Yolanda M. Fortenberry

Antimetastatic Potential of PAI-1–Specific RNA Aptamers

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin. This results in the detachment of tumor cells from the extracellular matrix, which is critical to the metastatic process. For this reason, we sought to isolate RNA aptamers that disrupt the interaction between PAI-1 and vitronectin. Through utilization of combinatorial chemistry techniques, aptamers have been selected that bind to PAI-1 with high affinity and specificity. We identified two aptamers, WT-15 and SM-20, that disrupt the interactions between PAI-1 and heparin, as well as PAI-1 and vitronectin, without affecting the antiprotease activity of PAI-1. Furthermore, SM-20 prevented the detachment of breast cancer cells (MDA-MB-231) from vitronectin in the presence of PAI-1, resulting in an increase in cellular adhesion. Therefore, the PAI-1 aptamer SM-20 demonstrates therapeutic potential as an antimetastatic agent and could possibly be used as an adjuvant to traditional chemotherapy for breast cancer.

Plasminogen activator inhibitor-1 inhibitors: a patent review (2006 – present)

Introduction: Plasminogen activator inhibitor-1 (PAI-1), the serine protease inhibitor (serpin), binds to and inhibits the plasminogen activators—tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA). This results in both a decrease in plasmin production and a decrease in the dissolution of fibrin clots. Elevated levels of PAI-1 are correlated with an increased risk for cardiovascular disease and have been linked to obesity and metabolic syndrome. Consequently, the pharmacological suppression of PAI-1 might prevent or treat vascular disease. Areas covered: This article provides an overview of the patenting activity on PAI-1 inhibitors. Patents filed by pharmaceutical companies or individual research groups are described, and the biological and biochemical evaluation of the inhibitors, including in vitro and in vivo studies, is discussed. An overview of patents pertaining to using these inhibitors for treating various diseases is also included. Expert opinion: Although there is still no PAI-1 inhibitor being evaluated in a clinical setting or approved for human therapy, research in this field has progressed, and promising new compounds have been designed. Most research has focused on improving the pharmacological profile of these compounds, which will hopefully allow them to proceed to clinical studies. Despite the need for further testing and research, the potential use of PAI-1 inhibitors for treating cardiovascular disease appears quite promising.

A Chemical Genetic Screen for Modulators of Asymmetrical 2,2′-Dimeric Naphthoquinones Cytotoxicity in Yeast

Background Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones. Methodology/Principal findings Exposure of wild type yeast strains to various BiQs demonstrated inhibition of yeast growth with IC50s in the µM range. Drug sensitivity and resistance screens were performed by exposing arrays of a haploid yeast deletion mutant library to BiQs at concentrations near their IC50. Sensitivity screens identified yeast with deletions affecting mitochondrial function and cellular respiration as having increased sensitivity to BiQs. Corresponding to this, wild type yeast grown in the absence of a fermentable carbon source were particularly sensitive to BiQs, and treatment with BiQs was shown to disrupt the mitochondrial membrane potential and lead to the generation of reactive oxygen species (ROS). Furthermore, baseline ROS production in BiQ sensitive mutant strains was increased compared to wild type and could be further augmented by the presence of BiQ. Screens for resistance to BiQ action identified the mitochondrial external NAD(P)H dehydrogenase, NDE1, as critical to BiQ toxicity and over-expression of this gene resulted in increased ROS production and increased sensitivity of wild type yeast to BiQ. Conclusions/Significance In yeast, binaphthoquinone cytotoxicity is likely mediated through NAD(P)H:quonine oxidoreductases leading to ROS production and dysfunctional mitochondria. Further studies are required to validate this mechanism in mammalian cells.

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration

BACKGROUND Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer. METHODS AND RESULTS We found that PCI inhibits cathepsin L with an inhibition rate (k(2)) of 3.0x10(5)M(-)(1)s(-)(1). Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells. CONCLUSIONS Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L. GENERAL SIGNIFICANCE Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.

Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases. This can potentially be accomplished by using small RNA molecules (aptamers). This studys goal is to develop RNA aptamers to a region of PAI-1 that will prevent the ability of PAI-1 to interact with the plasminogen activators. The aptamers were generated through a systematic evolution of ligands via exponential enrichment approach that ensures the creation of RNA molecules that bind to our target protein, PAI-1. In vitro assays were used to determine the effect of these aptamers on PAI-1s inhibitory activity. Three aptamers that bind to PAI-1 with affinities in the nanomolar range were isolated. The aptamer clones R10-4 and R10-2 inhibited PAI-1s antiproteolytic activity against tPA and disrupted PAI-1s ability to form a stable covalent complex with tPA. Increasing aptamer concentrations correlated positively with an increase in cleaved PAI-1. To the best of our knowledge, this is the first report of RNA molecules that inhibit the antiproteolytic activity of PAI-1.

Effects of Plasminogen Activator Inhibitor-1–Specific RNA Aptamers on Cell Adhesion, Motility, and Tube Formation

The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. The extracellular matrix protein vitronectin increases at sites of vessel injury and is also present in fibrin clots. Integrins present on the cell surface bind to vitronectin and anchor the cell to the extracellular matrix. However, the binding of PAI-1 to vitronectin prevents this interaction, thereby decreasing both cell adhesion and migration. We previously developed PAI-1-specific RNA aptamers that bind to (or in the vicinity of) the vitronectin binding site of PAI-1. These aptamers prevented cancer cells from detaching from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. In the current study, we used in vitro assays to investigate the effects that these aptamers have on human aortic smooth muscle cell (HASMC) and human umbilical vein endothelial cell (HUVEC) migration, adhesion, and proliferation. The PAI-1-specific aptamers (SM20 and WT15) increased attachment of HASMCs and HUVECs to vitronectin in the presence of PAI-1 in a dose-dependent manner. Whereas PAI-1 significantly inhibited cell migration through its interaction with vitronectin, both SM20 and WT15 restored cell migration. The PAI-1 vitronectin binding mutant (PAI-1AK) did not facilitate cell detachment or have an effect on cell migration. The effect on cell proliferation was minimal. Additionally, both SM20 and WT15 promoted tube formation on matrigel that was supplemented with vitronectin, thereby reversing the PAI-1s inhibition of tube formation. Collectively, results from this study show that SM20 and WT15 bind to the PAI-1s vitronectin binding site and interfere with its effect on cell migration, adhesion, and tube formation. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease.

Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis.

Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential.

Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin

Summary.  Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. Objective: Fifty‐five Ala‐scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. Results: Residues around the active site (Tyr50 and Glu202) and the sodium‐binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite‐2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin‐accelerated inhibition of thrombin by PCI. Exosite‐1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin–TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the ∼150‐fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite‐2 mutants that were essential for heparin‐catalyzed PCI–thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin–TM. Conclusions: Collectively, these results show that (i) similar thrombin exosite‐2 residues are critical for the heparin‐catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin–TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin–TM.

The role of serpins in tumor cell migration

Abstract Tumor cells are characterized by uncontrolled cell growth at a primary site that is caused by genetic alterations. Tumor cells that metastasize from their primary site to distant locations are commonly referred to as malignant. Cell migration is a critical step in this process. The ability of tumor cells to migrate and invade is partly controlled by proteolytic enzymes. These enzymes are secreted by either the tumor cells themselves or adjacent cells. They represent all classes of proteases, including serine and cysteine proteases. Serine proteases, in particular urokinase plasminogen activator (uPA), initiate a proteolytic cascade that culminates in degrading components of the extracellular matrix (ECM). Some serine proteases are controlled by a superfamily of proteins known as serpins. This minireview provides an overview of serpins that are vital in regulating tumor cell migration and progressing cancer.

Protein C inhibitor inhibits factor VIIa when bound to tissue factor

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