Building a Molecular Trap for a Serine Protease from Aptamer and Peptide Modules.

In drug development, molecular intervention strategies are usually based on interference with a single protein function, such as enzyme activity or receptor binding. However, in many cases, protein drug targets are multifunctional, with several molecular functions contributing to their pathophysiological actions. Aptamers and peptides are interesting synthetic building blocks for the design of multivalent molecules capable of modulating multiple functions of a target protein. Here, we report a molecular trap with the ability to interfere with the activation, catalytic activity, receptor binding, etc. of the serine protease urokinase-type plasminogen activator (uPA) by a rational combination of two RNA aptamers and a peptide with different inhibitory properties. The assembly of these artificial inhibitors into one molecule enhanced the inhibitory activity between 10- and 10,000-fold toward several functions of uPA. The study highlights the potential of multivalent designs and illustrates how they can easily be constructed from aptamers and peptides using nucleic acid engineering, chemical synthesis, and bioconjugation chemistry. By aptamer to aptamer and aptamer to peptide conjugation, we created, to the best of our knowledge, the first trivalent molecule which combines three artificial inhibitors binding to three different sites in a protein target. We hypothesize that by simultaneously preventing all of the functional interactions and activities of the target protein, this approach may represent an alternative to siRNA technology for a functional knockout.

A Conjugate of Two tPA-Binding RNA Aptamers Efficiently Inhibits Fibrinolysis.

Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.

Expected and unexpected features of protein-binding RNA aptamers.

RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 1016 different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. WIREs RNA 2016, 7:744-757. doi: 10.1002/wrna.1360 For further resources related to this article, please visit the WIREs website.

Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

[Pulmonary interstitial emphysema in a 15-month-old child treated with high frequency oscillatory ventilation]. / Pulmonalt interstitielt emfysem hos en 15 måneder gammel dreng behandlet med højfrekvent oscillerende ventilation.

Pulmonary interstitial emphysema (PIE) is a known complication of mechanical ventilation of preterm infants. PIE is associated with high mortality and treatment is controversial as both surgery and conservative treatment are possibilities. We present a case with a 15-month-old previously healthy child, hospitalized with severe respiratory distress. A computed tomography (CT) of the chest revealed localized PIE. He was successfully treated.

Transversus abdominis plane catheter bolus analgesia after major abdominal surgery.

Purpose. Transversus abdominis plane (TAP) blocks have been shown to reduce pain and opioid requirements after abdominal surgery. The aim of the present case series was to demonstrate the use of TAP catheter injections of bupivacaine after major abdominal surgery. Methods. Fifteen patients scheduled for open colonic resection surgery were included. After induction of anesthesia, bilateral TAP catheters were placed, and all patients received a bolus dose of 20 mL bupivacaine 2.5 mg/mL with epinephrine 5 µg/mL through each catheter. Additional bolus doses were injected bilaterally 12, 24, and 36 hrs after the first injections. Supplemental pain treatment consisted of paracetamol, ibuprofen, and gabapentin. Intravenous morphine was used as rescue analgesic. Postoperative pain was rated on a numeric rating scale (NRS, 0-10) at regular predefined intervals after surgery, and consumption of intravenous morphine was recorded. Results. The TAP catheters were placed without any technical difficulties. NRS scores were ≤3 at rest and ≤5 during cough at 4, 8, 12, 18, 24, and 36 hrs after surgery. Cumulative consumption of intravenous morphine was 28 (23-48) mg (median, IQR) within the first 48 postoperative hours. Conclusion. TAP catheter bolus injections can be used to prolong analgesia after major abdominal surgery.