Structure-guided development of Pb2+-binding DNA aptamers.
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ABSTRACT:
SUBMITTER: Liu H
PROVIDER: S-EPMC8748701 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.
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Project description:Despite the well-established significance of transcription factors (TFs) in pathogenesis, their utilization as pharmacological targets has been limited by the inherent challenges mainly associated with modulating their protein-protein and protein-DNA interactions. The lack of defined small-molecule binding pockets and the nuclear localization of TFs makes neither small molecule inhibitors nor neutral antibodies suitable in blocking TF interactions. Aptamers are short oligonucleotides exhibiting high affinity and specificity for a diverse range of targets. The large molecular weights, expansive blocking surfaces and efficient cellular internalization make aptamers as a compelling molecular tool for traditional TF interaction modulators. Here, we report a structure-guided design strategy called Blocker-SELEX for developing inhibitory aptamers (iAptamer) that selectively block TF interactions. Our approach led to the discovery of an iAptamer that cooperatively disrupts SCAF4/SCAF8-RNA Polymerase II (RNAP2) interactions, thus dysregulates RNAP2 dependent gene expression and splicing, leading to the impairing of cell proliferation. This approach was further applied to develop iAptamers efficiently block WDR5-MYC interaction. Together, our study highlights the potential of Blocker-SELEX in developing iAptamers that effectively disrupt TF interactions, and the generated iAptamers hold promising implications as chemical tools in studying biological functions of TF interactions and the potential for nucleic acids drug development.
2024-02-01 | GSE254651 | GEO
Project description:Following from the evaluation of different types of electrophiles, combined modeling and crystallographic analyses are used to generate potent boronic acid based inhibitors of a penicillin binding protein. The results suggest that a structurally informed approach to penicillin binding protein inhibition will be useful for the development of both improved reversibly binding inhibitors, including boronic acids, and acylating inhibitors, such as ?-lactams.
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Project description:Receptor-targeted image-guided Radionuclide Therapy (TRT) is increasingly recognized as a promising approach to cancer treatment. In particular, the potential for clinical translation of receptor-targeted alpha-particle therapy is receiving considerable attention as an approach that can improve outcomes for cancer patients. Higher Linear-energy Transfer (LET) of alpha-particles (compared to beta particles) for this purpose results in an increased incidence of double-strand DNA breaks and improved-localized cancer-cell damage. Recent clinical studies provide compelling evidence that alpha-TRT has the potential to deliver a significantly more potent anti-cancer effect compared with beta-TRT. Generator-produced 212Pb (which decays to alpha emitters 212Bi and 212Po) is a particularly promising radionuclide for receptor-targeted alpha-particle therapy. A second attractive feature that distinguishes 212Pb alpha-TRT from other available radionuclides is the possibility to employ elementallymatched isotope 203Pb as an imaging surrogate in place of the therapeutic radionuclide. As direct non-invasive measurement of alpha-particle emissions cannot be conducted using current medical scanner technology, the imaging surrogate allows for a pharmacologically-inactive determination of the pharmacokinetics and biodistribution of TRT candidate ligands in advance of treatment. Thus, elementally-matched 203Pb labeled radiopharmaceuticals can be used to identify patients who may benefit from 212Pb alpha-TRT and apply appropriate dosimetry and treatment planning in advance of the therapy. In this review, we provide a brief history on the use of these isotopes for cancer therapy; describe the decay and chemical characteristics of 203/212Pb for their use in cancer theranostics and methodologies applied for production and purification of these isotopes for radiopharmaceutical production. In addition, a medical physics and dosimetry perspective is provided that highlights the potential of 212Pb for alpha-TRT and the expected safety for 203Pb surrogate imaging. Recent and current preclinical and clinical studies are presented. The sum of the findings herein and observations presented provide evidence that the 203Pb/212Pb theranostic pair has a promising future for use in radiopharmaceutical theranostic therapies for cancer.
| S-EPMC10613023 | biostudies-literature
Project description:PurposeThe aims of this study were to develop a prostate-specific membrane antigen (PSMA) ligand for labelling with different radioisotopes of lead and to obtain an approximation of the dosimetry of a simulated 212Pb-based alpha therapy using its 203Pb imaging analogue.MethodsFour novel Glu-urea-based ligands containing the chelators p-SCN-Bn-TCMC or DO3AM were synthesized. Affinity and PSMA-specific internalization were studied in C4-2 cells, and biodistribution in C4-2 tumour-bearing mice. The most promising compound, 203Pb-CA012, was transferred to clinical use. Two patients underwent planar scintigraphy scans at 0.4, 4, 18, 28 and 42 h after injection, together with urine and blood sampling. The time-activity curves of source organs were extrapolated from 203Pb to 212Pb and the calculated residence times of 212Pb were forwarded to its unstable daughter nuclides. QDOSE and OLINDA were used for dosimetry calculations.ResultsIn vitro, all ligands showed low nanomolar binding affinities for PSMA. CA09 and CA012 additionally showed specific ligand-induced internalization of 27.4 ± 2.4 and 15.6 ± 2.1 %ID/106 cells, respectively. The 203Pb-labelled PSMA ligands were stable in serum for 72 h. In vivo, CA012 showed higher specific uptake in tumours than in other organs, and particularly showed rapid kidney clearance from 5.1 ± 2.5%ID/g at 1 h after injection to 0.9 ± 0.1%ID/g at 24 h. In patients, the estimated effective dose from 250-300 MBq of diagnostic 203Pb-CA012 was 6-7 mSv. Assuming instant decay of daughter nuclides, the equivalent doses projected from a therapeutic activity of 100 MBq of 212Pb-CA012 were 0.6 SvRBE5 to the red marrow, 4.3 SvRBE5 to the salivary glands, 4.9 SvRBE5 to the kidneys, 0.7 SvRBE5 to the liver and 0.2 SvRBE5 to other organs; representative tumour lesions averaged 13.2 SvRBE5 (where RBE5 is relative biological effectiveness factor 5). Compared to clinical experience with 213Bi-PSMA-617 and 225Ac-PSMA-617, the projected maximum tolerable dose was about 150 MBq per cycle.Conclusion212Pb-CA012 is a promising candidate for PSMA-targeted alpha therapy of prostate cancer. The dosimetry estimate for radiopharmaceuticals decaying with the release of unstable daughter nuclides has some inherent limitations, thus clinical translation should be done cautiously.
| S-EPMC6451745 | biostudies-literature
Project description:The mechanisms by which transcription factor (TF) protein AP-1 modulates amphetamine's effects on gene transcription in living brains are unclear. We describe here the first part of our studies to investigate these mechanisms, specifically, our efforts to develop and validate aptamers containing the binding sequence of TF AP-1 (5ECdsAP1), in order to elucidate its mechanism of action in living brains. This AP-1-targeting aptamer, as well as a random sequence aptamer with no target (5ECdsRan) as a control, was partially phosphorothioate modified and tagged with superparamagnetic iron oxide nanoparticles (SPIONs), gold, or fluorescein isothiothianate contrast agent for imaging. Optical and transmission electron microscopy studies revealed that 5ECdsAP1 is taken up by endocytosis and is localized in the neuronal endoplasmic reticulum. The results of magnetic resonance imaging (MRI) with SPION-5ECdsAP1 revealed that neuronal AP-1 TF protein levels were elevated in neurons of live male C57black6 mice after amphetamine exposure; however, pretreatment with SCH23390, a dopaminergic receptor antagonist, suppressed this elevation. As studies in transgenic mice with neuronal dominant-negative A-FOS mutant protein, which has no binding affinity for the AP-1 sequence, showed a completely null MRI signal in the striatum, we can conclude that the MR signal reflects specific binding between the 5ECdsAP1 aptamer and endogenous AP-1 protein. Together, these data lend support to the application of 5ECdsAP1 aptamer for intracellular protein-guided imaging and modulation of gene transcription, which will thus allow investigation of the mechanisms of signal transduction in living brains.
| S-EPMC3868842 | biostudies-literature
Project description:Aptamers have a spectrum of applications in biotechnology and drug design, because of the relative simplicity of experimental protocols and advantages of stability and specificity associated with their structural properties. However, to understand the structure-function relationships of aptamers, robust structure modeling tools are necessary. Several such tools have been developed and extensively tested, although most of them target various forms of biological RNA. In this study, we tested the performance of three tools in application to DNA aptamers, since DNA aptamers are the focus of many studies, particularly in drug discovery. We demonstrated that in most cases, the secondary structure of DNA can be reconstructed with acceptable accuracy by at least one of the three tools tested (Mfold, RNAfold, and CentroidFold), although the G-quadruplex motif found in many of the DNA aptamer structures complicates the prediction, as well as the pseudoknot interaction. This problem should be addressed more carefully to improve prediction accuracy.
| S-EPMC6975895 | biostudies-literature
Project description:BackgroundLead-212 (212Pb, t1/2 = 10.6 h) and lead-203 (203Pb, t1/2 = 51.9 h) are an element-equivalent, or a matched theranostic radioisotope pair that show great potential for application in targeted radionuclide therapy (TRT) and single-photon emission computed tomography (SPECT), respectively. At TRIUMF we have produced both 203Pb and 212Pb using TRIUMF's TR13 (13 MeV) and 500 MeV cyclotrons, and subsequently purified and evaluated both radioisotopes using a series of pyridine-modified DOTA analogues in comparison to the commercially available chelates DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and TCMC (1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane).ResultsProton irradiation (12.8 MeV) of natural and enriched thallium-203 (203Tl) targets gave 203Pb saturation yields of 134 ± 25 and 483 ± 3 MBq/μA, respectively. Thorium-228 (228Th, t1/2 = 1.9 y), a by-product of 232Th proton spallation on TRIUMF's main 500 MeV beamline (beamline 1A, BL1A), was recovered to build a 228Th/212Pb generator with the ability to deliver up to 9-10 MBq of 212Pb daily. Both lead isotopes were purified via solid phase extraction chromatography (Pb resin), and isolated in an acetate form ([203/212Pb]Pb(OAc)2) suitable for direct radiolabeling of chelators and bioconjugates. A series of cyclen-based chelators (herein referred to as DOTA-1Py, -2Py, and -3Py) along with established chelates DOTA and TCMC were evaluated for their ability to complex both 203Pb and 212Pb. All chelates incorporated 212Pb/203Pb efficiently, with higher radiolabeling yields observed for the 212Pb-complexes.ConclusionThe production of 203Pb and 212Pb was established using TRIUMF 13 MeV and 500 MeV cyclotrons, respectively. Both production methods provided radiometals suitable for subsequent radiolabeling reactions using known and novel chelates. Furthermore, the novel chelate DOTA-3Py may be a good candidate for biomolecule conjugation and further theranostic 212Pb/203Pb studies.
| S-EPMC7851237 | biostudies-literature
Project description:The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.
| S-EPMC3164881 | biostudies-literature
Project description:DNA aptamers are synthetic, single-stranded DNA oligonucleotides selected by SELEX methods for their binding with specific ligands. Here we present ethidium binding results for three related DNA aptamers (PDB code: 1OLD, 1DB6, and 2ARG)that bind L-argininamide (L-Arm). The ligand bound form of each aptamer's structure has been reported and each are found to be composed primarily of two domains consisting of a stem helical region and a loop domain that forms a binding pocket for the cognate ligand. Previous thermodynamic experiments demonstrated that the DNA aptamer 1OLD undergoes a large conformational ordering upon binding to L-Arm. Here we extend those linkage binding studies by examining the binding of the heterocyclic intercalator ethidium to each of the three aptamers by fluorescence and absorption spectrophotometric titrations. Our results reveal that ethidium binds to each aptamer with DeltaG degree's in the range of -8.7 to -9.4 kcal/mol. The stoichiometry of binding is 2:1 for each aptamer and is quantitatively diminished in the presence of L-Arm as is the overall fluorescence intensity of ethidium. Together, these results demonstrate that a portion of the bound ethidium is excluded from the aptamer in the presence of a saturating amount of L-Arm. These results demonstrate the utility of ethidium and related compounds for the probing of non-conventional DNA structures and reveal an interesting fundamental thermodynamic linkage in DNA aptamers. Results are discussed in the context of the thermodynamic stability and structure of each of the aptamers examined.
| S-EPMC6147400 | biostudies-literature