Enzyme occupancy measurement of intracellular protein tyrosine phosphatase 1B using photoaffinity probes.
Protein tyrosine phosphatase 1B (PTP1B) is believed to be one of the enzymes involved in down-regulating the insulin receptor and is a drug target for the treatment of type II diabetes. To better understand the in vitro and in vivo behavior of PTP1B inhibitors, a cell-based assay to directly measure enzyme occupancy of PTP1B by inhibitors using photoaffinity labeling was developed. Two photoaffinity probes were synthesized containing the photolabile diazirine moiety. These photoprobes were specific for PTP1B and T-cell protein tyrosine phosphatase over CD45, with the most potent photoprobe having an IC(50) value of 0.2nM for PTP1B. Activation of the photoprobes with a 40-W UV lamp in the presence of purified AspTyrLysAspAspAspAspLys (Flag)-PTP1B formed a 1:1 irreversible adduct with the enzyme. The photolabeling was competed by known PTP1B inhibitors, vanadate, and the peptide inhibitor N-benzoyl-l-glutamyl-[4-phosphono(difluoromethyl)]-l-phenylalanyl-[4-phosphono(difluoromethyl)]l-phenylalanineamide (BzN-EJJ-amide). In HepG2 (human hepatoma cell line) cells, endogenous PTP1B was labeled by the UV-activated photoprobes in both lysed and intact cells. Enzyme occupancy measurements were conducted with a series of PTP1B inhibitors using the photoprobe affinity assay. Several compounds were shown to bind to endogenous PTP1B in the HepG2 intact cells.
Skorey, K; Waddleton, D; Therien, M; Leriche, T
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