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

A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

“…The attachment of a coumarin fluorophore was accomplished by the introduction of the trifunctional amino acid lysine. Coumarins represent a widely used class of fluorescent dyes, distinguished by their small molecular size, low bleaching and large Stokes shifts …”

“…Coumarins represent aw idely used class of fluorescent dyes, distinguished by their small molecular size, lowb leaching and large Stokes shifts. [40][41][42][43][44] Scheme 1o utlines the convergent synthetic approach to the ABP 18,s imilar to the one that has previously been established for other benzguanidino phosphonates. [24] Details are given in the Supporting Information.…”

A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells

“…Furthermore, CTSS dysregulation is also involved in cancer progression (Sobotic et al, 2015). This involvement of CTSS in the pathogenesis of various diseases makes this protease an intensely studied drug target (Figueiredo et al, 2015;Kohl et al, 2015;Vazquez et al, 2015;Jadhav et al, 2014). In order to evaluate the precise role of CTSS function in this context, not only protein concentrations of CTSS should be detected but rather its catalytic activity as protein amount and enzymatic activity do not necessarily need to correlate.…”

Blue‐Fluorescent Probes for Lipid Droplets Based on Dihydrochromeno‐Fused Pyrazolo‐ and Pyrrolopyridines

“…This was not surprising because the pyrazolo skeleton can be found as a subunit together with other moieties such as pyridine, [9] coumarin, [10] quinoline, [11][12][13] and quinoxaline [14] in tri-and tetracyclic molecules that show fluorescent properties. [16,17] Moreover, there is a rich literature regarding fluorescent molecules based on oxygen heterocycles such as benzopyran and xanthene.…”