ABSTRACT
Background: Salvianolic acid B (SAB) is a major component of Salvia miltiorrhiza root (Danshen), widely used in East/Southeast Asia for centuries to treat cardiovascular diseases. Danshen depside salt, 85% of which is made up of SAB, is approved in China to treat chronic angina. Although clinical observations suggest that Danshen extracts inhibited arterial and venous thrombosis, the exact mechanism has not been adequately elucidated. Objective: To delineate the antithrombotic mechanisms of SAB. Methods: We applied platelet aggregation and coagulation assays, perfusion chambers, and intravital microscopy models. The inhibition kinetics and binding affinity of SAB to thrombin are measured by thrombin enzymatic assays, intrinsic fluorescence spectrophotometry, and isothermal titration calorimetry. We used molecular in silico docking models to predict the interactions of SAB with thrombin. Results: SAB dose-dependently inhibited platelet activation and aggregation induced by thrombin. SAB also reduced platelet aggregation induced by adenosine diphosphate and collagen. SAB attenuated blood coagulation by modifying fibrin network structures and significantly decreased thrombus formation in mouse cremaster arterioles and perfusion chambers. The direct SAB-thrombin interaction was confirmed by enzymatic assays, intrinsic fluorescence spectrophotometry, and isothermal titration calorimetry. Interestingly, SAB shares key structural similarities with the trisubstituted benzimidazole class of thrombin inhibitors, such as dabigatran. Molecular docking models predicted the binding of SAB to the thrombin active site. Conclusion: Our data established SAB as the first herb-derived direct thrombin catalytic site inhibitor, suppressing thrombosis through both thrombin-dependent and thrombin-independent pathways. Purified SAB may be a cost-effective agent for treating arterial and deep vein thrombosis.
ABSTRACT
The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of µM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.
Subject(s)
Amodiaquine , DNA , DNA/chemistry , DNA/metabolism , Amodiaquine/chemistry , Amodiaquine/metabolism , Amodiaquine/analogs & derivatives , Antimalarials/chemistry , Antimalarials/metabolism , Nucleic Acid Conformation , Binding Sites , Thermodynamics , CalorimetryABSTRACT
The COVID-19 pandemic caused by SARS-CoV-2 virus is an ongoing global health burden. Severe cases of COVID-19 and the rare cases of COVID-19 vaccine-induced-thrombotic-thrombocytopenia (VITT) are both associated with thrombosis and thrombocytopenia; however, the underlying mechanisms remain inadequately understood. Both infection and vaccination utilize the spike protein receptor-binding domain (RBD) of SARS-CoV-2. We found that intravenous injection of recombinant RBD caused significant platelet clearance in mice. Further investigation revealed the RBD could bind platelets, cause platelet activation, and potentiate platelet aggregation, which was exacerbated in the Delta and Kappa variants. The RBD-platelet interaction was partially dependent on the ß3 integrin as binding was significantly reduced in ß3-/- mice. Furthermore, RBD binding to human and mouse platelets was significantly reduced with related αIIbß3 antagonists and mutation of the RGD (arginine-glycine-aspartate) integrin binding motif to RGE (arginine-glycine-glutamate). We developed anti-RBD polyclonal and several monoclonal antibodies (mAbs) and identified 4F2 and 4H12 for their potent dual inhibition of RBD-induced platelet activation, aggregation, and clearance in vivo, and SARS-CoV-2 infection and replication in Vero E6 cells. Our data show that the RBD can bind platelets partially though αIIbß3 and induce platelet activation and clearance, which may contribute to thrombosis and thrombocytopenia observed in COVID-19 and VITT. Our newly developed mAbs 4F2 and 4H12 have potential not only for diagnosis of SARS-CoV-2 virus antigen but also importantly for therapy against COVID-19.
ABSTRACT
Electrochemical aptamer-based (E-AB) biosensors have demonstrated capabilities in monitoring molecules directly in undiluted complex matrices and in the body with the hopes of addressing personalized medicine challenges. This sensing platform relies on an electrode-bound, redox-reporter-modified aptamer. The electrochemical signal is thought to originate from the aptamer undergoing a binding-induced conformational change capable of moving the redox reporter closer to the electrode surface. While this is the generally accepted mechanism, it is notable that there is limited evidence demonstrating conformational change or distance-dependent change in electron transfer rates in E-AB sensors. In response, we investigate here the signal transduction of the well-studied cocaine-binding aptamer with different analytical methods and found that this sensor relies on a redox-reporter - ligand competition mechanism rather than a ligand-induced structure formation mechanism. Our results show that the covalently bound redox reporter, methylene blue, binds at or near the ligand binding site on the aptamer resulting in a folded conformation of the cocaine-binding aptamer. Addition of ligand then competes with the redox reporter for binding, altering its electron transfer rate. While we show this for the cocaine-binding aptamer, given the prevalence of methylene blue in E-AB sensors, a similar competition-based may occur in other systems.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Cocaine , Aptamers, Nucleotide/chemistry , Ligands , Methylene Blue , Oxidation-Reduction , Signal Transduction , Electrochemical Techniques/methods , ElectrodesABSTRACT
BACKGROUND: Platelet GPIbα-von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb-VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. OBJECTIVE: The objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. METHODS: Natural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα-VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX-induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. RESULTS: We found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbß3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF-mediated platelet aggregation, spreading, and thrombus growth at high shear. CONCLUSION: Fucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb-VWF interaction after fucoidan is hydrolyzed.
Subject(s)
Thrombosis , von Willebrand Factor , Humans , Animals , Mice , von Willebrand Factor/metabolism , Blood Platelets/metabolism , Platelet Aggregation , Platelet Glycoprotein GPIb-IX Complex/metabolism , Polysaccharides/pharmacology , Thrombosis/drug therapy , Thrombosis/prevention & control , Thrombosis/metabolism , Fibrinogen/metabolism , Protein BindingABSTRACT
Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.
Subject(s)
Antimalarials/metabolism , Aptamers, Nucleotide/metabolism , Artemisinins/metabolism , Antimalarials/chemistry , Aptamers, Nucleotide/chemistry , Artemisinins/chemistry , Binding, Competitive , Biosensing Techniques , Nucleic Acid Conformation , Proton Magnetic Resonance Spectroscopy , Spectrometry, Mass, Electrospray Ionization , Structure-Activity RelationshipABSTRACT
Levamisole is a common and harmful adulterant of street samples of cocaine and can cause electrochemical tests for cocaine to give false negative results. To see if levamisole would interfere with aptamer-based bioassays, we analyzed the binding of levamisole to the cocaine-binding DNA aptamer. At low aptamer concentrations (0.5 to 20 µM) using isothermal titration calorimetry methods and thermal stability measurements, no binding of levamisole to the cocaine-binding aptamer was observed. At higher levamisole concentrations (500 µM), weak binding to the cocaine-binding aptamer was detected using nuclear magnetic resonance (NMR) spectroscopy chemical shift perturbations. NMR-detected titrations show that levamisole binding is competitive with cocaine binding, indicating that both ligands share a common binding site. Finally, we show that the presence of levamisole does not interfere with the photochrome aptamer switch binding assay for cocaine. We conclude that assays using low concentrations of cocaine, and consequently low concentration of levamisole as an adulterant, should be unaffected by the weak binding of levamisole.
ABSTRACT
DNA three-way junctions (3WJs) consist of a Y-shaped hydrophobic branch point connecting three double-stranded stems and are viewed as druggable targets for cancer treatment. They are also important building blocks for the construction of DNA nanostructures and serve as recognition elements for DNA aptasensors for a wide variety of diagnostic applications. However, visible fluorescent light-up probes for specific staining of DNA 3WJs are currently lacking. Herein, we report that a merocyanine containing the N-methylbenzothiazolium (Btz) acceptor vinyl linked to a 2-fluorophenolic (FPhO) donor (FPhOBtz) serves as a universal fluorescent turn-on dye for DNA 3WJs. Our evidence is based on a multifaceted approach to define the specificity and affinity of FPhOBtz for 3WJ DNA aptamers; the cocaine binding aptamer MN4, the cholic acid binding aptamer (CABA), and four steroid aptamers (DOGS.1, DISS.1, BES.1, DCAS.1). FPhOBtz exhibits impressive turn-on (up to 730-fold) fluorescence at 580 nm upon aptamer binding with low micromolar affinity. Direct FPhOBtz displacement from the 3WJ binding domain through competitive alkaloid and steroid binding provides immediate fluorescent read out for host-guest detection strategies in human blood serum in the low micromolar regime. Our results present the first visible light-up fluorescent probe for DNA 3WJ detection strategies.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , DNA , Fluorescent Dyes/chemistry , Spectrometry, FluorescenceABSTRACT
The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods. This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer. Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. We have determined the binding affinity and thermodynamics for both ligand-binding sites. By separating the ligand-binding sites by an additional four base pairs, we engineered a variant of this aptamer that binds two adenosine ligands in an independent manner. Together with NMR and thermal stability experiments, these data indicate that the ATP-binding DNA aptamer follows a population-shift binding mechanism that is the source of the positive binding cooperativity by the aptamer.
Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Thermodynamics , Adenosine Triphosphate/metabolism , Binding Sites , Calorimetry , Magnetic Resonance SpectroscopyABSTRACT
The Photochrome Aptamer Switch Assay (PHASA) relies on ligand binding by an aptamer to alter the local environment of a stilbene compound covalently attached to the 5' end of the aptamer. We used the PHASA with both structure switching and non-structure switching versions of the cocaine-binding aptamer. We show that the largest change in fluorescence intensity and the lowest concentration limit of detection (CLooD) is obtained using the structure-switching cocaine-binding aptamer. Fluorescence anisotropy measurements were used to quantify the affinity of the conjugated aptamer to cocaine. We also used thermal melt analysis and Nuclear Magnetic Resonance (NMR) spectroscopy to show that the addition of the stilbene to the aptamer increases the melt temperature of the cocaine-bound structure-switching aptamer by (6.4 ± 0.3) °C compared to the unconjugated aptamer while the free form of the structure-switching aptamer-stilbene conjugate remains unfolded.
ABSTRACT
A challenge for the use of aptamers as biosensors is how to signal the occurrence of their ligand binding event into a signal that can be exploited in a detection scheme. Here, we present the concept of "aptachain" formation, where an aptamer is split into two overlapping or staggered strands and assembles into an extended oligomer upon ligand binding. This assembly of aptamers can then be used as a way to detect ligand binding by the aptamer. As an example of this concept, we employed the cocaine-binding aptamer as a model system, used its ability to tightly bind quinine and demonstrated its capability in a gold nanoparticle-based biosensing application. We used isothermal titration calorimetry to demonstrate that, when split into two overlapping DNA strands, the aptamer remains functional. Size-exclusion chromatography showed that the quinine-bound oligos form a larger assembly of aptamer units than in the absence of ligand. Finally, we used the oligomer forming ability of the aptachain oligos in a biosensor application for quinine that brings gold nanoparticles closer together resulting in a shift in their plasmonic resonance to a longer wavelength and an observed colour shift. We propose that splitting aptamers into overlapping strands that form oligomers in the presence of a ligand, aptachain formation, will be generally applicable to aptamers and prove useful in a variety of biotechnology applications.
ABSTRACT
We have developed a new cocaine-binding aptamer variant that has a significantly higher melt temperature when bound to a ligand than the currently used sequence. Retained in this new construct is the ligand-induced structure-switching binding mechanism that is important in biosensing applications of the cocaine-binding aptamer. Isothermal titration calorimetry methods show that the binding affinity of this new sequence is slightly tighter than the existing cocaine-binding aptamer. The improved thermal performance, a Tm increase of 4 °C for the cocaine-bound aptamer and 9 °C for the quinine-bound aptamer, was achieved by optimizing the DNA sequence in stem 2 of the aptamer to have the highest stability based on the nearest neighbor thermodynamic parameters and confirmed by UV and fluorescence spectroscopy. The sequences in stem 1 and stem 3 were unchanged in order to retain the structure switching and ligand binding functions. The more favorable thermal stability characteristics of the OR3 aptamer should make it a useful construct for sensing applications employing the cocaine-binding aptamer system.
Subject(s)
Aptamers, Nucleotide/chemistry , Cocaine/chemistry , Nucleic Acid Conformation , Calorimetry/methods , Cocaine/analysisABSTRACT
Understanding how aptamer structure and function are related is crucial in the design and development of aptamer-based biosensors. We have analyzed a series of cocaine-binding aptamers with different lengths of their stem 1 in order to understand the role that this stem plays in the ligand-induced structure-switching binding mechanism utilized in many of the sensor applications of this aptamer. In the cocaine-binding aptamer, the length of stem 1 controls whether the structure-switching binding mechanism for this aptamer occurs or not. We varied the length of stem 1 from being one to seven base pairs long and found that the structural transition from unfolded to folded in the unbound aptamer is when the aptamer elongates from 3 to 4 base pairs in stem 1. We then used this knowledge to achieve new binding selectivity of this aptamer for quinine over cocaine by using an aptamer with a stem 1 two base pairs long. This selectivity is achieved by means of the greater affinity quinine has for the aptamer compared with cocaine. Quinine provides enough free energy to both fold and bind the 2-base pair-long aptamer while cocaine does not. This tuning of binding selectivity of an aptamer by reducing its stability is likely a general mechanism that could be used to tune aptamer specificity for tighter binding ligands.