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bioRxiv · 10.64898/2026.08.22.746441

Subcellular pharmacology of a novel cell-permeable fluorescent phosphodiesterase inhibitor using advanced fluorescence microscopy

Banik, S.·Anselmi, M.·Satpathy, J.·Cozzi, P. G.·Schihada, H.·Goult, B. T.·Gualandi, A.·Annibale, P.
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Abstract

Direct visualisation of drug-target engagement within living cells remains a major challenge. Here we develop a cell-permeable fluorogenic analogue of the phosphodiesterase 4 (PDE4) inhibitor rolipram (MAN193), generated by conjugation to fluorescein diacetate. Following intracellular activation, the probe functions as a near-neutral PDE4 antagonist and enables direct visualisation of endogenous PDE4 populations. Using advanced fluorescence spectroscopy imaging approaches, including molecular brightness analysis and fluorescence anisotropy imaging, we demonstrate rolipram-displaceable binding of the analogue to cytosolic PDE4 and resolve thesubcellular distribution of binding sites. We further show that the probe enables visualisation of untagged PDE4 at focal adhesion complexes in cardiomyocyte-like cells. Together these findings establish fluorogenic drug conjugation combined with quantitative spectroscopy imaging as a generalisable strategy to map intracellular drug-target engagement with subcellular resolution, providing an effective framework for interrogating the spatial pharmacology of small molecules in living systems.

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Banik, S., Anselmi, M., Satpathy, J., Cozzi, P. G., Schihada, H., Goult, B. T., Gualandi, A., Annibale, P.. 2026-08-26. Subcellular pharmacology of a novel cell-permeable fluorescent phosphodiesterase inhibitor using advanced fluorescence microscopy. https://doi.org/10.64898/2026.08.22.746441

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