Figure 4.

NAZ2329, an allosteric inhibitor of R5 RPTP subfamily members. (A) Structure of NAZ2329. (B) Effect of preincubation of NAZ2329 on its inhibitory activity against PTPRZ. Recombinant human PTPRZ1 enzyme (the whole intracellular region) was premixed with NAZ2329 for the indicated times and followed by addition of a fluorogenic DiFMUP (non-specific PTP substrate, 6,8-difluoro-4-methylumbiliferyl phosphate) to measure PTP inhibitory activity. (C) Time dependence. The PTPRZ1 enzyme was added to the inhibitor–substrate mixture, and the inhibitory activity was determined at each time point relative to the vehicle control. (D) Reversibility of inhibition. PTPRZ1 enzymes were preincubated with NAZ2329 at the indicated concentrations for 30 min. The mixture was then 20-fold diluted, and recovered PTP activity was expressed as the relative activity compared with the vehicle-treated enzyme. (E) Lineweaver-Burk plot analysis. PTPRZ1 enzymes were preincubated with NAZ2329 for 60 min, and the PTP activity was measured using DiFMUP. (F) Concentration-inhibition curves of NAZ2329 for representative PTP members, including human PTPRZ and PTPRG (another R5 RPTP subfamily member), human PTPRS (R2A subfamily), human PTPRM (R2B subfamily), mouse PTPRB (R3 subfamily), human PTPRA (R4 subfamily), human PTPN1 (non-transmembrane PTP, NT1 subfamily), and human PTPN6 (NT2 subfamily), were generated. Assays were performed using DiFMUP. IC₅₀ values obtained are shown in the inset. Data represent the averages of two or three separate experiments.