in leukocyte accumulation, but without disruption of hemostasis. P-G6 potently inhibits the P-selectin/PSGL-1 pathway and represents a promising drug candidate for the prevention of venous thrombosis without increased bleeding risk.

Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities.

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1-P1' bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR(-/-)). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with -4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b(+) monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

Targeted antithrombotic protein micelles.

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.

Design of an Ultralow Molecular Weight Heparin That Resists Heparanase Biodegradation.

Heparanase, an endo-ß-d-glucuronidase produced by a variety of cells and tissues, cleaves the glycosidic linkage between glucuronic acid (GlcA) and a 3-O- or 6-O-sulfated glucosamine, typified by the disaccharide -[GlcA-GlcNS3S6S]-, which is found within the antithrombin-binding domain of heparan sulfate or heparin. As such, all current forms of heparin are susceptible to degradation by heparanase with neutralization of anticoagulant properties. Here, we have designed a heparanase-resistant, ultralow molecular weight heparin as the structural analogue of fondaparinux that does not contain an internal GlcA residue but otherwise displays potent anticoagulant activity. This heparin oligosaccharide was synthesized following a chemoenzymatic scheme and displays nanomolar anti-FXa activity yet is resistant to heparanase digestion. Inhibition of thrombus formation was further demonstrated after subcutaneous administration of this compound in a murine model of venous thrombosis. Thrombus inhibition was comparable to that observed for enoxaparin with a similar effect on bleeding time.

A rechargeable anti-thrombotic coating for blood-contacting devices.

Despite the potential of anti-thrombogenic coatings, including heparinized surfaces, to improve the performance of blood-contacting devices, the inevitable deterioration of bioactivity remains an important factor in device failure and related thrombotic complications. As a consequence, the ability to restore the bioactivity of a surface coating after implantation of a blood-contacting device provides a potentially important strategy to enhance its clinical performance. Here, we report the regeneration of a multicomponent anti-thrombogenic coating through use of an evolved sortase A to mediate reversible transpeptidation. Both recombinant thrombomodulin and a chemoenzymatically synthesized ultra-low molecular weight heparin were repeatedly and selectively immobilized or removed in a sequential, alternating, or simultaneous manner. The generation of activated protein C (aPC) and inhibition of activated factor X (FXa) was consistent with the molecular composition of the surface. The fabrication of a rechargeable anti-thrombogenic surface was demonstrated on an expanded polytetrafluoroethylene (ePTFE) vascular graft with reconstitution of the surface bound coating 4 weeks after in vivo implantation in a rat model.

Sulfated poly-amido-saccharides (sulPASs) are anticoagulants in vitro and in vivo.

Anticoagulant therapeutics are a mainstay of modern surgery and of clotting disorder management such as venous thrombosis, yet performance and supply limitations exist for the most widely used agent - heparin. Herein we report the first synthesis, characterization, and performance of sulfated poly-amido-saccharides (sulPASs) as heparin mimetics. sulPASs inhibit the intrinsic pathway of coagulation, specifically FXa and FXIa, as revealed by ex vivo human plasma clotting assays and serine protease inhibition assays. sulPASs activity positively correlates with molecular weight and degree of sulfation. Importantly, sulPASs are not degraded by heparanases and are non-hemolytic. In addition, their activity is reversed by protamine sulfate, unlike small molecule anticoagulants. In an in vivo murine model, sulPASs extend clotting time in a dose dependent manner with bleeding risk comparable to heparin. These findings support continued development of synthetic anticoagulants to address the clinical risks and shortages associated with heparin.

The serpin saga; development of a new class of virus derived anti-inflammatory protein immunotherapeutics.

Serine proteinase inhibitors, also called serpins, are an ancient grouping of proteins found in primitive organisms from bacteria, protozoa and horseshoe crabs and thus likely present at the time of the dinosaurs, up to all mammals living today. The innate or inflammatory immune system is also an ancient metazoan regulatory system, providing the first line of defense against infection or injury. The innate inflammatory defense response evolved long before acquired, antibody dependent immunity. Viruses have developed highly effective stratagems that undermine and block a wide variety of host inflammatory and immune responses. Some of the most potent of these immune modifying strategies utilize serpins that have also been developed over millions of years, including the hijacking by some viruses for defense against host immune attacks. Serpins represent up to 2-10 percent of circulating plasma proteins, regulating actions as wide ranging as thrombosis, inflammation, blood pressure control and even hormone transport. Targeting serpin-regulated immune or inflammatory pathways makes evolutionary sense for viral defense and many of these virus-derived inhibitory proteins have proven to be highly effective, working at very low concentrations--even down to the femptomolar to picomolar range. We are studying these viral anti-inflammatory proteins as a new class of immunomodulatory therapeutic agents derived from their native viral source. One such viral serpin, Serp-1 is now in clinical trial (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks), representing a 'first in class' therapeutic study. Several other viral serpins are also currently under investigation as anti-inflammatory or anti-immune therapeutics. This chapter describes these original studies and the ongoing analysis of viral serpins as a new class of virus-derived immunotherapeutic.

Analysis of In Vivo Serpin Functions in Models of Inflammatory Vascular Disease.

Serpins have a wide range of functions in regulation of serine proteases in the thrombotic cascade and in immune responses, representing up to 2-10% of circulating proteins in the blood. Selected serpins also have cross-class inhibitory actions for cysteine proteases in inflammasome and apoptosis pathways. The arterial and venous systems transport blood throughout the mammalian body representing a central site for interactions between coagulation proteases and circulating blood cells (immune cells) and target tissues, a very extensive and complex interaction. While analysis of serpin functions in vitro in kinetics or gel shift assays or in tissue culture provides very necessary information on molecular mechanisms, the penultimate assessment of biological or physiological functions and efficacy for serpins as therapeutics requires study in vivo in whole animal models (some also consider cell culture to be an in vivo approach).Mouse models of arterial transplant with immune rejection as well as models of inflammatory vasculitis induced by infection have been used to study the interplay between the coagulation and immune response pathways. We describe here three in vivo vasculitis models that are used to study the roles of serpins in disease and as therapeutics. The models described include (1) mouse aortic allograft transplantation, (2) human temporal artery (TA) xenograft into immunodeficient mouse aorta, and (3) mouse herpes virus (MHV68)-induced inflammatory vasculitis in interferon-gamma receptor (IFNγR) knockout mice.

Evaluation of a bioengineered construct for tissue engineering applications.

Effective biomaterial options for tissue repair and regeneration are limited. Current biologic meshes are derived from different tissue sources and are generally sold as decellularized tissues. This work evaluated two collagen based bioengineered constructs and a commercial product in a model of abdominal full thickness defect repair. To prepare the bioengineered construct, collagen type 1 from porcine skin was isolated using an acid solubilization method. After purification, the collagen was formed into collagen sheets that were physically bonded to form a mechanically robust construct that was subsequently laser micropatterned with pores as a means to promote tissue integration (collagen only construct). A second engineered construct consisted of the aforementioned collagen construct embedded in an RGD-functionalized alginate gel that serves as a bioactive interface (collagen-alginate construct). The commercial product is a biologic mesh derived from bovine pericardium (Veritas® ). We observed enhanced vascularization in the midportion of the engineered collagen-alginate construct 2 weeks after implantation. Overall, the performance of the bioengineered constructs was similar to that of the commercial product with comparable integration strength at 8 weeks. Bioengineered constructs derived from monomeric collagen demonstrate promise for a variety of load bearing applications in tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2345-2354, 2018.

Platelet-targeted dual pathway antithrombotic inhibits thrombosis with preserved hemostasis.

Despite advances in antithrombotic therapy, the risk of recurrent coronary/cerebrovascular ischemia or venous thromboembolism remains high. Dual pathway antithrombotic blockade, using both antiplatelet and anticoagulant therapy, offers the promise of improved thrombotic protection; however, widespread adoption remains tempered by substantial risk of major bleeding. Here, we report a dual pathway therapeutic capable of site-specific targeting to activated platelets and therapeutic enrichment at the site of thrombus growth to allow reduced dosing without compromised antithrombotic efficacy. We engineered a recombinant fusion protein, SCE5-TAP, which consists of a single-chain antibody (SCE5) that targets and blocks the activated GPIIb/IIIa complex, and tick anticoagulant peptide (TAP), a potent direct inhibitor of activated factor X (FXa). SCE5-TAP demonstrated selective platelet targeting and inhibition of thrombosis in murine models of both carotid artery and inferior vena cava thrombosis, without a significant impact on hemostasis. Selective targeting to activated platelets provides an attractive strategy to achieve high antithrombotic efficacy with reduced risk of bleeding complications.

Measurements of aneurysm morphology determined by 3-d micro-ultrasound imaging as potential quantitative biomarkers in a mouse aneurysm model.

Aneurysms remain a significant medical problem and our current understanding of aneurysm formation and developmental stages remains incomplete. Noninvasive 3-D micro-ultrasound (3-D micro-US) imaging technologies designed for noninvasive evaluation of small laboratory animals diminish risks associated with invasive examination and provide in-situ (live) analysis of vascular morphological changes, which enables quantitative measurements of live biological specimens. We demonstrate here that aneurysm morphology can be quantified using 3-D micro-US, and we validate this methodology through comparison of geometric measures with those obtained from 3-D serial histologic records in a mouse model of accelerated aneurysm formation. Aneurysms were induced in Balb/C mice after C57Bl/6 mouse aortic transplant with injections of a pro-inflammatory viral serpin with a mutated reactive site. Aortic transplant segments were imaged 28 days after transplant using 3-D micro-US. Upon sacrifice, the aortas were excised and histology sections (5-microm thick) were digitized, co-registered using mutual information and stacked to form 3-D images. Surfaces of the mouse aorta and aneurysm were manually segmented from the 3-D micro-US and histology images. Comparisons with 3-D histology images demonstrated that 3-D micro-US allowed in-vivo analysis of aneurysm morphology, including total aneurysm area, plaque growth and lumen size. Linear regression of 3-D US-derived aneurysm and plaque volumes vs. 3-D histology-derived volumes resulted in slopes of 1.30 (R(2) = 0.96) and 1.20 (R(2) = 0.98), respectively, demonstrating that 3-D micro-US measurements can be used to track aneurysm growth in a mouse aortic transplant model.

An immobilized liquid interface prevents device associated bacterial infection in vivo.

Virtually all biomaterials are susceptible to biofilm formation and, as a consequence, device-associated infection. The concept of an immobilized liquid surface, termed slippery liquid-infused porous surfaces (SLIPS), represents a new framework for creating a stable, dynamic, omniphobic surface that displays ultralow adhesion and limits bacterial biofilm formation. A widely used biomaterial in clinical care, expanded polytetrafluoroethylene (ePTFE), infused with various perfluorocarbon liquids generated SLIPS surfaces that exhibited a 99% reduction in S. aureus adhesion with preservation of macrophage viability, phagocytosis, and bactericidal function. Notably, SLIPS modification of ePTFE prevents device infection after S. aureus challenge in vivo, while eliciting a significantly attenuated innate immune response. SLIPS-modified implants also decrease macrophage inflammatory cytokine expression in vitro, which likely contributed to the presence of a thinner fibrous capsule in the absence of bacterial challenge. SLIPS is an easily implementable technology that provides a promising approach to substantially reduce the risk of device infection and associated patient morbidity, as well as health care costs.

Myxoma viral serpin, Serp-1, a unique interceptor of coagulation and innate immune pathways.

Serpins maintain haemostasis through regulation of serine proteinases in the thrombotic and thrombolytic pathways. Viruses encode serpins that can alter thrombotic and thrombolytic responses producing, in some cases, disseminated intravascular coagulation (DIC). However, it has not been precisely defined how viral serpins induce these profound responses. The rabbit myxoma viral serpin, Serp-1 inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA), plasmin and factor Xa in vitro and exhibits remarkable anti-inflammatory activity in various animal models. The effects of Serp-1 on activation of human platelets, endothelial cells, monocytes and T cells that mediate thrombosis and innate immune responses were therefore examined. We found that Serp-1 attenuated platelet and mononuclear cell adhesion to fibronectin and collagen. Serp-1 similarly inhibited monocyte migration into the peritoneum. Serp-1 inhibition of monocyte migration was lost in uPA receptor (uPAR) deficient mice. Serp-1 bound to the plasma membrane surface and altered uPA activation of endothelial cells (p=0.001), thrombin activation of platelets (p=0.021) and phorbol ester activation of endothelial (p=0.047), monocyte (p=0.011) and Jurkat T cells (p=0.012) as measured by intracellular calcium. Modulation of cellular activation was confirmed by membrane fluidity analysis. Microarray analysis of Serp-1 treated endothelial cells revealed alterations in Inositol 1,4,5-triphosphate receptor type II (ITPR2) a calcium-regulating gene. This study demonstrates the unique capacity of a viral serpin, Serp-1 to modify adhesion, activation, gene expression and calcium homeostasis in a wide range of cells that regulate coagulation and inflammation. Endothelial cells potentially represent a pivotal regulatory point for Serp-1 anti-inflammatory activity.

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A.

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.

In vivo optical analysis of quantitative changes in collagen and elastin during arterial remodeling.

Altered collagen and elastin content correlates closely with remodeling of the arterial wall after injury. Optical analytical approaches have been shown to detect qualitative changes in plaque composition, but the capacity for detection of quantitative changes in arterial collagen and elastin content in vivo is not known. We have assessed fluorescence spectroscopy for detection of quantitative changes in arterial composition in situ, in rabbit models of angioplasty and stent implant. Fluorescence emission intensity (FEI) recorded at sites remote from the primary implant site was correlated with immunohistochemical (IH) analysis and extracted elastin and collagen. FEI was significantly decreased (P<0.05) after treatment with anti-inflammatory agents, and plaque area decreased on comparison with saline-treated rabbits after stent implant or angioplasty (Por=0.961) analysis were detected by multiple regression (MR) analysis. Good correlations also were found for FEI with elastin and collagen measured by high-performance liquid chromatography; MR analysis provided highly predictive values for collagen and elastin (R2>or=0.994). Fluorescence spectroscopic analysis detects quantitative compositional changes in arterial connective tissue in vivo, demonstrating changes at sites remote from primary angioplasty and stent implant sites.

Engineered composite fascia for stem cell therapy in tissue repair applications.

A critical challenge in tissue regeneration is to develop constructs that effectively integrate with the host tissue. Here, we describe a composite, laser micromachined, collagen-alginate construct containing human mesenchymal stem cells (hMSCs) for tissue repair applications. Collagen type I was fashioned into laminated collagen sheets to form a mechanically robust fascia that was subsequently laser micropatterned with pores of defined dimension and spatial distribution as a means to modulate mechanical behavior and promote tissue integration. Significantly, laser micromachined patterned constructs displayed both substantially greater compliance and suture retention strength than non-patterned constructs. hMSCs were loaded in an RGD-functionalized alginate gel modified to degrade in vivo. Over a 7 day observation period in vitro, high cell viability was observed with constant levels of VEGF, PDGF-ß and MCP-1 protein expression. In a full thickness abdominal wall defect model, the composite construct prevented hernia recurrence in Wistar rats over an 8-week period with de novo tissue and vascular network formation and the absence of adhesions to underlying abdominal viscera. As compared to acellular constructs, constructs containing hMSCs displayed greater integration strength (cell seeded: 0.92 ± 0.19 N/mm vs. acellular: 0.59 ± 0.25 N/mm, p=0.01), increased vascularization (cell seeded: 2.7-2.1/hpf vs. acellular: 1.7-2.1/hpf, p<0.03), and increased infiltration of macrophages (cell seeded: 2021-3630 µm(2)/hpf vs. acellular: 1570-2530 µm(2)/hpf, p<0.05). A decrease in the ratio of M1 macrophages to total macrophages was also observed in hMSC-populated samples. Laser micromachined collagen-alginate composites containing hMSCs can be used to bridge soft tissue defects with the capacity for enhanced tissue repair and integration. STATEMENT OF SIGNIFICANCE: Effective restoration of large soft tissue defects caused by trauma or treatment complications represents a critical challenge in the clinic. In this study, a novel composite construct was engineered and evaluated for stem cell delivery and tissue repair. Laser micromachining was used to fabricate patterned, microporous constructs designed with pores of defined size and distribution as a means to tune mechanical responses, accommodate and protect incorporated cells, and enhance tissue integration. The construct was embedded within an engineered alginate gel containing hMSCs. Upon repair of a full thickness abdominal wall defect in a rat model, the composite construct modulated host innate immunity towards a reparative phenotypic response, promoted neovascularization and associated matrix production, and increased the strength of tissue integration.

Serpin treatment suppresses inflammatory vascular lesions in temporal artery implants (TAI) from patients with giant cell arteritis.

Giant cell arteritis (GCA) and Takayasu's disease are inflammatory vasculitic syndromes (IVS) causing sudden blindness and widespread arterial obstruction and aneurysm formation. Glucocorticoids and aspirin are mainstays of treatment, predominantly targeting T cells. Serp-1, a Myxomavirus-derived serpin, blocks macrophage and T cells in a wide range of animal models. Serp-1 also reduced markers of myocardial injury in a Phase IIa clinical trial for unstable coronary disease. In recent work, we detected improved survival and decreased arterial inflammation in a mouse Herpesvirus model of IVS. Here we examine Serp-1 treatment of human temporal artery (TA) biopsies from patients with suspected TA GCA arteritis after implant (TAI) into the aorta of immunodeficient SCID (severe combined immunodeficiency) mice. TAI positive for arteritis (GCApos) had significantly increased inflammation and plaque when compared to negative TAI (GCAneg). Serp-1 significantly reduced intimal inflammation and CD11b+ cell infiltrates in TAI, with reduced splenocyte Th1, Th17, and Treg. Splenocytes from mice with GCApos grafts had increased gene expression for interleukin-1 beta (IL-1ß), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1ß expression. In conclusion, GCApos TAI xenografts in mice provide a viable disease model and have increased intimal inflammation as expected and Serp-1 significantly reduces vascular inflammatory lesions with reduced IL-1ß.

Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo.

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Viral chemokine-binding proteins inhibit inflammatory responses and aortic allograft transplant vasculopathy in rat models.

BACKGROUND: Both CC and CXC chemokines direct monocyte and T-cell migration and activation at sites of vascular injury, but the relative contributions of each chemokine class to transplant vasculopathy development have not been defined. The nonselective C, CC, and CXC chemokine binding protein, M-T7, inhibits vasculopathy development after angioplasty and after renal transplant. We have assessed the effects of three viral chemokine-binding proteins with differing ranges of chemokine inhibition on plaque growth in rats after aortic allograft transplant. METHODS: One of two myxomaviral chemokine binding proteins, (1). M-T1, a selective CC chemokine inhibitor, or (2). M-T7, a nonselective chemokine-binding protein, was given immediately after transplant. A separate group was treated with the gamma68-herpesvirus protein, M3, a C, CC, CXC, and CX3C binding protein, with preferential CC binding. RESULTS: Intimal hyperplasia was significantly reduced at late times posttransplant after infusion of each chemokine-binding protein (P <0.05). Early inhibition of macrophage and T-cell invasion was associated with a late decrease in vasculopathy development. Infusion of an inactive myxomavirus protein did not inhibit plaque growth. Combined high-dose M-T1 and M-T7 did not reduce plaque growth or early cell invasion to a greater extent than either protein alone. Coinfusion of the CC chemokines macrophage chemoattractant protein-1 and macrophage inflammatory protein-1alpha neutralized M-T1 and M-T7 inhibition of monocyte invasion, respectively, suggesting a key role for CC chemokine-mediated cellular influx. CONCLUSION: Viral chemokine-modulating proteins effectively reduce aortic allograft vasculopathy, acting predominantly through inhibition of a CC chemokine-mediated response.