Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay.

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.

Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection.

We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The commercially available classic amyloid stain thioflavin T (Thio T) was used as the reference dye. T-284 and SH-516 dyes can be used for fluorometric quantification of fibrillar wild-type ASN at concentrations of approximately 1.5-20 microg/ml. Both dyes appeared suitable for step-wise monitoring of ASN variants (wild-type and mutants A30P and A53T) aggregation into fibrils in vitro, demonstrating good reproducibility, exceeding that for the commonly used Thio T. Our assay may be used for screening in vitro of agents capable of affecting the aggregation of ASN. In addition, T-284 and SH-516 cyanine dyes were shown to recognize amyloid proteins of various amino acid compositions selectively. T-284 demonstrated particular sensitivity to wild-type and A53T ASN, while for SH 516, the fluorescence response to fibrillar proteins was nearly the same except for lysozymes. T-284 and SH-516 cyanine dyes are sensitive and specific fluorescent probes for monitoring ASN fibril formation process in vitro, quantification of fibrillar ASN in solution, and fluorescent detection of various fibrillar protein species.

Specific fluorescent detection of fibrillar alpha-synuclein using mono- and trimethine cyanine dyes.

With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of alpha-synuclein (ASN) was studied. For the first time it was revealed that monomethine cyanines can specifically increase their fluorescence in aggregated ASN presence. Dialkylamino-substituted monomethine cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516 demonstrated the higher emission intensity and selectivity to aggregated ASN than classic amyloid stain Thioflavin T, and could be proposed as novel efficient fluorescent probes for fibrillar ASN detection. Studies of structure-function dependences have shown that incorporation of amino- or diethylamino- substituents into the 6-position of the benzothiazole heterocycle yields in a appearance of a selective fluorescent response to fibrillar alpha-synuclein presence. Performed calculations of molecular dimensions of studied cyanine dyes gave us the possibility to presume, that dyes bind with their long axes parallel to the fibril axis via insertion into the neat rows (so called 'channels') running along fibril.

Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures.

We ascertained the ability to detect fibrillar beta-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.

Fluorescent properties of pentamethine cyanine dyes with cyclopentene and cyclohexene group in presence of biological molecules.

A series of pentamethine cyanine dyes with cyclohexene or cyclopentene group in polymethyne chain, assumed as DNA groove-binders, were studied as fluorescent probes for nucleic acids as well as for native and denatured proteins. It was revealed that the presence of methyl or dimethyl substituent in 5 position of the cyclohexene group hinders the formation of dye-DNA fluorescent complex, while the methyl substituent in 2 position leads to the increasing of the dye-DNA complex fluorescence intensity. The dyes SL-251, SL-1041, and SL-1046 containing methyl group in the 2 position of the cyclic group, are reported as bright DNA-sensitive dyes. The study of the dyes DNA-binding specificity demonstrated significant AT-preference that points to the groove-binding interaction mode. At the same time, the dyes SL-251, SL-377, and SL-957 with the 2-methyl substituted cyclohexene group were shown to be sensitive fluorescent dyes both for nonspecific (in SDS presence) proteins detection and for native BSA.