A Peptidomimetic Fluorescent Probe to Detect the Trypsin β2 Subunit of the Human 20S Proteasome

This work describes the chemical synthesis, combinatorial selection, and enzymatic evaluation of peptidomimetic fluorescent substrates specific for the trypsin-like (&beta
2) subunit of the 20S human proteasome. After deconvolution of a library comprising nearly 6000 compounds composed of peg substituted diaminopropionic acid DAPEG building blocks, the sequence ABZ&ndash
Dap(O2(Cbz))&ndash
Dap(GO1)&ndash
Arg&ndash
ANB&ndash
NH2, where ABZ is 2-aminobenzoic acid, and ANB- 5 amino 2- nitro benzoic acid was selected. Its cleavage followed sigmoidal kinetics, characteristic for allosteric enzymes, with Km = 3.22 &plusmn
0.02 &mu
M, kcat = 245 s&minus
1, and kcat/Km = 7.61 &times
107 M&minus
1 s&minus
1. This process was practically halted when a selective inhibitor of the &beta
2 subunit of the 20S human proteasome was supplemented to the reaction system. Titration of the substrate resulting in decreased amounts of proteasome 20S produced a linear signal up to 10&minus
11 M. Using this substrate, we detected human proteasome 20S in human urine samples taken from the bladders of cancer patients. This observation could be useful for the noninvasive diagnosis of this severe disease.