PARP inhibitor cyanine dye conjugate with enhanced cytotoxic and antiproliferative activity in patient derived glioblastoma cell lines

We describe the synthesis and in vitro activity of drug-dye conjugate 1, which is a combination of the PARP inhibitor rucaparib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed strong cytotoxic activity with nanomolar potency (EC₅₀: 128 nM), which was a 780 fold improvement over rucaparib itself. We also observe a synergistic effect of 1 with temozolomide (TMZ), the standard drug for treatment for glioblastoma even though these cell lines were resistant to TMZ treatment. We envisage such conjugates to be worth exploring for their utility in the treatment of various brain cancers.     

Synthesis and antimicrobial activity of meso-substituted polymethine cyanine dyes

The condensation reaction of equivalent amounts of 2-cyanomethyl benzooxazole or its derivatives with variously substituted aromatic aldehydes gave 2-cyano-styryl benzooxazole or its derivatives. The subsequent reaction of the 2-cyano-styryl benzooxazoles with 2(4)-methyl substituted heterocyclic quaternary salts afforded meso-substituted styryl-2(4)-polymethine cyanines. The condensation reaction of 2-cyanomethyl benzooxazole or its derivatives with alpha-nitroso-beta-naphthol followed by reaction with 2(4)-methyl substituted heterocyclic quaternary salts gave meso-substituted aza-2(4)-polymethine cyanines. The reaction of 2-cyanomethyl benzooxazole or its derivatives with N-methyl heterocyclic quaternary salts followed by the reaction with 2-methylquinolinium methiodide afforded the corresponding meso-substituted trimethine cyanine dyes. Elemental analyses, visible absorption, IR, (1)H NMR spectroscopy, and mass spectra established the structures of these compounds. The relationship between the structure and properties of these dyes has been studied and the solvatochromic behavior of some selected cyanine dyes in organic solvents is discussed. Finally, the antimicrobial activity of selected novel dyes was investigated in vitro using a wide spectrum of microbial strains.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

The synthesis of a novel Crizotinib heptamethine cyanine dye conjugate that potentiates the cytostatic and cytotoxic effects of Crizotinib in patient-derived glioblastoma cell lines

We describe the synthesis of drug-dye conjugate 1 between anaplastic lymphoma kinase inhibitor Crizotinib and heptamethine cyanine dye IR-786. The drug-dye conjugate 1 was evaluated in three different patient-derived glioblastoma cell lines and showed potent cytotoxic activity with nanomolar potency (EC₅₀: 50.9 nM). We also demonstrate evidence for antiproliferative activity of 1 with single digit nanomolar potency (IC₅₀: 4.7 nM). Furthermore, the cytotoxic effects conveyed a dramatic, 110-fold improvement over Crizotinib. This improvement was even more pronounced (492-fold) when 1 was combined with Temozolomide, the standard drug for treatment for glioblastoma. This work lays the foundation for future exploration of similar tyrosine kinase inhibitor drug-dye conjugates for the treatment of glioblastoma.     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.     

Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis

We report the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 N‐hydroxysuccinimide ester (NHS) cyanine dyes, which have similar chemical properties as the CyDye? DIGE fluor minimal dyes for pre‐electrophoresis labelling. Multiple sample analyses in different laboratories indicate that the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 NHS ester cyanine dyes produces equivalent results to those obtained with DIGE CyDyes, and allows sample multiplexing and accurate quantitation for differential proteome analysis.     

Interaction of cyanine dyes with nucleic acids. XXI. Arguments for half-intercalation model of interaction

The spectral luminescent properties of two groups of monomethine cyanine dyes were studied in the presence of DNA. The first group included five dyes with 5,6-methylenedioxy-[d]-benzo-1,3-thiazole heterocycle and their unsubstituted analogs. Five monomethine pyrylium cyanines and their N-methyl-pyridine analogs were included in the second group. In each pair the pyrylium and pyridine dyes had similar geometry but differed in charge density distribution. The results presented some evidence in favor of the half-intercalation interaction mode between the studied dyes and DNA. When the benzothiazole residue had the lowest electron donor ability between the two heterocycles in the dye molecule, its substitution with the bulky methylenedioxy group led to a significant decrease in fluorescence enhancement of the dye-DNA complex. On the contrary, when the substituents that create steric hindrance (e.g., methylenedioxy and methyl groups) were introduced into the heterocycle with the higher electron donor ability, the fluorescence enhancement value of the dye-DNA complex was virtually unchanged. The changes in the Stock's shift values upon the formation of the dye-DNA complexes were in agreement with the proposed half-intercalation model. Interestingly, in the dye-DNA complexes the pyrylium dyes probably resided in a place similar to the pyridine ones. It is possible that the benzothiazole (or benzooxazole) ring intercalated between the DNA bases and the pyrylium (or pyridine) residue was located in the DNA groove closer to the phosphate backbone.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

Ab initio (CASPT2) excited state calculations, including circular dichroism, of helically twisted cyanine dyes

Ab initio calculations at the CASSCF/CASPT2 level were performed on helically twisted mono-, tri-, and pentamethine cyanine dyes in the all-Z-configurations. Excitation energies and oscillator and rotatory strengths were calculated for the five lowest energy singlet states. Both the long wavelength methine band and the cis-band could be identified unambiguously from their configurational parentage. The calculated state energies are within 0.09 eV of the experimental value for the methine band and within 0.16 eV for the cis-band. The calculated rotatory strengths of the methine band shows sign inversion as the length of the chromophore increases: negative for the short monomethine, strongly positive for the pentamethine. The trimethine presents a borderline case: the measured rotatory strength is almost nil, the calculated one depends on the geometry. There is good agreement between rotatory strengths calculated in the velocity and in the length formalism.     

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N′-substituents

The effect of various N,N′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42.     

Photoinactivation of bacteriophage PM2 by cyanine dyes]

Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.     

Synthesis,G-Quadruplex DNA binding and cytotoxic properties of naphthalimide substituted styryl dyes

G-Quadruplex DNAs, formed by G-rich DNA sequences in human genes, are promising targets for design of cancer drugs. In this study, two naphthalimide substituted styryl dyes with different sizes of aromatic groups were synthesized. The spectral analysis showed that the dye X-2 with a large aromatic group formed aggregates in buffer solution displaying very weak fluorescence intensity, and disaggregated in the presence of G-Quadruplex DNAs with large intensity enhancements (up to ~1800 fold). Moreover, X-2 displayed good selectivity to G-Quadruplex DNAs. In contrast, dye X-3 with the smaller aromatic group had much lower fluorescence enhancements and poor selectivity to G-Quadruplex DNAs, suggesting that the suitably sized aromatic ring was essential for the interaction with G-Quadruplex. Further binding studies suggested that X-2 mainly bound on G-quartet surface through end-stacking mode. Cytotoxicity assay showed that both of the two dyes showed good anti-proliferative activities against the cancer cell lines and less cytotoxicity in non-malignant cell lines, which were better than a standard drug 5-fluorouracil. In addition, living cell imaging was also studied and demonstrated the potential applications of the new dye X-2 in bioassays and cell imaging.     

Mechanism of potential-dependent light absorption changes of lipid bilayer membranes in the presence of cyanine and oxonol dyes

Summary The mechanism by which the light absorption of cyanine and oxonol dyes changes in response to changes in transmembrane electrical potential has been studied. Trains of membrane potential steps produce changes in the intensity of light passing through glycerylmonooleate (GMO) bilayer lipid membranes (BLM) in the presence of these dyes. The size of the signal-averaged absorbance change for one of the cyanine dyes diS-C₂-(5) is 10^–5. The response time for the absorbance change of all of the dyes was 10 sec. In order for an absorption signal to be observed, the concentration of dye on both sides of the membrane must be different. Since GMO bilayer membranes are permeable to the charged dyes that were studied, the dye concentration asymmetry necessary for the optical signal had to be maintained with a constant dc membrane potential, onto which the trains of potential steps were superimposed. The more hydrophobic dyes were the most permeant. Inclusion of cholesterol in the GMO bilayers decreased the permeance of the positively charged cyanine dyes, but increased the permeance of the negatively charged oxonol dyes. The magnitude and the size of the BLM absorbance change depended on the wavelength of illumination. Comparisons of the wavelength dependence of the BLM spectra with absorption difference spectra obtained with model membrane systems allow us to postulate a mechanism for a BLM absorbance change. For the cyanine and oxonol dyes, the data are consistent with an ON-OFF mechanism where a quantity of dye undergoes a rapid potential-dependent movement between a hydrocarbon-like binding site on the membrane and the aqueous salt solution near the membrane. For some dyes, which readily aggregate on the membrane, part of the absorbance change may possibly be explained by a potential dependent change in the state of aggregation of dye molecules localized on the membrane. Mechanisms involving a potential dependent change in the polarizability of the environment of membrane-localized dye molecules cannot be excluded, but seem unlikely.     

Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA

Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.     

Interaction of cyanine dyes with nucleic acids. Part 19: new method for the covalent labeling of oligonucleotides with pyrylium cyanine dyes

New chemistry for the fluorescent labeling of oligonucleotides with cyanine dyes is proposed. It is based on the use of pyrylium salts as amine-specific reagents. Monomethyne pyrylium cyanine dye 1 was covalently linked to 5'-aminoalkyl modified oligonucleotide, with simultaneous conversion of the non-fluorescent dye 1 into fluorescent pyridinium cyanine structure 2.     

Binding of symmetrical cyanine dyes into the DNA minor groove

Optical methods, such as fluorescence, circular dichroism and linear flow dichroism, were used to study the binding to DNA of four symmetrical cyanine dyes, each consisting of two identical quinoline, benzthiazole, indole, or benzoxazole fragments connected by a trimethine bridge. The ligands were shown to form a monomer type complex into the DNA minor groove. The complex of quinoline-containing ligand with calf thymus DNA appeared to be the most resistant to ionic strength, and it did not dissociate completely even in 1 M NaCl. Binding of cyanine dyes to DNA could also be characterized by possibility to form ligand dimers into the DNA minor groove, by slight preference of binding to AT pairs, as well as by possible intercalation between base pairs of poly(dG)-poly(dC). The correlation found between the binding constants to DNA and the extent of cyanine dyes hydrophobicity estimated as the n-octanol/water partition coefficient is indicative of a significant role of hydrophobic interactions for the ligand binding into the DNA minor groove.     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.     

Facile synthesis of symmetric, monofunctional cyanine dyes for imaging applications

Efficient syntheses of several members of a new class of symmetric, monocarboxylate-functionalized cyanine dyes have been developed. The synthesis is a simple two-step method, typically with greater than 60% yield and easy final product purification. The new monocarboxylate-functionalized cyanine dyes exhibit excellent water solubility and similar excitation and emission properties to those of Cy5 and Alexa Fluor 647. The application of the new dyes in cellular imaging has been demonstrated through direct conjugating of the dye with an antibody, then imaging of microtubules inside cells, visualized by near-infrared fluorescence microscopy.