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Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications. Inspired by the biologics of hematophagous organisms such as leeches, the authors in this work design and create inhibtors of thrombin and factor Xa by linking exosite-binding aptamers with small molecule active site inhibtors. They coin these inhibitors EXACT inhibitors. [ABSTRACT FROM AUTHOR]

Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer–drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone. [ABSTRACT FROM AUTHOR]

The anticoagulant properties of a novel RNA aptamer that binds FIXa depend collectively on the intensity of surface contact activation of human blood plasma, aptamer concentration, and its binding affinity for FIXa. Accordingly, anticoagulation efficiency of plasma containing any particular aptamer concentration is low when coagulation is strongly activated by hydrophilic surfaces compared to the anticoagulation efficiency in plasma that is weakly activated by hydrophobic surfaces. Anticoagulation efficiency is lower at hypothermic temperatures possibly because aptamer-FIXa binding decreases with decreasing temperatures. Experimental results demonstrating these trends are qualitatively interpreted in the context of a previously established model of anticoagulation efficiency of thrombin-binding DNA aptamers that exhibit anticoagulation properties similar to the FIXa aptamer. In principle, FIXa aptamer anticoagulants should be more efficient and therefore more clinically useful than thrombin-binding aptamers because aptamer binding to FIXa competes only with FX that is at much lower blood concentration than fibrinogen (FI) that competes with thrombin-binding aptamers. Our findings may have translatable relevance in the application of aptamer anticoagulants for clinical conditions in which blood is in direct contact with non-biological surfaces such as those encountered in cardiopulmonary bypass circuits. [ABSTRACT FROM AUTHOR]

Understanding inherent differences between thrombosis and hemostasis in the vascular system are critical to developing safe and effective anticoagulants. To this end, constituents of the contact activated and intrinsic pathway of coagulation appear to be involved in pathological thrombus formation, but are not required for normal hemostasis. In addition to coagulation, activation of the contact system is involved in fibrinolytic, inflammatory, and angiogenic processes that can also contribute to the thrombotic environment. This review discusses the role of the contact system in these processes, and highlights the potential of FXII and FXI as safer targets for antithrombotic therapy. [ABSTRACT FROM AUTHOR]

Nucleic acid aptamers offer several distinct advantages for the selective inhibition of protein targets within the coagulation cascade. A highly attractive feature of aptamers as antithrombotics is their ability to encode for complementary 'controlling agents' which selectively bind to and neutralize their active counterparts via Watson-Crick base pairing or, in a less selective and clinically characterized manner, cationic polymers that can counteract the activity of an aptamer or free/protein-complexed nucleic acid. The former property allows aptamer-based antithrombotic therapies to be administered with a goal of selective, high intensity target inhibition, knowing that rapid drug reversal is readily available. In addition, by purposefully varying the ratio of active agent to a specific controlling agent administered, the intensity of antithrombotic therapy can be regulated with precision according to patient needs and the accompanying clinical conditions. REG1, currently undergoing phase 2B clinical investigation, consists of an RNA aptamer (RB006; pegnivacogin) which targets factor IXa and its complementary controlling agent (RB007; anivamersen). Aptamers directed against other serine coagulation proteases, some with and some without parallel controlling agents, have been designed. Aptamers directed against platelet surface membrane receptor targets are in preclinical development. The following review offers a contemporary summary of nucleic acid aptamers as a translatable platform for regulatable antithrombotic drugs expanding the paradigm of patient- and disease-specific treatment in clinical practice. [ABSTRACT FROM AUTHOR]

The contribution of thrombosis to the natural history and clinical expression of advanced atherosclerotic coronary artery disease is well established. Less well understood is the biochemical and pathobiological distinction between normal hemostasis and thrombosis as the proximate cause of acute coronary syndrome. In this article, we summarize an evolving area of interest within the field of antithrombotic therapy—the contact system and the intrinsic pathway of coagulation, focusing our discussion on factors XI, XII, and IX to include their biochemical properties, relationship to arterial thrombosis phenotypes, and rational for future investigation of targeted pharmacotherapy. [ABSTRACT FROM AUTHOR]

With an ever increasing number of people taking numerous medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs, but, unfortunately, it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Here we describe the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents based upon aptamers. These universal antidotes exploit the fact that, when systemically administered, aptamers are the only free extracellular oligonucleotides found in circulation. We show that protein- and polymer-based molecules that capture oligonucleotides can reverse the activity of several aptamers in vitro and counteract aptamer activity in vivo. The availability of universal antidotes to control the activity of any aptamer suggests that aptamers may be a particularly safe class of therapeutics. [ABSTRACT FROM AUTHOR]

Tie2 is a receptor tyrosine kinase that is expressed predominantly in the endothelium and plays key roles in both physiological and pathological angiogenesis. The ligands for Tie2, the angiopoietins (Ang), perform opposing functions in vascular maintenance and angiogenesis; Ang1 regulates vascular quiescence, while Ang2 is thought to promote vascular destabilization and facilitate angiogenesis. However, the mechanisms responsible for these differences are not understood. To begin to elucidate the molecular differences between the angiopoietins, we previously developed a specific RNA aptamer inhibitor of Ang2. Here, we used the same iterative in vitro selection process, termed SELEX ( Systematic Evolution of Ligands by EXponential enrichment), to screen a library of 2′-fluoro-modified ribonucleotides for Ang1-binding aptamers. After nine rounds of selection, we identified a single clone, ANG9-4, that bound with high affinity to human Ang1 ( K d 2.8 nM) but not Ang2 ( K d > 1 μM), demonstrating specificity for Ang1. ANG9-4 blocked Ang1-mediated Tie2 phosphorylation and downstream Akt activation. Moreover, ANG9-4 inhibited Ang1-induced endothelial cell survival. Together, these findings demonstrate the feasibility of developing an Ang1-inhibitory aptamer. ANG9-4 and its derivatives may provide useful tools for elucidating the biology of Ang1 and for treating certain angiogenic diseases. [ABSTRACT FROM AUTHOR]

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type–specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics. [ABSTRACT FROM AUTHOR]

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals. [ABSTRACT FROM AUTHOR]

Investigates whether drug-antidote pairs could be rationally designed by taking advantage of properties inherent to nucleic acids to make antidote-controlled anticoagulant agents. Potency of the protein-binding oligonucleotides against coagulation factor IXa; Ability of oligonucleotides complementary to the aptamers to act as antidotes capable of efficiently reversing the activity of the anticoagulants.

Focuses on the use of RNA molecules as therapeutic agents. Capability of RNA in interpreting genetic information; Discussion of RNA as a biological macromolecule; Immunotherapy using messenger RNA-transfected dendritic cells.

Reports on a ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Potential of ribozymes as gene inhibitors; Use of self-splicing intron from Tetrahymena thermophila; Model system for messenger RNA repair.

The ability to target ribozymes to specifically cleave viral RNAs in vitro has led to much speculation about their potential therapeutic value as antiviral agents in vivo. To transfer a ribozyme's potential as an antiviral agent from test tubes to cells and organisms successfully, the characteristics that distinguish these settings must be considered. In vitro, ribozymes and substrate RNAs freely diffuse in solution in test tubes, and trans-cleavage reactions are dependent on a diffusive step. In eukaryotic cells, by contrast, many RNAs do not appear to diffuse freely. Instead, they appear to be highly compartmentalized and actively sorted to specific cellular locations. Such RNA trafficking may result in localization of substrate RNAs in a different compartment than ribozymes, which would effectively reduce substrate RNA availability to ribozymes and therefore limit the effectiveness of ribozymes as gene inhibitors. [ABSTRACT FROM AUTHOR]

In an effort to target the in vivo context of tumor-specific moieties, we screened a large library of nuclease-resistant RNA oligonucleotides in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to p68, an RNA helicase that has been shown to be upregulated in colorectal cancer. [ABSTRACT FROM AUTHOR]