ABSTRACT
Current anticancer therapies suffer from issues such as off-target side effects and the emergence of drug resistance; therefore, the discovery of alternative therapeutic approaches is vital. These can include the development of drugs with different modes of action, and the exploration of new biomolecular targets. For the former, there has been increasing interest in drugs that are activated by an external stimulus to generate cytotoxic species. For the latter, significant efforts are being directed to explore non-canonical DNA and RNA structures (e.g. guanine-quadruplexes), as alternative biomolecular targets. Herein we report the synthesis of a library of 21 new platinum(II)-Salphen complexes, investigation of their photophysical and photochemical properties, their interactions with duplex and quadruplex DNA, and their cytotoxicity against HeLa cancer cells in the dark and upon light irradiation. Thanks to the intrinsic phosphorescence of the platinum(II) complexes, confocal microscopy was used for six of the complexes to determine their cellular permeability and localisation in two cancer cell lines. These studies have allowed us to identify two lead platinum(II) complexes with high guanine-quadruplex DNA affinity and selectivity, good cell permeability and nuclear localisation, and high cytotoxicity against HeLa cancer cells upon irradiation with no detected cytotoxicity in the dark.
ABSTRACT
The discovery of new compounds with pharmacological properties is usually a lengthy, laborious and expensive process. Thus, there is increasing interest in developing workflows that allow for the rapid synthesis and evaluation of libraries of compounds with the aim of identifying leads for further drug development. Herein, we apply combinatorial synthesis to build a library of 90â iridium(III) complexes (81 of which are new) over two synthesise-and-test cycles, with the aim of identifying potential agents for photodynamic therapy. We demonstrate the power of this approach by identifying highly active complexes that are well-tolerated in the dark but display very low nM phototoxicity against cancer cells. To build a detailed structure-activity relationship for this class of compounds we have used density functional theory (DFT) calculations to determine some key electronic parameters and study correlations with the experimental data. Finally, we present an optimised semi-automated synthesise-and-test protocol to obtain multiplex data within 72â hours.
Subject(s)
Antineoplastic Agents , Coordination Complexes , Photochemotherapy , Iridium/pharmacology , Antineoplastic Agents/pharmacology , Photochemotherapy/methods , Structure-Activity Relationship , Coordination Complexes/pharmacologyABSTRACT
The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.
Subject(s)
Deep Learning , Microscopy , Single Molecule Imaging , MotionABSTRACT
Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.
Subject(s)
Fluorescent Dyes , G-Quadruplexes , Fluorescent Dyes/chemistry , RNA , Microscopy, Fluorescence , Cytoplasm/metabolismABSTRACT
G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).
Subject(s)
G-Quadruplexes , Platinum , Platinum/chemistry , Microscopy , DNA/chemistryABSTRACT
G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.
Subject(s)
Coordination Complexes , G-Quadruplexes , Humans , Coordination Complexes/chemistry , DNA/chemistry , Polymers , Guanine/chemistry , Telomere , Circular DichroismABSTRACT
A number of Gram-negative bacteria such as Pseudomonas aeruginosa are becoming resistant to front-line antibiotics. Consequently, there is a pressing need to find alternative bio-molecular targets for the development of new drugs. Since non-canonical DNA structures such as guanine-quadruplexes (G4s) have been implicated in regulating transcription, we were interested in determining whether there are putative quadruplex-forming sequences (PQS) in the genome of Pseudomonas aeruginosa. Using bioinformatic tools, we screened 36 genes potentially relevant to drug resistance for the presence of PQS and 10 of these were selected for biophysical characterisation (i.e. circular dichroism and thermal difference UV/Vis spectroscopy). These studies showed that three of these G-rich sequences (linked to murE, ftsB and mexC genes) form stable guanine-quadruplexes which were studied by NMR spectroscopy; detailed analysis of one of the sequences (mexC) confirmed that it adopts a two-quartet antiparallel quadruplex structure in the presence of K+ ions. We also show by FRET melting assays that small molecules can stabilise these three new G4 DNA structures under physiological conditions. These initial results could be of future interest in the development of new antibiotics with alternative bio-molecular targets which in turn would help tackle antimicrobial resistance.
ABSTRACT
G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).
ABSTRACT
Guanine-rich sequences of DNA and RNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target G4s. Amongst those are metal complexes with Schiff base ligands. Herein, we report the functionalisation of one of these well-established G4 DNA binders (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies highlighted the accumulation of this Pt-salphen-111In conjugate in the tumour.
Subject(s)
G-Quadruplexes , Animals , Mice , Click Chemistry , DNAABSTRACT
Citrate (Cit) and Deferoxamine B (DFOB) are two important organic ligands coexisting in soils with distinct different affinities for metal ions. It has been theorized that siderophores and weak organic ligands play a synergistic role during the transport of micronutrients in the rhizosphere, but the geochemical controls of this process remain unknown. Here we test the hypothesis that gradients in pH and ion strength regulate and enable the cooperation. To this end, first we use potentiometric titrations to identify the dominant Zn(II)-Cit and Zn(II)-DFOB complexes and to determine their ionic strength dependent stability constants between 0 and 1 mol dm-3. We parametrise the Extended Debye-Hückel (EDH) equation and determine accurate intrinsic association constants (logß0) for the formation of the complexes present. The speciation model developed confirms the presence of [Zn(Cit)]-, [Zn(HCit)], [Zn2(Cit)2(OH)2]4-, and [Zn(Cit)2]4-, with [Zn(Cit)]- and [Zn2(Cit)2(OH)2]4- the dominant species in the pH range relevant to rhizosphere. We propose the existence of a new [Zn(Cit)(OH)3]4- complex above pH 10. We also verify the existence of two hexadentate Zn(II)-DFOB species, i.e., [Zn(DFOB)]- and [Zn(HDFOB)], and of one tetradentate species [Zn(H2DFOB)]+. Second, we identify the pH and ionic strength dependent ligand exchange points (LEP) of Zn with citrate and DFOB and the stability windows for Zn(II)-Cit and Zn(II)-DFOB complexes in NaCl and rice soil solutions. We find that the LEPs fall within the pH and ionic strength gradients expected in rhizospheres and that the stability windows for Zn(II)-citrate and Zn(II)-DFOB, i.e., low and high affinity ligands, can be distinctly set off. This suggests that pH and ion strength gradients allow for Zn(II) complexes with citrate and DFOB to dominate in different parts of the rhizosphere and this explains why mixtures of low and high affinity ligands increase leaching of micronutrients in soils. Speciation models of soil solutions using newly determined association constants demonstrate that the presence of dissolved organic matter and inorganic ligands (i.e., bicarbonate, phosphate, sulphate, or chlorides) do neither affect the position of the LEP nor the width of the stability windows significantly. In conclusion, we demonstrate that cooperative and synergistic ligand interaction between low and high affinity ligands is a valid mechanism for controlling zinc transport in the rhizosphere and possibly in other environmental reservoirs such as in the phycosphere. Multiple production of weak and strong ligands is therefore a valid strategy of plants and other soil organisms to improve access to micronutrients.
Subject(s)
Rhizosphere , Siderophores , Citrates , Citric Acid/chemistry , Hydrogen-Ion Concentration , Ligands , Micronutrients , Osmolar Concentration , Soil/chemistry , Zinc/chemistryABSTRACT
The important role that G-quadruplex DNA (G4 DNA) structures play in regulating biological processes is becoming widely recognised. These structures have also been proposed to be attractive drug targets. Therefore, there has been significant interest in developing small molecules that can selectively bind to G4 DNA over other topologies. In this paper we investigate the interaction between DNA and helical compounds (helicenes) based on a central carbocation trisubstituted with aromatic rings. We show that the non-planar structure of these helicenes results in a significantly reduced affinity for dsDNA when compared to their planar analogues, whilst maintaining a high affinity for G4 DNA. Additionally, the right- and left-handed enantiomers of one of these helicenes recognise the chiral DNA environments of G4 and dsDNA differently. We show that upon DNA binding the helicenes display a fluorescence switch-on effect, which we have successfully used for cellular imaging in live and fixed U2OS cells, staining mitochondria and the nucleus, respectively.
ABSTRACT
Bacteria, fungi and grasses use siderophores to access micronutrients. Hence, the metal binding efficiency of siderophores is directly related to ecosystem productivity. Salinization of natural solutions, linked to climate change induced sea level rise and changing precipitation patterns, is a serious ecological threat. In this study, we investigate the impact of salinization on the zinc(II) binding efficiency of the major siderophore functional groups, namely the catecholate (for bacterial siderophores), α-hydroxycarboxylate (for plant siderophores; phytosiderophores) and hydroxamate (for fungal siderophores) bidentate motifs. Our analysis suggests that the order of increasing susceptibility of siderophore classes to salinity in terms of their zinc(II) chelating ability is: hydroxamate < catecholate < α-hydroxycarboxylate. Based on this ordering, we predict that plant productivity is more sensitive to salinization than either bacterial or fungal productivity. Finally, we show that previously observed increases in phytosiderophore release by barley plants grown under salt stress in a medium without initial micronutrient deficiencies, are in line with the reduced zinc(II) binding efficiency of the α-hydroxycarboxylate ligand and hence important for the salinity tolerance of whole-plant zinc(II) status.
ABSTRACT
One of the key hallmarks of Alzheimer's disease is the aggregation of the amyloid-ß peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-ß aggregation. While a handful of molecules have been shown to inhibit amyloid-ß aggregation in vitro, there remains a lack of in vivo data reported due to their inability to cross the blood-brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-ß aggregation in vitro. We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π-π stacking and metal coordination to amyloid-ß (confirmed via a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood-brain barrier to specific locations in the brains of mice using focused ultrasound.
ABSTRACT
Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).
Subject(s)
Copper/analysis , Boron Compounds/chemistry , Boron Compounds/toxicity , Cell Line, Tumor , Copper/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Humans , Lysosomes/chemistry , Microscopy, Fluorescence/methodsABSTRACT
Transition and lanthanide metal complexes have rich photophysical properties that can be used for cellular imaging, biosensing and phototherapy. One of the applications of such luminescent compounds is the detection and visualisation of nucleic acids. In this brief review, we survey the recent literature on the use of luminescent metal complexes (including ReI, RuII, OsII, IrIII, PtII, EuIII and TbIII) as DNA optical probes, including examples of compounds that bind selectively to non-duplex DNA topologies such as quadruplex, i-motif and DNA mismatches. We discuss the applications of metal-based luminescent complexes in cellular imaging, including time-resolved microscopy and super-resolution techniques. Their applications in biosensing and phototherapy are briefly mentioned in the relevant sections.
Subject(s)
Coordination Complexes/chemistry , DNA/chemistry , Metals/chemistryABSTRACT
The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and "off-target" interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to "cage" a DNA-targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G-quadruplex DNA binder, namely a PtII -salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII -salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII -salphen to the nucleus and its binding to DNA.
Subject(s)
DNA/drug effects , Rotaxanes/chemistry , Small Molecule Libraries/pharmacology , Binding Sites/drug effects , Cell Survival/drug effects , DNA/chemistry , Humans , Molecular Structure , Rotaxanes/chemical synthesis , Small Molecule Libraries/chemistryABSTRACT
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
Subject(s)
DNA/metabolism , G-Quadruplexes , Intravital Microscopy/methods , Molecular Imaging/methods , Animals , Cell Line, Tumor , DNA/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Fibroblasts , Fluorescent Dyes/chemistry , Gene Knockdown Techniques , Humans , Indoles/chemistry , Mice , Microscopy, Fluorescence/methods , RNA Helicases/genetics , RNA Helicases/metabolismABSTRACT
Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.
Subject(s)
DNA/analysis , DNA/chemistry , Fluorescence , Fluorescent Dyes/chemistry , G-Quadruplexes , Electron Transport , Fluorescent Dyes/analysis , Molecular Probes/analysis , Molecular Probes/chemistryABSTRACT
Phosphatidylinositol phosphates (PIPs) are membrane phospholipids that play crucial roles in a wide range of cellular processes. Their function is dictated by the number and positions of the phosphate groups in the inositol ring (with seven different PIPs being active in the cell). Therefore, there is significant interest in developing small-molecule receptors that can bind selectively to these species and in doing so affect their cellular function or be the basis for molecular probes. However, to date there are very few examples of such molecular receptors. Towards this aim, herein we report a novel tripodal molecule that acts as receptor for mono- and bis-phosphorylated PIPs in a cell free environment. To assess their affinity to PIPs we have developed a new cell free assay based on the ability of the receptor to prevent alkaline phosphatase from hydrolysing these substrates. The new receptor displays selectivity towards two out of the seven PIPs, namely PI(3)P and PI(3,4)P2. To rationalise these results, a DFT computational study was performed which corroborated the experimental results and provided insight into the host-guest binding mode.