Nevertheless, it is worth noting that MNPC does not directly block EGFRvIII; instead, it acts downstream of EGFRvIII signaling via directly blocking both NQO1 and GSTP1 reductases
August 9, 2021
Nevertheless, it is worth noting that MNPC does not directly block EGFRvIII; instead, it acts downstream of EGFRvIII signaling via directly blocking both NQO1 and GSTP1 reductases. provides the primers used for mutagenesis. (PDF 3953 kb) 13045_2020_979_MOESM1_ESM.pdf (3.8M) GUID:?7B3DB495-A011-456E-91D4-F7F465583C74 Data Availability StatementThe datasets generated and analyzed during the current study are not publicly available due to the ongoing study but are available from the corresponding author on reasonable request. Abstract Background Glioblastoma (GBM) is a universally lethal tumor with frequently overexpressed or mutated epidermal growth factor receptor (EGFR). NADPH quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase Pi 1 (GSTP1) are commonly upregulated in GBM. NQO1 and GSTP1 decrease the formation of reactive oxygen species (ROS), which mediates the oxidative stress and promotes GBM cell proliferation. Methods High-throughput screen was used for agents selectively active against GBM cells with EGFRvIII mutations. Co-crystal structures were revealed molecular details of target recognition. Pharmacological and gene knockdown/overexpression approaches were used to investigate the oxidative stress in vitro and in vivo. Results We identified a small molecular inhibitor, MNPC, that binds to both NQO1 and GSTP1 with high affinity and selectivity. MNPC inhibits NQO1 and GSTP1 enzymes and induces apoptosis in GBM, specifically inhibiting the growth of cell lines and primary GBM bearing the EGFRvIII mutation. Co-crystal structures between MNPC and NQO1, and molecular docking of MNPC with GSTP1 reveal that it Eriodictyol binds the active sites and acts as a potent dual inhibitor. Inactivation of both NQO1 and GSTP1 with siRNA or MNPC results in imbalanced redox homeostasis, leading to apoptosis and mitigated cancer proliferation in vitro and Eriodictyol in vivo. Conclusions Thus, MNPC, a dual inhibitor for both NQO1 and GSTP1, provides a novel lead compound for treating GBM via the exploitation of specific vulnerabilities created by mutant EGFR. strain BL21 (DE3). Bacterial culture was grown in LB medium with 35?g/ml of kanamycin at 37?C until OD600 reached 0.6 to 0.8 and then induced by adding 0.4?mM isopropyl-L-thio-B-D-galactopyranoside (IPTG) for 16?h at 20?C. Recombinant NQO1 proteins were purified as follows: after harvest by centrifugation, cells were lysed in 10% glycerol, 1% TritonX-100, 200?mM NaCl, 10?mM imidazole and 100?mM Tris (pH 7.6) supplemented with 1?mM phenylmethanesulfonyl fluoride (PMSF). Soluble protein was separated from the cleared cell lysate by centrifugation at 21,000?g 40?min, then submitted to NiCNTA resin (Qiagen) with an elution buffer of 200?mM NaCl, 150?mM imidazole and 20?mM Tris (pH 7.6). Protein was then concentrated and loaded onto a Superdex 200 10/300 GL (GE Healthcare) pre-equilibrated with 200?mM NaCl, 20?mM Tris (pH 7.6). Recombinant GSTP1 protein was purified as described above, except with a slight difference in buffer composition. For GSTP1, -mercaptoethanol was added to all buffers to a final concentration of 2?mM. The purity of NQO1 and GSTP1 was confirmed by SDS-PAGE and Coomassie blue staining. Crystallization and structure determination Crystals of the NQO1 complex with MNPC were obtained by co-crystallization with the sitting drop vapor diffusion method. Purified NQO1 was concentrated to 12?mg/mL and then incubated with MNPC at a molar ratio of 1 1:3 over ice for 1?h. One microliter of NQO1-MNPC solution was mixed with 1 L of mother liquor and further equilibrated with reservoir solution at 20?C. Crystals appeared in a week, with a crystallization condition of 0.2?M lithium sulfate, 1.8?M ammonium sulfate, 0.1?M imidazole pH 7.0. The crystals were cryoprotected using the crystallization solution with 20% glycerol and then flash-frozen directly into liquid nitrogen. The attempt was also made to obtain crystals of the GSTP1CMNPC complex. GSTP1 having a concentration of 10?mg/ml was utilized for crystallization, and the perfect solution is of the GSTP1CMNPC combination was generated just as NQO1-MNPC. Crystals appeared in one day time or two Eriodictyol in the condition of 0.1?M MES PH5.4, 30% PEG8000, 10?mM DTT, 20?mM CaCl2, and grew in a week to the maximum size at 20?C. After crystals grew to the full size, the crystallization condition was supplemented with MNPC of final concentration 3?mM. After soaked for 4?h, crystals were then Rabbit Polyclonal to PCNA flash-frozen in liquid nitrogen until data collection. Diffraction data were collected in the Shanghai Synchrotron Radiation Facility (SSRF) at beamline 17U1, 18U1 and 19U1. The data were.