Spectroscopic and Biophysical Characterization of Novel Fluorescent Drug Analogues

Abstract

Since the pioneering work of Herschel in 1820, fluorescence spectroscopy and microscopy have been the most informative and sensitive techniques and are widely used to characterize biomolecular functions. This thesis presents the methodology and application of different fluorescent spectroscopy techniques to characterize the novel fluorescent drug analogues. Fluorescence-based drug analogues have played a key role in understanding the functionality of cell receptors and their associated signaling complexes at a molecular level. Fluorescent drug analogues are the building block of biological macromolecules used to understand the complexity of the cell membrane and signaling. Our study aims to determine the behaviour of novel drugs by exploiting the properties of fluorescent analogs.

The Receptor Signaling Group at the MDC-Berlin recently displayed how to use 8-FDA-cAMP, a Fluorescein Diacetate (FDA)-conjugate of cAMP, a key cell 2nd messenger, to study its intracellular dynamics. Here we studied two drug analogues generated based upon the same principle of FDA-conjugation. Highly sensitive Time Correlated Single Photon Counting (TCSPC) and confocal laser scanning microscopy were employed to characterize the novel fluorescent drug analogues. In this thesis, the construction and demonstration of TCSPC, UV-Vis spectrophotometer and a laser scanning confocal microscope setup have been explained.

Moreover, the cellular uptake and subcellular import kinetics behaviour of 8-FDA-cAMP, MAN193, and AG3457 fluorescent molecules were investigated using FDA as a control. The subcellular localization in the region of mitochondria was observed and analyzed for MAN193 and 8-FDA-cAMP molecules, although no subcellular localization was observed for the AG3457 molecule. A competition binding experiment was employed to understand the behaviour of cellular uptake with or without preincubation with a competitor binding molecule. We further characterized the effect of dimethyl sulfoxide (DMSO) on the membrane permeability of FDA and 8-FDA-cAMP molecules by measuring the cellular uptake kinetics on HEK293T cells.