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1.
Chem Commun (Camb) ; 58(58): 8049-8052, 2022 Jul 19.
Article in English | MEDLINE | ID: covidwho-1908311

ABSTRACT

A universal aptamer against spike-proteins of diverse SARS-CoV-2 variants was discovered via DNA SELEX towards the wild-type (WT) spike-protein. This aptamer, A1C1, binds to the WT spike-protein or other variants of concern such as Delta and Omicron with low nanomolar affinities. A1C1 inhibited the interaction between hACE2 and various spike-proteins by 85-89%. This universal A1C1 aptamer can be used to design diagnostic and therapeutic molecular tools to target SARS-CoV-2 and its variants.


Subject(s)
Aptamers, Nucleotide , COVID-19 , Angiotensin-Converting Enzyme 2 , Aptamers, Nucleotide/pharmacology , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
3.
Nucleic Acid Ther ; 32(3): 139-150, 2022 06.
Article in English | MEDLINE | ID: covidwho-1774317

ABSTRACT

Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. We take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets factor X/Xa, with UFH (or low molecular weight heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. This aptamer-UFH combination (1) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, (2) allows for a reduced level of UFH to be employed, (3) more effectively limits thrombin generation compared to UFH alone, and (4) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation.


Subject(s)
Aptamers, Nucleotide , COVID-19 , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/pharmacology , Aptamers, Nucleotide/therapeutic use , Cardiopulmonary Bypass/adverse effects , Factor X , Heparin , Humans , Thrombin
4.
Cell Chem Biol ; 29(2): 215-225.e5, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1664751

ABSTRACT

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.


Subject(s)
Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Blood Coagulation/drug effects , Factor V/antagonists & inhibitors , Factor Va/antagonists & inhibitors , Amino Acid Sequence , Anticoagulants/chemistry , Anticoagulants/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Pairing , Binding Sites , COVID-19/blood , COVID-19/drug therapy , Cell Membrane/chemistry , Cell Membrane/metabolism , Factor V/chemistry , Factor V/genetics , Factor V/metabolism , Factor Va/chemistry , Factor Va/genetics , Factor Va/metabolism , Heparin, Low-Molecular-Weight/chemistry , Heparin, Low-Molecular-Weight/metabolism , Humans , Immune Sera/chemistry , Immune Sera/metabolism , Models, Molecular , Nucleic Acid Conformation , Protamines , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/growth & development , SARS-CoV-2/pathogenicity , SELEX Aptamer Technique , Substrate Specificity
5.
Int J Mol Sci ; 23(3)2022 Jan 26.
Article in English | MEDLINE | ID: covidwho-1648333

ABSTRACT

The SARS-CoV-2 virus is currently the most serious challenge to global public health. Its emergence has severely disrupted the functioning of health services and the economic and social situation worldwide. Therefore, new diagnostic and therapeutic tools are urgently needed to allow for the early detection of the SARS-CoV-2 virus and appropriate treatment, which is crucial for the effective control of the COVID-19 disease. The ideal solution seems to be the use of aptamers-short fragments of nucleic acids, DNA or RNA-that can bind selected proteins with high specificity and affinity. They can be used in methods that base the reading of the test result on fluorescence phenomena, chemiluminescence, and electrochemical changes. Exploiting the properties of aptamers will enable the introduction of rapid, sensitive, specific, and low-cost tests for the routine diagnosis of SARS-CoV-2. Aptamers are excellent candidates for the development of point-of-care diagnostic devices and are potential therapeutic tools for the treatment of COVID-19. They can effectively block coronavirus activity in multiple fields by binding viral proteins and acting as carriers of therapeutic substances. In this review, we present recent developments in the design of various types of aptasensors to detect and treat the SARS-CoV-2 infection.


Subject(s)
Aptamers, Nucleotide/therapeutic use , COVID-19 Testing/methods , COVID-19/therapy , Aptamers, Nucleotide/pharmacology , COVID-19/diagnosis , COVID-19/economics , COVID-19/virology , COVID-19 Testing/economics , Genetic Therapy/methods , Genetic Therapy/trends , Humans , Point-of-Care Testing/economics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity
6.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Article in English | MEDLINE | ID: covidwho-1559358

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.


Subject(s)
Aptamers, Nucleotide/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Angiotensin-Converting Enzyme 2/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Humans , Mutation , Neutralization Tests , Nucleic Acid Conformation , Protein Binding/drug effects , Protein Interaction Domains and Motifs , SARS-CoV-2/physiology , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
7.
Pharmacol Res ; 175: 105982, 2022 01.
Article in English | MEDLINE | ID: covidwho-1527828

ABSTRACT

All the different coronavirus SARS-CoV-2 variants isolated so far share the same mechanism of infection mediated by the interaction of their spike (S) glycoprotein with specific residues on their cellular receptor: the angiotensin converting enzyme 2 (ACE2). Therefore, the steric hindrance on this cellular receptor created by a bulk macromolecule may represent an effective strategy for the prevention of the viral spreading and the onset of severe forms of Corona Virus disease 19 (COVID-19). Here, we applied a systematic evolution of ligands by exponential enrichment (SELEX) procedure to identify two single strand DNA molecules (aptamers) binding specifically to the region surrounding the K353, the key residue in human ACE2 interacting with the N501 amino acid of the SARS-CoV-2 S. 3D docking in silico experiments and biochemical assays demonstrated that these aptamers bind to this region, efficiently prevent the SARS-CoV-2 S/human ACE2 interaction and the viral infection in the nanomolar range, regardless of the viral variant, thus suggesting the possible clinical development of these aptamers as SARS-CoV-2 infection inhibitors. Our approach brings a significant innovation to the therapeutic paradigm of the SARS-CoV-2 pandemic by protecting the target cell instead of focusing on the virus; this is particularly attractive in light of the increasing number of viral mutants that may potentially escape the currently developed immune-mediated neutralization strategies.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Aptamers, Nucleotide/pharmacology , COVID-19/drug therapy , Receptors, Virus/antagonists & inhibitors , SARS-CoV-2/pathogenicity , Virus Internalization/drug effects , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Aptamers, Nucleotide/genetics , Aptamers, Nucleotide/metabolism , COVID-19/enzymology , COVID-19/genetics , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Mutation , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SELEX Aptamer Technique
8.
Viruses ; 13(10)2021 10 02.
Article in English | MEDLINE | ID: covidwho-1444335

ABSTRACT

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , Arabinonucleotides/pharmacology , SARS-CoV-2/drug effects , Virus Attachment/drug effects , COVID-19/drug therapy , Humans , Protein Binding/drug effects , Protein Domains/drug effects , Protein Domains/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
9.
Theranostics ; 11(18): 9133-9161, 2021.
Article in English | MEDLINE | ID: covidwho-1410987

ABSTRACT

During an epidemic or pandemic, the primary task is to rapidly develop precise diagnostic approaches and effective therapeutics. Oligonucleotide aptamer-based pathogen detection assays and control therapeutics are promising, as aptamers that specifically recognize and block pathogens can be quickly developed and produced through simple chemical synthesis. This work reviews common aptamer-based diagnostic techniques for communicable diseases and summarizes currently available aptamers that target various pathogens, including the SARS-CoV-2 virus. Moreover, this review discusses how oligonucleotide aptamers might be leveraged to control pathogen propagation and improve host immune system responses. This review offers a comprehensive data source to the further develop aptamer-based diagnostics and therapeutics specific for infectious diseases.


Subject(s)
Aptamers, Nucleotide , Bacteria/genetics , Communicable Diseases/diagnosis , Molecular Diagnostic Techniques/methods , Viruses/genetics , Aptamers, Nucleotide/pharmacology , Biosensing Techniques , COVID-19 Testing/methods , Communicable Disease Control , Communicable Diseases/microbiology , Communicable Diseases/virology , Enzyme-Linked Immunosorbent Assay/methods , Host-Pathogen Interactions/immunology , Humans , SELEX Aptamer Technique , Virus Internalization
11.
Viruses ; 13(5)2021 05 18.
Article in English | MEDLINE | ID: covidwho-1234833

ABSTRACT

COVID-19 is a pandemic respiratory disease that is caused by the highly infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Anti-SARS-CoV-2 antibodies are essential weapons that a patient with COVID-19 has to combat the disease. When now repurposing a drug, namely an aptamer that interacts with SARS-CoV-2 proteins for COVID-19 treatment (BC 007), which is, however, a neutralizer of pathogenic autoantibodies in its original indication, the possibility of also binding and neutralizing anti-SARS-CoV-2 antibodies must be considered. Here, the highly specific virus-neutralizing antibodies have to be distinguished from the ones that also show cross-reactivity to tissues. The last-mentioned could be the origin of the widely reported SARS-CoV-2-induced autoimmunity, which should also become a target of therapy. We, therefore, used enzyme-linked immunosorbent assay (ELISA) technology to assess the binding of well-characterized publicly accessible anti-SARS-CoV-2 antibodies (CV07-209 and CV07-270) with BC 007. Nuclear magnetic resonance spectroscopy, isothermal calorimetric titration, and circular dichroism spectroscopy were additionally used to test the binding of BC 007 to DNA-binding sequence segments of these antibodies. BC 007 did not bind to the highly specific neutralizing anti-SARS-CoV-2 antibody but did bind to the less specific one. This, however, was a lot less compared to an autoantibody of its original indication (14.2%, range 11.0-21.5%). It was also interesting to see that the less-specific anti-SARS-CoV-2 antibody also showed a high background signal in the ELISA (binding on NeutrAvidin-coated or activated but noncoated plastic plate). These initial experiments suggest that the risk of binding and neutralizing highly specific anti-SARS CoV-2 antibodies by BC 007 should be low.


Subject(s)
Antibodies, Neutralizing/immunology , Aptamers, Nucleotide/pharmacology , SARS-CoV-2/immunology , Antibodies, Blocking/immunology , Antibodies, Viral/immunology , Autoantibodies/immunology , COVID-19/immunology , COVID-19/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/immunology , Neutralization Tests/methods , Pandemics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology
12.
Angew Chem Int Ed Engl ; 60(18): 10279-10285, 2021 04 26.
Article in English | MEDLINE | ID: covidwho-1122115

ABSTRACT

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.


Subject(s)
Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , Binding Sites/drug effects , COVID-19/metabolism , Drug Discovery , HEK293 Cells , Humans , Protein Binding/drug effects , Protein Interaction Domains and Motifs/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , SELEX Aptamer Technique , Spike Glycoprotein, Coronavirus/chemistry
13.
Angew Chem Int Ed Engl ; 60(18): 10266-10272, 2021 04 26.
Article in English | MEDLINE | ID: covidwho-1074294

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 is threating global health. Inhibiting interaction of the receptor-binding domain of SARS-CoV-2 S protein (SRBD ) and human ACE2 receptor is a promising treatment strategy. However, SARS-CoV-2 neutralizing antibodies are compromised by their risk of antibody-dependent enhancement (ADE) and unfavorably large size for intranasal delivery. To avoid these limitations, we demonstrated an aptamer blocking strategy by engineering aptamers' binding to the region on SRBD that directly mediates ACE2 receptor engagement, leading to block SARS-CoV-2 infection. With aptamer selection against SRBD and molecular docking, aptamer CoV2-6 was identified and applied to prevent, compete with, and substitute ACE2 from binding to SRBD . CoV2-6 was further shortened and engineered as a circular bivalent aptamer CoV2-6C3 (cb-CoV2-6C3) to improve the stability, affinity, and inhibition efficacy. cb-CoV2-6C3 is stable in serum for more than 12 h and can be stored at room temperature for more than 14 days. Furthermore, cb-CoV2-6C3 binds to SRBD with high affinity (Kd =0.13 nM) and blocks authentic SARS-CoV-2 virus with an IC50 of 0.42 nM.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Antiviral Agents/chemistry , Aptamers, Nucleotide/chemistry , COVID-19/metabolism , Drug Discovery , HEK293 Cells , Humans , Molecular Docking Simulation , Protein Binding/drug effects , Protein Interaction Domains and Motifs/drug effects , SARS-CoV-2/chemistry , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry
14.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Article in English | MEDLINE | ID: covidwho-767668

ABSTRACT

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Subject(s)
RNA/drug effects , RNA/pharmacology , Aptamers, Nucleotide/pharmacology , Aptamers, Nucleotide/therapeutic use , Betacoronavirus , COVID-19 , Chemistry Techniques, Analytical/methods , Chemistry Techniques, Analytical/standards , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus Infections/drug therapy , Drug Delivery Systems/methods , Drug Development/organization & administration , Drug Discovery , Humans , MicroRNAs/pharmacology , MicroRNAs/therapeutic use , Oligonucleotides, Antisense/pharmacology , Oligonucleotides, Antisense/therapeutic use , Pandemics , Pneumonia, Viral/drug therapy , RNA/adverse effects , RNA, Antisense/pharmacology , RNA, Antisense/therapeutic use , RNA, Guide/pharmacology , RNA, Guide/therapeutic use , RNA, Messenger/drug effects , RNA, Messenger/pharmacology , RNA, Ribosomal/drug effects , RNA, Ribosomal/pharmacology , RNA, Small Interfering/pharmacology , RNA, Small Interfering/therapeutic use , RNA, Viral/drug effects , Ribonucleases/metabolism , Riboswitch/drug effects , SARS-CoV-2
15.
Infect Genet Evol ; 85: 104497, 2020 11.
Article in English | MEDLINE | ID: covidwho-704005

ABSTRACT

COVID-19 pandemic is rapidly advancing among human population. Development of new interventions including therapeutics and vaccines against SARS-CoV-2 will require time and validation before it could be made available for public use. Keeping in view of the emergent and evolving situation the motive is to repurpose and test the immediate efficacy of available drugs and therapeutics against COVID-19. Through this article we propose and discuss the possibility of repurposing the available nuclease resistant RNA aptamer against the nucleocapsid protein of SARS-CoV as a potential therapeutic agent for COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Aptamers, Nucleotide/pharmacology , SARS-CoV-2/metabolism , Viral Structural Proteins/antagonists & inhibitors , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/therapeutic use , Coronavirus Nucleocapsid Proteins/antagonists & inhibitors , Coronavirus Nucleocapsid Proteins/chemistry , Drug Repositioning , Humans , Models, Molecular , Molecular Conformation , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Spike Glycoprotein, Coronavirus/chemistry , Viral Structural Proteins/chemistry
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