Good Cell Permeability Research Articles

A reversible water-soluble naphthalimide-based chemosensor for imaging of cellular copper(II) ion and cysteine

Cellular free copper ion is essential yet potentially toxic to living orgasms, thus cells have evolved mechanisms to balance global copper content including the chelation reactions by cysteine. To better understand the delicate counter-balance of free copper(II) ion and cysteine in the living cells, we developed a water-soluble naphthalimide-based derivative NC as a chemosensor for reversible dual detection of copper(II) ion and cysteine. The chemosensor NC exhibits a rapid, sensitive and quantitative response for copper(II) ion in aqueous solution due to the formation of the corresponding complex NC-Cu(II) in the range of 0–5μM with the detection limit of 7.11nM. Afterwards, NC is released from NC-Cu(II) complex accompanied with a “turn-on” fluorescence in the presence of cysteine ranging from 0 to 160μM. It's also worth noting that the linear range of the NC-Cu(II) complex for the cysteine is identical with the intracellular level of cysteine under physiological conditions (30–200μM). To achieve its practical application, we further demonstrated that the chemosensor NC exhibits good cell permeability and could be employed to monitor free copper(II) ion and cysteine in the living cells.

Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe.

In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

A novel substrate-inspired fluorescent probe to monitor native albumin in human plasma and living cells

Human albumin (HA) displays crucial roles in maintaining health and fighting diseases. Accurate determination of native HA in plasma or non-plasma samples are of immense significance in both basic research and clinical practice. Herein, a novel ratiometric two-photon fluorescent probe (N-butyl-4-(4-phenyl-benzoyloxy) 1,8-naphthalimide, BPBN) has been designed and developed for highly selective and sensitive sensing the enzymatic activities of HA, on the basis of its unique pseudo-esterase feature. BPBN exhibits excellent selectivity, high sensitivity and good reactivity under physiological conditions. As an enzymatic activity-based probe, BPBN can distinguish between native HA and denatured HA, while the currently used dye-binding method cannot. The probe has been successfully applied to measure native HA in plasma samples and the secreted HA in the hepatocyte culture supernatant. BPBN has also been used for two-photon imaging of HA reabsorption in living renal cells, and the results demonstrate that this probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. All these findings suggest that BPBN can be reliably used for the highly selective and sensitive detection of native HA in complex biological samples, as well as for exploring HA-associated biological processes and the physiological functions of native HA in living cells.

Benzothiazole-Based Fluorescent Sensor for Ratiometric Detection of Zn(II) Ions and Secondary Sensing PPi and Its Applications for Biological Imaging and PPase Catalysis Assays

In this paper, we designed and synthesized three benzothiazole-based fluorescent probes L1, L2, and L3 for zinc ion detection. Among various metal ions, only the zinc ion exhibited fluorescence enhancement at 475 nm accompanied by the blue-shift emission wavelength in HEPES buffer solution containing probes. Through titration experiment, the detection limit of L1 for zinc ion sensing was calculated to be as low as 7 nM, which showed a high sensitivity. Furthermore, the confocal laser scanning micrographs of HeLa cells demonstrate good cell permeability of probe L1 and selective detection of zinc ion in living cells. The L1–Zn2+ complex was further used for pyrophosphate (PPi) sensing in HEPES buffer solution, the limit of detection was calculated to be as low as 60 nM. L1–Zn2+ can monitor the enzyme catalyzed degradation process of PPi, thus providing a meaningful way for tracking of zinc ion and pyrophosphate in biological systems.

A solvent-tuning fluorescence sensor for In(III) and Al(III) ions and its bioimaging application

A fluorescence-enhanced probe PY, which is based on integration of pyrene moiety and amino acid group, was developed for the orthogonal recognition of In3+ and Al3+ by the quick formation of 1:1 complex. PY shows high selectivity and sensitivity for In3+ in NaOAc/HOAc (pH 4.8) buffer solution but rather for Al3+ in methanol. Moreover, this probe shows good cell permeability, and the recognition of In3+ by PY was successfully applied in bioimaging, which was the first example for detection of this metal ion in living cells.

Abstract 5226: Novel class of potent and selective inhibitors efface MTH1 as broad-spectrum cancer target

Abstract Malignant transformation is accompanied by increased reactive oxygen species (ROS) known to promote carcinogenesis and damage free nucleotides and DNA. During replication, damaged nucleotides are incorporated into DNA resulting in DNA breaks and mutations, which can ultimately lead to cell death. Cancer cells may evade this process via overexpression of MTH1 (also known as NUDT1), a member of nudix phosphohydrolase protein family, which converts the oxidized nucleotides 8-oxo-dGTP and 2-OH-dATP into the corresponding monophosphates thus preventing their incorporation into DNA and avoiding cell death. Initial RNAi-mediated knockdown of MTH1 and tool compounds (TH588, (S)-crizotinib) inhibiting MTH1 supported this model. As MTH1 is not essential for non-transformed cell survival, MTH1 was hypothesized to be a non-oncogenic cancer addiction and a potential broad-spectrum cancer target. Attractive target rationale combined with previous success in identifying potent and cellularly active MTH1 inhibitors prompted us to develop new cancer therapeutics inhibiting MTH1. By using fragment-based screening and structure-based drug design, a series of 4-amino-2-carboxamide-7-azaindoles was identified. We developed biochemically potent and selective MTH1 inhibitors with good cell permeability and metabolic stability. These MTH1 inhibitors demonstrated target engagement in cellular thermal shift assay (CETSA), and a strong positive correlation between cellular and biochemical potency was observed. One promising MTH1 inhibitor from this structural class was BAY-707. Unexpectedly however, these properties did not translate into accumulation of oxidized nucleotides within DNA and consequent induction of γH2AX and DNA damage response. Moreover, while tool compounds (TH588, (S)-crizotinib) were confirmed to be biochemically potent MTH1 inhibitors which stunted the proliferation of a range of cancer cell lines, our more potent and cellularly active MTH1 inhibitors, including BAY-707, demonstrated no significant effect on cancer cell survival. Furthermore, we were unable to demonstrate in vivo efficacy using xenograft models of human cancers or syngeneic mouse tumor models. Finally, our in vitro and in vivo combination studies with pro-oxidants, standard-of-care drugs or radiation also failed to result in significant additive or synergistic growth inhibitory effects on cancer cells. Thus, our findings support the recently published observations made with other potent and selective MTH1 chemical probes (AZ compound 15, IACS-4759, NPD7155) and CRISPR/Cas9-mediated MTH1 knockout. Based on these observations and our additional target validation experiments, we concluded that MTH1 is not essential for cancer cell survival or for the sanitization of damaged nucleotides within cells and thus not a viable target for development of novel anticancer agents. Citation Format: Manuel Ellermann, Anja Giese, Ashley Eheim, Stefanie Bunse, Roland Neuhaus, Jörg Weiske, Maria Quanz, Andrea Glasauer, Fredrik Rahm, Jenny Viklund, Martin Andersson, Tobias Ginman, Rickard Forsblom, Johan Lindström, Lionel Trésaugues, Matyas Gorjanacz. Novel class of potent and selective inhibitors efface MTH1 as broad-spectrum cancer target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2017-5226

Curcumin-Based Fluorescent and Colorimetric Probe for Detecting Cysteine in Living Cells and Zebrafish

We synthesized a new curcumin-based fluorescent and colorimetric probe (CAC) and described its application for the specific detection of cysteine (Cys), including in solution, in living cells, and in a living vertebrate organism. The probe CAC itself showed a strong fluorescence emission, the conjugate addition–cyclization reaction with Cys resulted in the fluorescence quenching at 490 nm and the absorbance decreasing at 400 nm, accompanied by the appearance of a new absorption band at 427 nm. The probe CAC exhibited higher properties such as acting as a “naked eye” probe, readily synthesized, highly selective for Cys, and a lower detection limit of 0.19 μM. In addition, the probe CAC can be successfully applied to detect Cys in living cells and zebrafish with good cell and organism permeability, indicating its significant potential in living biological systems.

An Optimized Two-Photon Fluorescent Probe for Biological Sensing and Imaging of Catechol-O-Methyltransferase.

A practical two-photon fluorescent probe was developed for highly sensitive and selective sensing of the activities of catechol-O-methyltransferase (COMT) in complex biological samples. To this end, a series of 3-substituted 7,8-dihydroxycoumarins were designed and synthesized. Among them, 3-BTD displayed the best combination of selectivity, sensitivity, reactivity, and fluorescence response following COMT-catalyzed 8-O-methylation. The newly developed two-photon fluorescent probe 3-BTD can be used for determining the activities of COMT in complex biological samples and bio-imaging of endogenous COMT in living cells and tissue slices with good cell permeability, low cytotoxicity, and high imaging resolution. All these findings suggest that 3-BTD holds great promise for developing therapeutic molecules that target COMT, as well as for exploring COMT-associated biological processes and its biological functions in living systems. Furthermore, the strategy also sheds new light on the development of fluorescent probes for other conjugative enzymes.

In Silico Identification of Mimicking Molecules as Defense Inducers Triggering Jasmonic Acid Mediated Immunity against Alternaria Blight Disease in Brassica Species.

Alternaria brassicae and Alternaria brassicicola are two major phytopathogenic fungi which cause Alternaria blight, a recalcitrant disease on Brassica crops throughout the world, which is highly destructive and responsible for significant yield losses. Since no resistant source is available against Alternaria blight, therefore, efforts have been made in the present study to identify defense inducer molecules which can induce jasmonic acid (JA) mediated defense against the disease. It is believed that JA triggered defense response will prevent necrotrophic mode of colonization of Alternaria brassicae fungus. The JA receptor, COI1 is one of the potential targets for triggering JA mediated immunity through interaction with JA signal. In the present study, few mimicking compounds more efficient than naturally occurring JA in terms of interaction with COI1 were identified through virtual screening and molecular dynamics simulation studies. A high quality structural model of COI1 was developed using the protein sequence of Brassica rapa. This was followed by virtual screening of 767 analogs of JA from ZINC database for interaction with COI1. Two analogs viz. ZINC27640214 and ZINC43772052 showed more binding affinity with COI1 as compared to naturally occurring JA. Molecular dynamics simulation of COI1 and COI1-JA complex, as well as best screened interacting structural analogs of JA with COI1 was done for 50 ns to validate the stability of system. It was found that ZINC27640214 possesses efficient, stable, and good cell permeability properties. Based on the obtained results and its physicochemical properties, it is capable of mimicking JA signaling and may be used as defense inducers for triggering JA mediated resistance against Alternaria blight, only after further validation through field trials.

Peptide-Decorated Tunable-Fluorescence Graphene Quantum Dots.

We report here the synthesis of graphene quantum dots with tunable size, surface chemistry, and fluorescence properties. In the size regime 15-35 nm, these quantum dots maintain strong visible light fluorescence (mean quantum yield of 0.64) and a high two-photon absorption (TPA) cross section (6500 Göppert-Mayer units). Furthermore, through noncovalent tailoring of the chemistry of these quantum dots, we obtain water-stable quantum dots. For example, quantum dots with lysine groups bind strongly to DNA in solution and inhibit polymerase-based DNA strand synthesis. Finally, by virtue of their mesoscopic size, the quantum dots exhibit good cell permeability into living epithelial cells, but they do not enter the cell nucleus.

INCB24360 (Epacadostat), a Highly Potent and Selective Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-oncology.

A data-centric medicinal chemistry approach led to the invention of a potent and selective IDO1 inhibitor 4f, INCB24360 (epacadostat). The molecular structure of INCB24360 contains several previously unknown or underutilized functional groups in drug substances, including a hydroxyamidine, furazan, bromide, and sulfamide. These moieties taken together in a single structure afford a compound that falls outside of “drug-like” space. Nevertheless, the in vitro ADME data is consistent with the good cell permeability and oral bioavailability observed in all species (rat, dog, monkey) tested. The extensive intramolecular hydrogen bonding observed in the small molecule crystal structure of 4f is believed to significantly contribute to the observed permeability and PK. Epacadostat in combination with anti-PD1 mAb pembrolizumab is currently being studied in a phase 3 clinical trial in patients with unresectable or metastatic melanoma.

A fluorescent probe based upon anthrancene-dopamine thioether for imaging Hg2+ ions in living cells

A novel anthrancene-dopamine thioether L compound was designed as fluorescent probe for detecting Hg2+ in living cells sample. L exhibits a good sensitive and selective recognition towards Hg2+ ions in the presence of other important relevant metal ions and amino acids in HEPES solution. The addition of Hg2+ causes a marked enhancement in the fluorescence emission intensity with the detection limit as low as 1.1×10−6M, combining with obvious colormetric change from colorless to pale brown. Mechanistic studies show that catechol group and sulfur atom in L all participate in the coordination with Hg2+, though catechol group contributes mainly to chelation-enhanced fluorescence enhancement and sulfur atom to selectivity. Furthermore, L demonstrates good cell permeability and compatibility for sensitive fluorescent detection of Hg2+ in HepG2 cells. This present probe will have broad applications in biological imaging.

A 2-(2′-hydroxyphenyl)quinazolin-4(3H)-one derived fluorescence ‘turn on’ probe for recognition of Hg2+ in water solution and its live cell imaging

Abstract A new fluorescence “turn on” Hg 2+ probe 3,3′-(((2-hydroxy-5-methyl-3-(4-oxo-3,4-dihydroquinazolin-2-yl)phenyl)methylene)bis(sulfanediyl))dipropionic acid ( QBDA ) based on Hg 2+ -triggered transformation of dithioacetal to aldehyde has been developed. Probe QBDA exhibits high selectivity and sensitivity to Hg 2+ over other metal ions in PBS buffered (20 mM, pH = 7.4) solution, the detection limit of QBDA for Hg 2+ was estimated to be 2.04 × 10 −8 M and the potential interference from Ag + can be suppressed in PBS buffered normal saline solution. Living cell imaging investigations show that QBDA possesses good cell permeability, and can act as a light-up imaging probe for Hg 2+ ions in MCF-7 cell.

An excited-state intramolecular photon transfer fluorescence probe for localizable live cell imaging of cysteine

Small molecule probes suitable for selective and specific fluorescence imaging of some important but low-concentration intracellular reactive sulfur species such as cysteine (Cys) pose a challenge in chemical biology. We present a readily available, fast-response fluorescence probe CHCQ-Ac, with 2-(5′-chloro-2-hydroxyl-phenyl)-6-chloro-4(3 H)-quinazolinone (CHCQ) as the fluorophore and acrylate group as the functional moiety, that enables high-selectivity and high-sensitivity for detecting Cys in both solution and biological system. After specifically reacted with Cys, the probe undergoes a seven-membered intramolecular cyclization and released the fluorophore CHCQ with excited-state intramolecular photon transfer effect. A highly fluorescent, insoluble aggregate was then formed to facilitate high-sensitivity and high-resolution imaging. The results showed that probe CHCQ-Ac affords a remarkably large Stokes shift and can detect Cys under physiological pH condition with no interference from other analytes. Moreover, this probe was proved to have excellent chemical stability, low cytotoxicity and good cell permeability. Our design of this probe provides a novel potential tool to visualize and localize cysteine in bioimaging of live cells that would greatly help to explore various Cys-related physiological and pathological cellular processes in cell biology and diagnostics.

Facile green synthesis of nitrogen-doped carbon dots using Chionanthus retusus fruit extract and investigation of their suitability for metal ion sensing and biological applications

Nitrogen-doped carbon dots (N-CDs) were synthesized from Chionanthus retusus (C. retusus) fruit extract using a simple hydrothermal-carbonization method. Their ability to sense metal ions, and their biological activity in terms of cell viability and bioimaging applications were evaluated. The resulting N-CDs were characterized by various physicochemical techniques such as high-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and Raman spectroscopy. The optical properties were characterized by ultraviolet visible (UV-vis) fluorescence spectroscopy techniques. The average size of the N-CDs was approximately 5±2nm with an interlayer distance of 0.21nm, as calculated from the HRTEM images. The presence of phytoconstituent functionalities and the percentages of components in the N–CDs were confirmed by XPS studies, and a nitrogen content of 5.3% was detected. The N–CDs demonstrated highly durable fluorescence properties and low cytotoxicity with a quantum yield of 9%. The synthesized N–CDs were then used as probes for the detection of metal ions. The N–CDs exhibited high sensitivity and selectivity towards Fe3+, with a linear relationship between 0 and 2μM and a detection limit of 70μM. The synthesized N–CDs are anticipated to have diverse biomedical applications, particularly for bioimaging, given their high fluorescence, excellent water solubility, good cell permeability, and negligible cytotoxicity. Finally, the potential of N–CDs as biological probes was investigated using fungal (Candida albicans and Cryptococcus neoformans) strains via fluorescent microscopy. We found that N–CDs were suitable candidates for differential staining applications in yeast cells with good cell permeability, localization with negligible cytotoxicity. Hence, N–CDs may find dual utility as probes for the detection of cellular pools of metal ions (Fe3+) and also for early detection of opportunistic yeast infections in biological samples.

A Single and Simple Receptor as a Multifunctional Chemosensor for the Al3+/Cu2+ ions and Its Live Cell Imaging Applications.

AbstractA simple and smart chemosensor (DSA) has been developed using Schiff base ligand for highly sensitive and selective colorimetric detection of Al3+/ Cu2+ in partial aqueous DMSO mixture (1:1) as a solvent medium. It showed a rapid fluorescence response (∼ 10 Sec) with Al3+ as greenish yellow from weak orange fluorescence while, Cu2+ ions affords yellow colour from fade orange colour and it can be sensed even by naked eyes. Besides showing great selectivity, the probe gives a lower sensitivity of 20 nM for Al3+ and 0.27 μM for Cu2+ in the fluorescence and UV‐vis. spectral studies respectively. The solid state colorimetric and fluorescence changes of the probe were examined in silica supported TLC plates. The sensing mechanism of the proposed probe in the presence of Al3+ and Cu2+ was determined with the support of a job's plot, 1H‐NMR, ESI‐Mass and theoretical (DFT) analysis. Based on the observed results from the absorbance and fluorescence spectroscopic studies, a logic circuit was designed with the combination of OR, NOT and AND logic gates. Moreover, the probe DSA has been employed for live cell imaging to detect Al3+ ions in C6 glioma cells and it showed a good cell permeability and efficiency in the detection of Al3+ ions.

Discovery of a PCAF Bromodomain Chemical Probe.

The p300/CBP‐associated factor (PCAF) and related GCN5 bromodomain‐containing lysine acetyl transferases are members of subfamily I of the bromodomain phylogenetic tree. Iterative cycles of rational inhibitor design and biophysical characterization led to the discovery of the triazolopthalazine‐based L‐45 (dubbed L‐Moses) as the first potent, selective, and cell‐active PCAF bromodomain (Brd) inhibitor. Synthesis from readily available (1R,2S)‐(−)‐norephedrine furnished L‐45 in enantiopure form. L‐45 was shown to disrupt PCAF‐Brd histone H3.3 interaction in cells using a nanoBRET assay, and a co‐crystal structure of L‐45 with the homologous Brd PfGCN5 from Plasmodium falciparum rationalizes the high selectivity for PCAF and GCN5 bromodomains. Compound L‐45 shows no observable cytotoxicity in peripheral blood mononuclear cells (PBMC), good cell‐permeability, and metabolic stability in human and mouse liver microsomes, supporting its potential for in vivo use.

Benzothiazole-based fluorescent sensor for hypochlorite detection and its application for biological imaging

We have developed a ratiometric fluorescent probe for hypochlorite detection with high selectivity and sensitivity. The probe 1 can rapidly turn into an oxidized form accompanied by the fluorescence changes from yellow to blue only upon the addition of hypochlorite among various reactive oxygen species (ROS) and reactive nitrogen species (RNS). The limit of detection was calculated to be as low as 8.9nM, which showed good sensitivity to hypochlorite. Furthermore, the confocal laser scanning micrographs of HeLa cells confirmed good cell permeability of probe 1 and its application to selective detecting hypochlorite in living cells.

A near-infrared fluorescent probe for Cu2+ in living cells based on coordination effect

A near-infrared (NIR) fluorescence probe for copper(Ⅱ) ions (Cu2+) has been synthesized and demonstrated that it could selectively and sensitively react with Cu2+ through specific coordination reaction. ESI-Mass and Job’s plot confirmed a stoichiometric ratio of 2–1 between the probe molecules and Cu2+. This NIR fluorescence probe shows a fast response, high sensitivity and anti-interference performance for Cu2+ and has been successfully applied for the detection of Cu2+ in aqueous solution. The detection limit was calculated as low as 14nM. It has been further demonstrated that the probe exhibited good cell permeability and could be applied for monitoring the fluctuation of Cu2+ in living cells by NIR fluorescence imaging.

A benzothiazole-based fluorescent probe for selective detection of H2S in living cells and mouse hippocampal tissues

This study reports a benzothiazole-based fluorescent probe EPS-HS for real-time detection of H2S. The probe is characterized in a large red-shift, good selectivity, high sensitivity and favorable biocompatibility. We measured the detection limit of EPS-HS in PBS buffer and fetal bovine serum. Moreover, in HT22 living cell imaging studies, EPS-HS showed good cell permeability and H2S could be detected within the cells. Furthermore, in situ visualization of H2S was performed on the hippocampal slices of normal mice with EPS-HS. We were also able to estimate the concentration of sulfide in mouse hippocampus tissues. The probe will be applied for better treatment of neurological deficits caused by damage to the hippocampus.