Sriram Ambadapadi

Interactions of cytosolic sulfotransferases with xenobiotics.

Abstract Cytosolic sulfotransferases are a superfamily of enzymes that catalyze the transfer of the sulfonic group from 3′-phosphoadenosine-5′-phosphosulfate to hydroxy or amine groups in substrate molecules. The human cytosolic sulfotransferases that have been most studied, namely SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1, are expressed in different tissues of the body, including liver, intestine, adrenal, brain and skin. These sulfotransferases play important roles in the sulfonation of endogenous molecules such as steroid hormones and neurotransmitters, and in the elimination of xenobiotic molecules such as drugs, environmental chemicals and natural products. There is often overlapping substrate selectivity among the sulfotransferases, although one isoform may exhibit greater enzyme efficiency than other isoforms. Similarly, inhibitors or enhancers of one isoform often affect other isoforms, but typically with different potency. This means that if the activity of one form of sulfotransferase is altered (either inhibited or enhanced) by the presence of a xenobiotic, the sulfonation of endogenous and xenobiotic substrates for other isoforms may well be affected. There are more examples of inhibitors than enhancers of sulfonation. Modulators of sulfotransferase enzymes include natural products ingested as part of the human diet as well as environmental chemicals and drugs. This review will discuss recent work on such interactions.

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.

Celecoxib influences steroid sulfonation catalyzed by human recombinant sulfotransferase 2A1

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17β-dihydroequilenin (17β-Eqn), 17β-dihydroequilin (17β-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17β-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17β-Eq was stimulated at celecoxib concentrations below 40 μM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

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-1beta (IL-1β), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1β 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-1β.

Myxomavirus anti-inflammatory chemokine binding protein reduces the increased plaque growth induced by chronic Porphyromonas gingivalis oral infection after balloon angioplasty aortic injury in mice.

Thrombotic occlusion of inflammatory plaque in coronary arteries causes myocardial infarction. Treatment with emergent balloon angioplasty (BA) and stent implant improves survival, but restenosis (regrowth) can occur. Periodontal bacteremia is closely associated with inflammation and native arterial atherosclerosis, with potential to increase restenosis. Two virus-derived anti-inflammatory proteins, M-T7 and Serp-1, reduce inflammation and plaque growth after BA and transplant in animal models through separate pathways. M-T7 is a broad spectrum C, CC and CXC chemokine-binding protein. Serp-1 is a serine protease inhibitor (serpin) inhibiting thrombotic and thrombolytic pathways. Serp-1 also reduces arterial inflammation and improves survival in a mouse herpes virus (MHV68) model of lethal vasculitis. In addition, Serp-1 demonstrated safety and efficacy in patients with unstable coronary disease and stent implant, reducing markers of myocardial damage. We investigate here the effects of Porphyromonas gingivalis, a periodontal pathogen, on restenosis after BA and the effects of blocking chemokine and protease pathways with M-T7 and Serp-1. ApoE−/− mice had aortic BA and oral P. gingivalis infection. Arterial plaque growth was examined at 24 weeks with and without anti-inflammatory protein treatment. Dental plaques from mice infected with P. gingivalis tested positive for infection. Neither Serp-1 nor M-T7 treatment reduced infection, but IgG antibody levels in mice treated with Serp-1 and M-T7 were reduced. P. gingivalis significantly increased monocyte invasion and arterial plaque growth after BA (P<0.025). Monocyte invasion and plaque growth were blocked by M-T7 treatment (P<0.023), whereas Serp-1 produced only a trend toward reductions. Both proteins modified expression of TLR4 and MyD88. In conclusion, aortic plaque growth in ApoE−/− mice increased after angioplasty in mice with chronic oral P. gingivalis infection. Blockade of chemokines, but not serine proteases significantly reduced arterial plaque growth, suggesting a central role for chemokine-mediated inflammation after BA in P. gingivalis infected mice.

Serpins, Viruses, and the Virome: New Directions in Therapy

Serine protease inhibitors, termed serpins, regulate myriad physiological processes in the mammalian body from thrombotic and thrombolytic pathways to inflammation, angiogenesis, hormone transport, and hypertension. The large percentage of serpins among the plasma proteins in the circulating blood as in the case of plasminogen activators, the functional redundancy of serpins, and also the debilitating serpinopathies of antithrombin III (SERPINC1), neuroserpin (SERPINI1), and alpha-1 antitrypsin (SERPINA1) provide evidence of the importance of serpins and their widespread impact in normal physiological homeostasis. Inflammation, also termed innate immunity, interacts closely with and both regulates and is regulated by thrombotic and thrombolytic serine proteases. Activation of the coagulation proteases is, in turn, controlled by serpins. Apoptosis is also modulated by serpins with cross-class inhibitory activity for cysteine and serine proteases. Excessive inflammation and cell death processes are now recognized as interacting with the thrombotic and thrombolytic proteases. Viruses have evolved to communicate and control these processes by encoding their own serpins, which confer on them the ability to evade host immune defenses. This chapter provides an introduction to the viral serpins derived from poxvirus origins that have been shown not only to be essential for successful viral infection but, in some cases, as for Serp-1 and Serp-2, to have the potential to mitigate inflammatory disease in animal models. Serp-1 has in fact been successfully tested in a small phase 2A clinical trial in unstable angina patients with coronary stent implants. A tandem discussion of mammalian serpins with actions similar to those of the viral serpins is also presented to emphasize potential evolutionary relationships between viral and mammalian serpins. The anti-inflammatory serpins hold the potential to be effective in disease states such as atheroma, sepsis, cancer, and wound healing given that these conditions are all associated with aberrant inflammatory responses and with dysregulation of thrombotic, thrombolytic, and apoptotic protease cascades. The capacity of viral serpins to provide antiviral protection by modulating the virome as well as possible therapeutic effects of serpin metabolites in inflammation will also be discussed. In summary, viral serpins have evolved over many millions of years and provide a unique and highly potent reservoir for both the study of serpin modulation of inflammatory responses and for new therapeutic approaches to inflammatory and even infectious diseases.

Selective Deletion of Heparan Sulfotransferase Enzyme, Ndst1, in Donor Endothelial and Myeloid Precursor Cells Significantly Decreases Acute Allograft Rejection

Early damage to transplanted organs initiates excess inflammation that can cause ongoing injury, a leading cause for late graft loss. The endothelial glycocalyx modulates immune reactions and chemokine-mediated haptotaxis, potentially driving graft loss. In prior work, conditional deficiency of the glycocalyx-modifying enzyme N-deacetylase-N-sulfotransferase-1 (Ndst1f/f TekCre+) reduced aortic allograft inflammation. Here we investigated modification of heparan sulfate (HS) and chemokine interactions in whole-organ renal allografts. Conditional donor allograft Ndst1 deficiency (Ndst1−/−; C57Bl/6 background) was compared to systemic treatment with M-T7, a broad-spectrum chemokine-glycosaminoglycan (GAG) inhibitor. Early rejection was significantly reduced in Ndst1−/− kidneys engrafted into wildtype BALB/c mice (Ndst1+/+) and comparable to M-T7 treatment in C57Bl/6 allografts (P < 0.0081). M-T7 lost activity in Ndst1−/− allografts, while M-T7 point mutants with modified GAG-chemokine binding displayed a range of anti-rejection activity. CD3+ T cells (P < 0.0001), HS (P < 0.005) and CXC chemokine staining (P < 0.012), gene expression in NFκB and JAK/STAT pathways, and HS and CS disaccharide content were significantly altered with reduced rejection. Transplant of donor allografts with conditional Ndst1 deficiency exhibit significantly reduced acute rejection, comparable to systemic chemokine-GAG inhibition. Modified disaccharides in engrafted organs correlate with reduced rejection. Altered disaccharides in engrafted organs provide markers for rejection with potential to guide new therapeutic approaches in allograft rejection.

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.

Overview of Serpins and Their Roles in Biological Systems

Serine protease inhibitors are ubiquitous regulators for a multitude of pathways in humans. The serpins represent an ancient pathway now known to be present in all kingdoms and often regulating central pathways for clotting, immunity, and even cancer in man. Serpins have been present from the time of the dinosaurs and can represent a large proportion of circulating blood proteins. With this introductory chapter, we present an overview of serpins as well as an introduction and overview of the chapters describing the methodology used in the new approaches to understanding their molecular mechanisms of action and their roles in health and disease.

Kinetic Measurement of Serpin Inhibitory Activity by Real-Time Fluorogenic Biochemical Assay

Biochemical fluorogenic and chromogenic assays facilitate real-time study of enzyme function. Based on the principle of enzymatic inhibition, these kinetic assays can be adapted to measure the function of serpins. Compared to traditional, electrophoretic study of serpin inhibitory complex formation, kinetic assays allow for finer temporal resolution as well as more quantitative comparisons between different conditions. This chapter describes methodology for performing real-time, kinetic measurement of serpin inhibitory activity by fluorogenic substrate conversion assay. Specifically, the methods covered include measurement of alpha-1-antitrypsin inhibitory activity against trypsin and heparin-dependent anti-thrombin III inhibitory activity against thrombin. These methods are scalable to small-volume, high-density format and can be applied for high-throughput screening of serpin activity modulators.