a-high-throughput-compatible-fret-based-platform-for-identification

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

The Scripps Research Institute

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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.

Development of universal antidotes to control aptamer activity.

Avec un nombre toujours croissant de personnes qui prennent de nombreux m&eacute;dicaments, la n&eacute;cessit&eacute; d&#39;administrer des m&eacute;dicaments en toute s&eacute;curit&eacute; et de limiter des effets secondaires involontaires n&#39;a jamais &eacute;t&eacute; plus grande. Antidote contr&ocirc;le demeure le moyen le plus direct pour contrer les effets secondaires aigus des m&eacute;dicaments, mais, malheureusement, il a &eacute;t&eacute; difficile et le co&ucirc;t prohibitif pour g&eacute;n&eacute;rer des antidotes pour agents th&eacute;rapeutiques plus. Nous d&eacute;crivons ici l&#39;&eacute;laboration d&#39;un ensemble de mol&eacute;cules d&#39;antidote qui sont capables de contrer les effets de toute une classe d&#39;agents th&eacute;rapeutiques bas&eacute; sur aptam&egrave;res. Ces antidotes universels exploitent le fait que, lorsqu&#39;il est administr&eacute; de fa&ccedil;on syst&eacute;mique, aptam&egrave;res sont les oligonucl&eacute;otides extracellulaires libres seulement en circulation. Nous montrons que les mol&eacute;cules de base de prot&eacute;ine et de polym&egrave;re qui captent les oligonucl&eacute;otides peuvent inverser l&#39;activit&eacute; de plusieurs aptam&egrave;res in vitro et contrer aptam&egrave;re activit&eacute; in vivo. La disponibilit&eacute; des antidotes universelles pour contr&ocirc;ler l&#39;activit&eacute; de n&#39;importe quel aptam&egrave;re sugg&egrave;re que les aptam&egrave;res peuvent &ecirc;tre une classe particuli&egrave;rement s&ucirc;re de la th&eacute;rapeutique.

D&eacute;veloppement des antidotes universelles pour contr&ocirc;ler l&#39;activit&eacute; aptam&egrave;re.

Mit einer st&auml;ndig wachsenden Zahl von Menschen, die die zahlreichen Medikamente war sicher verwalten Drogen und unbeabsichtigte Nebenwirkungen zu begrenzen m&uuml;ssen nie gr&ouml;&szlig;er. Gegengift Steuerung bleibt die direkteste Mittel zur akuten Nebenwirkungen Medikamente entgegenzuwirken, aber leider hat es sind schwierig und Kosten unerschwinglich Gegenmittel f&uuml;r die meisten Therapeutika zu generieren. Hier beschreiben wir die Entwicklung einer Reihe von Gegengift-Molek&uuml;le, die Bek&auml;mpfung der Auswirkungen einer gesamten Klasse von Therapeutika, die auf der Grundlage Aptamere sind. Diese universelle Gegenmittel nutzen die Tatsache, dass systemisch verabreicht, Aptamere die einzige freie extrazellul&auml;re Oligonucleotides gefunden im Umlauf sind. Wir zeigen, dass Protein und Polymer-basierten Molek&uuml;le, die Oligonukleotide erfassen k&ouml;nnen die Aktivit&auml;t von in-vitro-mehrere Aptamere umgekehrt und Aptamer Aktivit&auml;t in vivo entgegenzuwirken. Die Verf&uuml;gbarkeit von universal Gegenmittel gegen die T&auml;tigkeit jeder Aptamer Steuern zufolge Aptamere eine besonders sichere Klasse von Therapeutika sein kann.

Entwicklung von universal Gegenmittel gegen Aptamer-Aktivit&auml;ten zu kontrollieren.

Con un numero sempre crescente di persone che assumono farmaci numerosi, la necessit&agrave; di tranquillamente somministrare farmaci e limitare gli effetti collaterali indesiderati non &egrave; mai stato maggiore. Antidoto controllo resta il mezzo pi&ugrave; diretto per contrastare acuti effetti collaterali dei farmaci, ma, purtroppo, &egrave; stato impegnativo e costo proibitivo per generare gli antidoti per gli agenti terapeutici pi&ugrave;. Qui descriviamo lo sviluppo di un insieme di molecole di antidoto che sono in grado di contrastare gli effetti di un&#39;intera classe di agenti terapeutici basati su aptameri. Questi antidoti universali che sfruttano il fatto che, quando somministrate sistematicamente, aptameri sono i oligonucleotides extracellulare solo libero trovato in circolazione. Mostriamo che molecole proteiche e polimero che catturano i oligonucleotides possono invertire l&#39;attivit&agrave; di diversi aptameri in vitro e contrastare aptamer attivit&agrave; in vivo. La disponibilit&agrave; di antidoti universali per controllare l&#39;attivit&agrave; di qualsiasi aptamer suggerisce che aptameri possono essere una classe particolarmente sicura di terapie.

Sviluppo degli antidoti universali per controllare attivit&agrave; aptamer.

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&#xC218;&#xB9CE;&#xC740; &#xC57D;&#xD488;&#xC744; &#xBCF5;&#xC6A9; &#xD558;&#xB294; &#xC0AC;&#xB78C;&#xB4E4;&#xC758; &#xC99D;&#xAC00; &#xC22B;&#xC790;&#xC640; &#xD568;&#xAED8; &#xC548;&#xC804; &#xD558; &#xAC8C; &#xC57D;&#xBB3C;&#xC744; &#xAD00;&#xB9AC; &#xD558; &#xACE0; &#xC758;&#xB3C4; &#xD558;&#xC9C0; &#xC54A;&#xC740; &#xBD80;&#xC791;&#xC6A9;&#xC744; &#xC81C;&#xD55C;&#xD560; &#xD544;&#xC694;&#xAC00; &#xD070; &#xC801; &#xD569;&#xB2C8;&#xB2E4;. &#xD574;&#xB3C5; &#xC81C; &#xC81C;&#xC5B4; &#xC57D;&#xBB3C;&#xC758; &#xC2EC;&#xAC01;&#xD55C; &#xBD80;&#xC791;&#xC6A9;&#xC744; &#xC911;&#xD654; &#xD558;&#xB294; &#xAC00;&#xC7A5; &#xC9C1;&#xC811;&#xC801;&#xC778; &#xC218;&#xB2E8; &#xB0A8;&#xC544; &#xC788;&#xC9C0;&#xB9CC;, &#xBD88;&#xD589; &#xD558; &#xAC8C;&#xB3C4;, &#xADF8;&#xAC83;&#xC740; &#xB3C4;&#xC804; &#xB418; &#xACE0; &#xBE44;&#xC6A9;&#xC744; &#xAC00;&#xC7A5; &#xCE58;&#xB8CC; &#xC5D0;&#xC774;&#xC804;&#xD2B8; antidotes &#xC0DD;&#xC131; &#xAE08;&#xC9C0;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC; aptamers &#xAE30;&#xBC18; &#xCE58;&#xB8CC;&#xC81C;&#xC758; &#xC804;&#xCCB4; &#xD074;&#xB798;&#xC2A4;&#xC758; &#xC601;&#xD5A5;&#xC744; counteracting &#xC218; &#xC788;&#xB294; &#xD574;&#xB3C5; &#xC81C; &#xBD84;&#xC790; &#xC9D1;&#xD569;&#xC758; &#xAC1C;&#xBC1C;&#xC744; &#xC124;&#xBA85; &#xD569;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xBCF4;&#xD3B8;&#xC801;&#xC778; antidotes&#xC744; &#xCCB4;&#xACC4;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xB9AC; &#xD558;&#xB294; &#xACBD;&#xC6B0; aptamers&#xB294; &#xC21C;&#xD658;&#xC5D0; &#xC788;&#xB294; &#xC720;&#xC77C; &#xD558; &#xAC8C; &#xC790;&#xC720;&#xB85C;&#xC6B4; &#xC138;&#xD3EC; &#xC678; oligonucleotides &#xC0AC;&#xC2E4; &#xC545;&#xC6A9;. &#xC6B0;&#xB9AC; oligonucleotides &#xCEA1;&#xCC98; &#xB2E8;&#xBC31;&#xC9C8; &#xBC0F; &#xD3F4;&#xB9AC;&#xBA38; &#xAE30;&#xBC18; &#xBD84;&#xC790; &#xC5EC;&#xB7EC; aptamers &#xC0DD;&#xCCB4; &#xC678;&#xC5D0;&#xC11C;&#xC758; &#xD65C;&#xB3D9;&#xC744; &#xBC18;&#xB300; &#xD558; &#xACE0; aptamer &#xD65C;&#xB3D9; vivo&#xC5D0;&#xC11C; &#xC911;&#xD654; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xC5B4;&#xB5A4; aptamer&#xC758; &#xD65C;&#xB3D9;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xBCF4;&#xD3B8;&#xC801;&#xC778; antidotes &#xAC00;&#xC6A9;&#xC131; aptamers &#xCE58;&#xB8CC;&#xC81C;&#xC758; &#xD2B9;&#xD788; &#xC548;&#xC804; &#xD074;&#xB798;&#xC2A4;&#xC77C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

Aptamer &#xD65C;&#xB3D9;&#xC744; &#xC81C;&#xC5B4; &#xD558;&#xB294; &#xBCF4;&#xD3B8;&#xC801;&#xC778; &#xD574;&#xB3C5;&#xC758; &#xAC1C;&#xBC1C;&#xC785;&#xB2C8;&#xB2E4;.

Con un creciente n&uacute;mero de personas que toman medicamentos numerosos, nunca ha sido mayor de la necesidad para administrar medicamentos y limitar los efectos secundarios no deseados de forma segura. Control de ant&iacute;doto sigue siendo el medio m&aacute;s directo para contrarrestar los efectos secundarios agudos de las drogas, pero, lamentablemente, ha sido un reto y costo prohibitivo para generar ant&iacute;dotos para agentes terap&eacute;uticos m&aacute;s. Aqu&iacute; describimos el desarrollo de un conjunto de mol&eacute;culas de ant&iacute;doto que son capaces de contrarrestar los efectos de una clase entera de agentes terap&eacute;uticos basados en apt&aacute;meros. Estos ant&iacute;dotos universales explotan el hecho de que, cuando se administra sist&eacute;micamente, apt&aacute;meros son los oligonucle&oacute;tidos extracelulares s&oacute;lo libres encontradas circulaci&oacute;n. Demostramos que las mol&eacute;culas basadas en prote&iacute;na y pol&iacute;mero que capturan oligonucle&oacute;tidos pueden revertir la actividad de varios apt&aacute;meros in vitro y contrarrestar apt&aacute;meros actividad in vivo. La disponibilidad de los ant&iacute;dotos universales para controlar la actividad de cualquier apt&aacute;meros sugiere que apt&aacute;meros pueden ser una clase particularmente segura de la terap&eacute;utica.

Desarrollo de ant&iacute;dotos universales para controlar la actividad de apt&aacute;meros.

Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin.

Architectural designs for DNA nanostructures typically fall within one of two broad categories: tile-based designs (assembled from chemically synthesized oligonucleotides) and origami designs (woven structures employing a biological scaffold strand and synthetic staple strands). Both previous designs typically contain many Holliday-type multi-arm junctions. Here we describe the design, implementation, and testing of a unique architectural strategy incorporating some aspects of each of the two previous design categories but without multi-arm junction motifs. Goals for the new design were to use only chemically synthesized DNA, to minimize the number of component strands, and to mimic the back-and-forth, woven strand routing of the origami architectures. The resulting architectural strategy employs "weave tiles" formed from only two oligonucleotides as basic building blocks, thus decreasing the burden of matching multiple strand stoichiometries compared to previous tile-based architectures and resulting in a structurally flexible tile. As an example application, we have shown that the four-helix weave tile can be used to increase the anticoagulant activity of thrombin-binding aptamers in vitro.

Weave tile architecture construction strategy for DNA nanotechnology.

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.

Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism.

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over blood coagulation.

Kristin M. Bompiani

Department of Chemistry

The Scripps Research Institute

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Kristin M. Bompiani

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemistry

The Scripps Research Institute

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Department of Chemistry