Journal: Journal of genetics and genomics = Yi chuan xue bao

Article Title: A facile and sensitive method of quantifying glutaminase binding to its inhibitor CB-839 in tissues

doi: 10.1016/j.jgg.2020.06.001

Figure Lengend Snippet: (A) Schematic of AlphaLISA-based CETSA. GLS engagement by CB-839 protects it from thermal denaturation, which allows it to be recognized by GLS antibodies in 384-well plates. Secondary antibodies conjugated to donor and acceptor beads recognize the Fc domain of GLS antibodies, and all together form a complex. When donor beads are excited at 680nm, they emit singlet oxygen; the close complex formed by donor bead-antibody-GLS-antibody-acceptor bead allows the singlet oxygen to excite the acceptor bead, and an Alpha signal is emitted at 615nm. In contrast, if no CB-839 is present to engage with GLS, GLS is thermally denatured and precipitates, and cannot be recognized by the anti-GLS antibodies to generate the close complex necessary for singlet oxygen to excite the acceptor bead, and no Alpha signal is emitted. (B) Optimization of Antibodies. HCT116 cells were trypsinized, resuspended in PBS, and heated for 3 minutes at 54°C prior to being lysed. Lysates were added to the noted anti-GLS antibodies in 384-well plates with a mixture of donor and acceptor beads. (C) Optimization of temperatures. HCT116 cells were treated with 10 μM CB-839 for 2 hours, then exposed to the noted temperatures for 3 minutes. (D): Characterization of concentration-dependent engagement of CB-839 to GLS. Unheated samples have baseline alpha signal, because GLS remains intact. This alpha signal does not significantly change with increasing concentrations of CB-839. However, heated samples have no alpha signal with very low CB-839 concentrations; the alpha signal increases with increased CB-839 concentrations, suggesting thermal protective effect with drug engagement of GLS. (E) Optimal duration of cell treatment. Cells were treated with 10 μM CB-839 for the noted durations; heating conditions: 52°C for 3 minutes. (F) CB-839 specifically engages GLS to provide thermal stability. HCT116 cells were treated with 10uM of compounds noted, all known to inhibit other enzymes; only CB-839 provided thermal protection of GLS.

Article Snippet: For AlphaLisa-based CETSA, cells were lysed in CETSA Cell Lysis Buffer 2 (PerkinElmer, USA, CETSA-BUF2-100ML).

Techniques: Concentration Assay