Novel probe provides enzymatic marker for brain disease
The enzyme 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) has several functions, such as the metabolism of fatty acids, amino acids and steroids. Furthermore, it seems to be associated with the mechanisms involved in Alzheimer’s and Parkinson’s diseases. However, no imaging agents are available to help visualize the enzyme in live cells or tissues.
Now researchers at Columbia University in New York have developed a fluorogenic substrate for 17β-HSD10 that provides a bright, switchable probe that permits noninvasive real-time imaging under a fluorescence microscope of the enzyme in action within live cells.
The investigators report in the Nov. 21 Journal of the American Chemical Society that they designed the probe as a mimic of the enzyme’s natural substrate, choosing 2,6-disubstituted naphthalene as the core. Because 17β-HSD10 is a dehydrogenase that operates in the direction of oxidation, they devised the probe such that it converts from an alcohol to a ketone upon oxidation.
Ketones, however, typically quench fluorescence, so the scientists addressed the problem by adding an electron-donating methyl group in the 6-position to counteract the ketone’s electron-withdrawing tendencies, which lead to quenching.
They created several early versions of the probe, adjusting various parameters until they achieved an optimal balance of redshift and quenching. The ketone version of the final probe is fluorescent in neither water nor in a potassium phosphate buffer, but it emits a bright green-yellow fluorescence in chloroform and in cells. Tested in live human kidney cells, the probe showed a linear increase in fluorescence during the first 5 h of incubation, indicating an increase in metabolism of the probe by the cells.
The researchers believe that the probe will enable the further elucidation of the role 17β-HSD10 plays in Alzheimer’s and Parkinson’s diseases.
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