(Representative data from 3 experiments with n=3 per group) Small Molecule GLUT Inhibitors Display Limited Toxicity to Normal Human Astrocytes and Neurons. As GLUT1 and GLUT3 are extremely important Cilliobrevin D to normal brain function, we assessed the toxicity of these compounds on normal human astrocytes (NHAs) which express high levels of GLUT1, and on neurons which express high levels of GLUT3. other tumor types, and the recognized novel inhibitors provide an excellent starting point for further lead development. which suggests the possibility of targeting glucose uptake as a therapy (3). Silencing of GLUT1 or pharmacological inhibition with WZB117 has also been shown to decrease the PIK3CB tumor formation capabilities of TICs (32). Currently, you will find few GLUT inhibitors and no GLUT3 specific inhibitors. The GLUT inhibitors that have been recognized have not been extensively assessed for efficacy in GBM or for potential toxicity to normal cell types (5). As such, there is still a need to identify potent inhibitors of glucose transporters with strong efficacy but limited toxicity for potential novel therapeutic applications. Both GLUT1 and GLUT3 are transmembrane proteins that belong to the major facilitator superfamily (MFS) (33). Each transporter molecule consists of a 12 transmembrane helices (TH) segment and an intracellular helices (ICH) bundle. The transmembrane segment is further divided into an N-terminal domain name (TH 1C6) and a C-terminal domain name (TH 7C12) (Physique 1a, b). Substrates are thought to be transported through an alternating access mechanism (34) that involves multiple conformational changes of the transporter (Physique 1c) (35). A recently published crystal structure of human GLUT1 bound to a sugar analog compound adopted an in-ward open conformation and provided detailed structural information regarding sugar-transporter interactions and a template for structure based drug discovery (Physique 1)(36). In the present study, we applied homology modeling and structure-based virtual screening (SBVS) to select putative small molecule GLUT3 inhibitors. Our investigation led to the identification of several compounds that blocked the uptake of glucose and decreased the growth of GBM patient-derived xenograft (PDX) cells hit compounds will then be used to re-evaluate modeling. b) Library construction and assessment using structure-based virtual screening. Open in a separate window Physique 3. Potential GLUT3 inhibitors recognized by SBVS.a) indolinones/imidazolinones b) dihydroquinolinones. c) Isoflavones d) miscellaneous core structures. Small Molecules Inhibit GBM PDX Spheroid Growth < 0.001 one-ay ANOVA and Dunnetts multiple comparisons between D456 and NHA treated with 5 M and 20 M) (b) SRI-37684 (**< 0.001 one-ay ANOVA and Dunnetts multiple Cilliobrevin D comparisons between D456 and NHA treated with 5 M) (c) SRI- 37218 (**< 0.005 one-ay ANOVA and Dunnetts multiple comparisons between D456 and NHA treated with 50 M) (d) SRI-39260 (e) SRI-39256 (f) Cilliobrevin D SRI-39257 Cilliobrevin D at indicated concentrations for 7 days (g) Representative images of D456 GBM PDX cells on day 7 after treatment with indicated concentrations of antagonists. (Representative data from 3 experiments with n=3 per group) Small Molecule GLUT Inhibitors Display Limited Toxicity to Normal Human Astrocytes and Neurons. As GLUT1 and GLUT3 are extremely important to normal brain function, we assessed the toxicity of these compounds on normal human astrocytes (NHAs) which express high levels of GLUT1, and on neurons which express high levels of GLUT3. NHAs were treated in the same manner as the GBM PDX cells mentioned above to identify compounds with a potentially favorable therapeutic index. Both dihydroquinolinone compounds (SRI- 37683 and SRI-37684) and one indolinone compound (SRI-37218) displayed significantly stronger growth inhibition of GBM PDX cells compared to NHAs and thus were selected for further biological evaluation (Shape 4 a-c). An added compound (SRI-39257) shown hook difference in development between D456 GBM PDX cells and NHAs but had not been selected for even more analysis because of an insignificant reduction in growth in the 5 M focus in JX12 cells (Shape 4f and Assisting Information Shape 2d). Substances without considerable IC50 variations between NHAs and GBM PDX cells weren't evaluated additional. Representative pictures of D456 GBM PDX cells treated with SRI-37683 and SRI-37684, probably the most efficacious substances with reduced toxicity to NHAs, are demonstrated in Shape 4g. To measure the potential toxicity from the determined substances against neurons, we examined SRI-37683, SRI- 37684, and SRI-37218 at 5 M, 20 M,.