Fluorescence anisotropy
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Fluorescence anisotropy measurements were performed on a set of multichromophoric compounds, which contain a different number of aminopyrenyl moieties linked to a triazine ring, in order to reveal the nature of both the electronic excited states and relaxation pathways of the compounds. Our experimental results complement quantum chemical calculations. We propose that the lowest excited state from which fluorescence proceeds is localized on a single individual aminopyrene moiety. In contrast, excitation to a higher excited state is likely followed by a migration of energy to another nearby aminopyrene chromophore before the internal conversion to the emitting state takes place. We suggest that this migration is responsible for the experimentally measured decrease of fluorescence anisotropy of the studied compounds.
DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.
- MeSH
- DNA chemická syntéza chemie MeSH
- fluorescenční barviva chemická syntéza chemie MeSH
- fluorescenční polarizace MeSH
- knihovny malých molekul chemická syntéza chemie farmakologie MeSH
- ligandy MeSH
- objevování léků MeSH
- preklinické hodnocení léčiv MeSH
- techniky kombinatorické chemie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
2nd ed. xxiii, 698 s. : il.
- Klíčová slova
- Biochemie, Laboratorní technika,
- MeSH
- biochemie MeSH
- fluorescence MeSH
- fluorescenční protilátková technika MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- fyzika, biofyzika
1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.
- MeSH
- antifungální látky chemie MeSH
- difenylhexatrien chemie MeSH
- fluorescenční barviva chemie MeSH
- fluorescenční polarizace MeSH
- fosfatidylcholiny chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- itrakonazol chemie MeSH
- lipidové dvojvrstvy chemie MeSH
- povrchové vlastnosti MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.
- MeSH
- antigeny povrchové genetika metabolismus MeSH
- fluorescenční barviva chemická syntéza MeSH
- fluorescenční polarizace metody MeSH
- glutamátkarboxypeptidasa II antagonisté a inhibitory genetika metabolismus MeSH
- knihovny malých molekul farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- rychlé screeningové testy metody MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
