title: Design, characterization and cellular uptake studies of fluorescence-labeled prototypic cathepsin inhibitors.
abstract: 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
authors: Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany. guetschow@uni-bonn.de.
authors: Franziska
authors: F
authors: Kohl
authors: Janina
authors: J
authors: Schmitz
authors: Norbert
authors: N
authors: Furtmann
authors: Anna-Christina
authors: AC
authors: Schulz-Fincke
authors: Matthias D
authors: MD
authors: Mertens
authors: Jim
authors: J
authors: Küppers
authors: Marcel
authors: M
authors: Benkhoff
authors: Edda
authors: E
authors: Tobiasch
authors: Ulrike
authors: U
authors: Bartz
authors: Jürgen
authors: J
authors: Bajorath
authors: Marit
authors: M
authors: Stirnberg
authors: Michael
authors: M
authors: Gütschow
mesh_terms: D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002477:Cells / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D013329:Structure-Activity Relationship
chemical_list: D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D002403:Cathepsins