No abstract

The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation-like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATPsensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor-treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 Å resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.

Allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs) is considered to be one of the most promising approaches for therapeutics. We have previously reported on the pharmacological activity of several compounds that act as negative allosteric modulators (NAMs) of nAChRs. In the following studies, the effects of 30 NAMs from our small chemical library on both human ␣4␤2 (H␣4␤2) and human ␣3␤4 (H␣3␤4) nAChRs expressed in human embryonic kidney ts201 cells were investigated. During calcium accumulation assays, these NAMs inhibited nAChR activation with IC 50 values ranging from 2.4 M to more than 100 M. Several NAMs showed relative selectivity for H␣4␤2 nAChRs with IC 50 values in the low micromolar range. A lead molecule, KAB-18, was identified that shows relative selectivity for H␣4␤2 nAChRs. This molecule contains three phenyl rings, one piperidine ring, and one ester bond linkage. Structure-activity relationship (SAR) analyses of our data revealed three regions of KAB-18 that contribute to its relative selectivity. Predictive three-dimensional quantitative SAR (comparative molecular field analysis and comparative molecular similarity indices analysis) models were generated from these data, and a pharmacophore model was constructed to determine the chemical features that are important for biological activity. Using docking approaches and molecular dynamics on a H␣4␤2 nAChR homology model, a binding mode for KAB-18 at the ␣/␤ subunit interface that corresponds to the predicted pharmacophore is described. This binding mode was supported by mutagenesis studies. In summary, these studies highlight the importance of SAR, computational, and molecular biology approaches for the design and synthesis of potent and selective antagonists targeting specific nAChR subtypes.