Potential Molecular Probes Research Articles

Marine alkaloid oroidin analogues with antiviral potential: A novel class of synthetic compounds targeting the cellular chaperone Hsp90.

Marine organisms and their metabolites are a diverse source of scaffolds for potential pharmacological molecular probes and, less frequently, for pharmaceutical lead compounds. In this study, 157 synthetic analogues of marine sponge-derived alkaloids clathrodin and oroidin were screened against replicon models of two RNA viruses, hepatitis C (HCV) and Chikungunya virus (CHIKV) as part of a larger screening project. Four compounds were found to selectively inhibit the HCV replicon (IC50 1.6-4.6μm). These belong to the 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole class of compounds originally designed to target the ATP-binding site of bacterial DNA gyrase. The ATP-binding site of this bacterial protein has high structural similarity to the ATP-binding site of heat-shock protein 90 (Hsp90), a host cell chaperone universally required for viral replication, which led us to examine inhibition of Hsp90 as the compounds' potential mechanism of action. Binding of the four hit compounds to Hsp90 was evaluated through microscale thermophoresis and molecular modeling, which supported our hypothesis of interaction with Hsp90 (Kd 18-79μm) as basis for the compounds' antiviral activity. The presented novel structural class of small molecules that target the Hsp90 ATP-binding site has excellent potential for further antiviral drug development because of the compounds' low toxicity and synthetic accessibility.

Primary study on fluro [ 19F] berberine derivative for human hepatocellular carcinoma targetting in vitro

[ 18F]HX-01, a Fluorine-18 labeled berberine derivative, is a potential positron emission tomography (PET) tumor imaging agent, while [ 19F]HX-01 is a nonradioactive reference substance with different energy state and has the same physical and chemical properties. In order to collect data for further study of [ 18F]HX-01 PET imaging of hepatocellular carcinoma in vivo, this study compared the uptake of [ 19F]HX-01 by human hepatocellular carcinoma and normal hepatocytes in vitro. The target compound, [ 19F]HX-01, was synthesized in one step using berberrubine and 3-fluoropropyl 4-methylbenzenesulfonate. Cellular uptake and localization of [ 19F]HX-01 were performed by a fluorescence microscope in human hepatocellular carcinoma HepG2, SMMC-7721 and human normal hepatocyte HL-7702. Cellular proliferation inhibition and cell cytotoxicity assay of the [ 19F]HX-01 were conducted using cell counting kit-8 (CCK-8) on HepG2, SMMC-7721 and HL-7702 cells. Fluorescent microscopy showed that the combining ability of [ 19F]HX-01 to the carcinoma SMMC-7721 and HepG2 was higher than that to the normal HL-7702. Cellular proliferation inhibition assay demonstrated that [ 19F]HX-01 leaded to a dose-dependent inhibition on SMMC-7721, HepG2, and HL-7702 proliferation. Cell cytotoxicity assay presented that the cytotoxicity of [ 19F]HX-01 to SMMC-7721 and HepG2 was obviously higher than that to HL-7702. This in vitro study showed that [ 19F]HX-01 had a higher selectivity on human hepatocellular carcinoma cells (SMMC-7721, HepG2) but has less toxicity to normal hepatocytes (HL-7702). This could set up the idea that the radioactive reference substance [ 18F]HX-01 may be worthy of further development as a potential molecular probe targeting human hepatocellular carcinoma using PET.

Synthesis and evaluation of benzothiazole-triazole and benzothiadiazole-triazole scaffolds as potential molecular probes for amyloid-β aggregation

Fluorescent scaffolds that can be easily modifiedviaclick chemistry were investigated as probes for Aβ-plaque deposits in mouse tissue.

Synthesis of acridine-1,8-dione substituted (E)-5-(3-aminoallyl)-uridine-5′-triphosphate: a new potential fluorogenic molecular probe

The first synthesis of highly fluorescent acridine-1,8-dione dye labeled aminoallyl-uridine-5′-triphosphate is described. The coupling reaction of acridine-1,8-dione benzoic acid succinimidyl ester with aminoallyl-uridine-5′-triphosphate, followed by anion exchange purification results in the final compound with good yield and high purity. The acridine-1,8-dione benzoic acid succinimidyl ester is obtained by the reaction of acridine-1,8-dione benzoic acid with N-hydroxysuccinimide. This fluorogenic acridine-1,8-dione dye substituted uridine nucleotide has potential to generate labeled nucleic acids for molecular biology and molecular cytogenetic applications.

Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α.

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 μM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein-protein binding events.

Solvatochromism of a tricycloquinazoline based disk-shaped liquid crystal: a potential molecular probe for fluorescence imaging

Fluorescence microscopy images of a non-fluorescent discotic liquid crystal (DLC) using tricycloquinazoline (TCQ) based DLC as a molecular probe.

Droplet electrochemical study of the pH dependent redox behavior of novel ferrocenyl-carborane derivatives and its application in specific cancer cell recognition

Novel ferrocenyl based carboranes (FcCBs) and their distinguish behavior for cancer cell recognition have been explored in this contribution. The voltammetric study in a droplet of 10μL placed on the surface of a glassy carbon electrode demonstrates the excellent electrochemical behavior of FcCBs, which could be further exploited for establishing the promising and sensitive biosensors. The FcCBs’ redox behavior is examined in a wide pH range, and square wave voltammetry revealed the reversible and irreversible nature of first and second anodic peaks. The obvious shifts in peak potentials corresponding with the change of pH values demonstrate the abstraction of electrons to be accompanied with the transfer of protons. By using the droplet electrochemical technique, FcCBs can be employed to distinguish normal and cancer cells with a linear range from 1.0×103 to 3.0×104cellsmL−1 and the limit of detection at 800cellsmL−1. The novel carborane derivatives could be utilized as important potential molecular probes for specific recognition of cancer cells like leukemia cells from normal cells.

CdTe quantum dots conjugated to concanavalin A as potential fluorescent molecular probes for saccharides detection in Candida albicans

Semiconductor colloidal quantum dots (QDs) have been applied in biological analysis due to their unique optical properties and their versatility to be conjugated to biomolecules, such as lectins and antibodies, acquiring specificity to label a variety of targets. Concanavalin A (Con A) lectin binds specifically to α-d-mannose and α-d-glucose regions of saccharides that are usually expressed on membranes of mammalian cells and on cell walls of microbials. Candida albicans is the most common fungal opportunistic pathogen present in humans. Therefore, in this work, this fungus was chosen as a model for understanding cells and biofilm-forming organisms. Here, we report an efficient bioconjugation process to bind CdTe (Cadmium Telluride) QDs to Con A, and applied the bioconjugates to label saccharide structures on the cellular surface of C. albicans suspensions and biofilms. By accomplishing hemagglutination experiments and circular dichroism, we observed that the Con A structure and biochemical properties were preserved after the bioconjugation. Fluorescence microscopy images of yeasts and hyphae cells, as well as biofilms, incubated with QDs-(Con A) showed a bright orange fluorescence profile, indicating that the cell walls were specifically labeled. Furthermore, flow cytometry measurements confirmed that over 93% of the yeast cells were successfully labeled by QD-(Con A) complex. In contrast, non-conjugated QDs or QDs-(inhibited Con A) do not label any kind of biological system tested, indicating that the bioconjugation was specific and efficient. The staining pattern of the cells and biofilms demonstrate that QDs were effectively bioconjugated to Con A with specific labeling of saccharide-rich structures on C. albicans. Consequently, this work opens new possibilities to monitor glucose and mannose molecules through fluorescence techniques, which can help to optimize phototherapy protocols for this kind of fungus.

Design and Synthesis of 24-Fluorinated Bafilomycin Analogue as an NMR Probe with Potent Inhibitory Activity to Vacuolar-type ATPase

Abstract A fluorine-labeled bafilomycin analogue was designed and convergently synthesized from three segments via the Stille coupling, macrolactonization, and diastereoselective aldol reaction. The V-ATPase inhibitory activity of the analogue was comparable to that of the natural product, indicating its utility as a potential molecular probe for investigating the inhibition mechanism of bafilomycin by NMR.

68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET

Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine-glycine-arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a (68)Ga-labeled NGR peptide as a new molecular probe that binds to APN. NGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with (68)Ga at 95°C for 10 min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of (68)Ga-DOTA-NGR was determined in normal mice by dissection method. (68)Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry. The radiochemical purity of (68)Ga-DOTA-NGR was 98.0% ± 1.4% with a specific activity of about 17.49 MBq/nmol. The uptake of (68)Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that (68)Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1 h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58 ± 1.26 at 1.5 h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature. (68)Ga-DOTA-NGR was easily synthesized and showed favorable biodistribution and kinetics. (68)Ga-DOTA-NGR could also specifically bind to the APN receptor in vitro and in vivo, and might be a potential molecular probe for the noninvasive detection of APN-positive tumors and neovasculature.

Water as a potential molecular probe for functional groups on carbon surfaces

A new and simple method, using water as a potential molecular probe, is proposed for the determination of the concentration of surface oxygen groups on carbon adsorbents. The procedure is based on a determination of the Henry constant between a water molecule and a functional group from the volume integration of the Boltzmann factor over the accessible space around the functional group. Three porous carbons are used in this study to test the new method: A-5, RF-100 and RF-200. The results obtained are in good agreement with those measured by Boehm titration. This new method can be applied to adsorbents containing small concentrations of oxygen groups where the Boehm titration method may give unreliable results.

A two-step stimulus-response cell-SELEX method to generate a DNA aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection

Aptamers are single-stranded oligonucleotides that are capable of binding wide classes of targets with high affinity and specificity. Their unique three-dimensional structures present numerous possibilities for recognizing virtually any class of target molecules, making them a promising alternative to antibodies used as molecular probes in biomedical analysis and clinical diagnosis. In recent years, cell-systematic evolution of ligands by exponential enrichment (SELEX) has been used extensively to select aptamers for various cell targets. However, aptamers that have evolved from cell-SELEX to distinguish the "stimulus-response cell" have not previously been reported. Moreover, a number of cumbersome and time-consuming steps involved in conventional cell-SELEX reduce the efficiency and efficacy of the aptamer selection. Here, we report a "two-step" methodology of cell-SELEX that successfully selected DNA aptamers specifically against "inflamed" endothelial cells. This has been termed as stimulus-response cell-SELEX (SRC-SELEX). The SRC-SELEX enables the selection of aptamers to distinguish the cells activated by stimulus of healthy cells or cells isolated from diseased tissue. We report a promising aptamer, N55, selected by SRC-SELEX, which can bind specifically to inflamed endothelial cells both in cell culture and atherosclerotic plaque tissue. This aptamer probe was demonstrated as a potential molecular probe for magnetic resonance imaging to target inflamed endothelial cells and atherosclerotic plaque detection.

Recognition and capture of metastatic hepatocellular carcinoma cells using aptamer-conjugated quantum dots and magnetic particles

Metastatic recurrence is the most important biological behavior of hepatocellular carcinoma (HCC) and the main cause of treatment failure. Early prediction of metastasis is currently impossible due to the lack of specific molecular probes to recognize metastatic HCC cells. Aptamers have recently emerged as promising potential molecular probes for biomedical applications. Two well-matched HCC cell lines including HCCLM9 with high metastatic potential and MHCC97-L with low metastatic potential, were used to select aptamers for HCC metastasis. With a whole-cell-SELEX strategy, in which HCCLM9 cells were used as target cells and MHCC97-L cells as subtractive cell, 6 potential aptamers had been generated. Detailed study on selected aptamer LY-1 revealed that it could bind metastatic HCC cells with high affinity and specificity, not only in cells culture and animal models of HCC metastasis, but also in clinical HCC specimens. Moreover, the aptamer LY-1 and magnetic particles conjugates could efficiently capture the HCC cells from complex mixture whole blood. These studies demonstrated that this HCC specific aptamer LY-1 could be a promising molecular probe to recognize metastatic HCC cells.

Study of Fluorescence Quenching in Aluminum-Doped Ceria Nanoparticles: Potential Molecular Probe for Dissolved Oxygen

This work investigates a novel usage of aluminum-doped ceria nanoparticles (ADC-NPs), as the molecular probe in optical fluorescence quenching for sensing the dissolved oxygen (DO). Cerium oxide (ceria) nanoparticles can be considered one of the most unique nanomaterials that are being studied today due to the diffusion and reactivity of oxygen vacancies in ceria, which contributes to its high oxygen storage capability. Aluminum can be considered a promising dopant to increase the oxygen ionic conductivity in ceria nanoparticles which can improve the sensitivity of ceria nanoparticles to DO. The fluorescence intensity of ADC-NPs, synthesized via chemical precipitation, is found to have a strong inverse relationship with the DO concentration in aqueous solutions. Stern-Volmer constant of ADC-NPs at room temperature is determined to be 454.6 M(-1), which indicates that ADC-NPs have a promising sensitivity to dissolved oxygen, compared to many presently used fluorophores. In addition, Stern-Volmer constant is found to have a relatively small dependence on temperature between 25 °C to 50 °C, which shows excellent thermal stability of ADC-NPs sensitivity. Our work suggests that ADC-NPs, at 6 nm, are the smallest diameter DO molecular probes between the currently used optical DO sensors composed of different nanostructures. This investigation can improve the performance of fluorescence-quenching DO sensors for industrial and environmental applications.

Silicon nanotweezers for biomechanical and bioelectrical assays

In modern life, technologies enabling detection of biological molecules at a low threshold, for health and ecological concerns, are in high demand. Directly interrogating the molecules is a promising direction to clarify the noisy response of conventional assays arising from simultaneous different reactions. Besides sophisticated biophysical instrument such as the atomic force microscope, this paper proposes silicon nanotweezers (SNT) as a new microsystem for molecular manipulation. SNT can trap molecules and sense their biomechanical and bioelectrical response in minute operations. In this review SNT characteristics are overviewed; their operation modes are illustrated by molecule and cell trapping, manipulation and characterization in air and in solution. As they are tiny and can be mass produced by highly parallel microsystem technology, SNT can be seen as a potential molecular and cellular probe for routine analysis and bio detection.

In vitro study of SPIO‐labeled human pancreatic cancer cell line BxPC‐3

The survivin gene is highly expressed in pancreatic cancer. The purpose of this study was to design and synthesize functionalized magnetic iron oxide nanoparticles (MNPs) targeting survivin gene for the detection of pancreatic cancer. The pancreatic cancer cell line BxPC-3 with survivin gene expression was selected in this study. The healthy lung fibroblast cell was used as a control. Chitosan-coated MNPs (CS@MNPs) and antisense oligodeoxynucleotide of survivin gene were conjugated to MNPs to give Sur-MNPs. Fourier transform infrared spectroscopy was performed to confirm the conjunction of chitosan. The interactions of MNPs, CS@MNPs, and Sur-MNPs in BxPC-3 cells were observed, recorded and analyzed. The size, morphology, cell uptake, cytotoxicity and stability of those particles were assessed by transmission electron microscope, Prussian blue staining, MTT assay and agarose gel electrophoresis. The magnetic resonance signal intensities of pancreatic cells labeled with CS@MNPs and MNPs, and Sur-MNPs, were compared on T₂-weighted images. The results demonstrated that the level of cellular uptake of CS@MNPs was higher than that of naked MNPs. The Sur-MNPs had a suitable size (12 nm sized core), high stability, no cytotoxicity and good water dispersion. Sur-MNPs did not accumulate in healthy lung fibroblast cells, while being taken up by BxPC-3 cells. The Sur-MNPs in BxPC-3 cells could be visualized on T₂-weighted images, which suggested that Sur-MNPs could be used to detect the expression of survivin gene. Thus, Sur-MNPs may be a potential molecular imaging probe targeting survivin gene for early detection of pancreatic cancer cells.

Visual detection of single-base mismatches in DNA using hairpin oligonucleotide with double-target DNA binding sequences and gold nanoparticles

We describe a hairpin oligonucleotide (HO) with double-target DNA binding sequences in the loop and 11-base in the stem for visual detection of single-base mismatches (SBM) in DNA with highly specificity. The thiol-modified HO was immobilized on gold nanoparticle (Au-NP) surface through a self-assembling process. The strategy of detecting SBM depends on the unique molecular recognition properties of HO to the perfect-matched DNA and SBM to generate different quantities of duplex DNA on the Au-NP surface, which are captured on the test zone of lateral flow test strip via the DNA hybridization reaction between the duplex DNA and preimmobilized DNA probe. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SBM. It was found that the ability of HO to differentiate perfect-matched DNA and SBM was increased dramatically by incorporating double-target DNA binding sequences in the loop of HO. The signal ratio between perfect-matched DNA and SBM was up to 28, which is much higher than that of conventional HO or molecular beacon. The approach was applied to detect the mutation sites, Arg142Cys and Gly529Ile, of transglutaminase 1 gene in autosomal recessive congenital ichthyosis. The results presented here show that the new HO is a potential molecular recognition probe for the future development of nucleic acid-based biosensors and bioassays. The approach can be used for point-of-care diagnosis of genetic diseases and detecting infectious agents or warning against bio-warfare agents.

Synthesis and characterization of fluorinated homocysteine derivatives as potential molecular probes for 19 F magnetic resonance spectroscopy and imaging

A N-trifluoroacetyl-protected amino acid containing a thioester function, 2,2,2-trifluoro-N-(2-oxo-tetrahydrothiophen-3-yl)acetamide (TFA-tHcy), has been synthesized and characterized. It was then used to prepare a fluorine-labeled N-homocysteinylated protein, (19)F-Hcy-εN-Lys-albumin, that was characterized by SDS-PAGE, MALDI-TOF-MS, UV-vis and (19)F NMR spectroscopy. On average, four N-trifluoroacetylhomocysteine residues were covalently conjugated to human serum albumin through the N-substituted homocysteine thiolactone. The in situ homocysteinylation of human plasma proteins with TFA-tHcy has also been performed and has led to the formation of N-homocysteinylated proteins, with albumin modification accounting for ca. 75% of all fluorine-labeled human plasma proteins. The synthesized fluorinated molecular probes can be potentially used as informative molecular probes for in vivo (19)F magnetic resonance spectroscopy and imaging.

Synthesis of bi-substrate state mimics of dihydropteroate synthase as potential inhibitors and molecular probes

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillusanthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.

A 2-Helix Small Protein Labeled with 68Ga for PET Imaging of HER2 Expression

Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix-based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one alpha-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation. In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, (68)Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model. The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that (68)Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of (68)Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo. This proof-of-concept research clearly demonstrated that synthetic 2-helix (68)Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.