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Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors

  • Besides their extracellular activity crucial for several pathophysiological conditions, human cysteine cathepsins, in particular cathepsins K and S, represent important intracellular targets for drug development. In the present study, a prototypic dipeptide nitrile inhibitor structure was equipped with a coumarin moiety to function as a fluorescent reporter group. In a second inhibitor, a PEG linker was introduced between the dipeptide nitrile and the fluorophore. These tool compounds 6 and 7 were characterized by kinetic investigations as covalent reversible inhibitors of human cathepsins L, S, K and B. Probe 6 showed a pronounced inhibitory activity against cathepsins K and S, which was corroborated by modeling of inhibition modes. Probe 7 was highly potent (Ki = 93 nM) and selective for cathepsin S. To examine the ability of both probes to enter living cells, human embryonic kidney 293 cells were targeted. At a concentration of 10 µM, cellular uptake of probe 6 was demonstrated by fluorescence measurement after an incubation time of 30 min and 3 h, respectively. The probe’s concentration in cell lysates was ascertained on the basis of the emission at 492 nm upon excitation at 450 nm, and the results were expressed as concentrations of probe 6 relative to the protein concentration originating from the lysate. After incubation of 10 µM of probe 6 for 3 h, the cellular uptake was confirmed by fluorescence microscopy. HPLC was used to assess the probes’ lipophilicity, and the obtained log D7.4 value of 2.65 for probe 6 was in agreement with the demonstrated cellular uptake.

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Document Type:Article
Author:Franziska Kohl, Janina Schmitz, Norbert Furtmann, Anna-Christina Schulz-Fincke, Matthias D. Mertens, Jim Küppers, Marcel Benkhoff, Edda Tobiasch, Ulrike Bartz, Jürgen Bajorath, Marit Stirnberg, Michael Gütschow
Parent Title (English):Org Biomol Chem. (Organic & Biomolecular Chemistry)
First Page:10310
Last Page:10323
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=26307943
Publisher:Royal Society of Chemistry
Date of first publication:2015/08/19
Embargo Date:2016/09/01
Departments, institutes and facilities:Fachbereich Angewandte Naturwissenschaften
Entry in this database:2015/09/09