Notch signaling is an area of great interest in oncology. RO4929097 is a potent and selective inhibitor of γ-secretase, producing inhibitory activity of Notch signaling in tumor cells. The RO4929097 IC50 in cell-free and cellular assays is in the low nanomolar range with >100-fold selectivity with respect to 75 other proteins of various types (receptors, ion channels, and enzymes). RO4929097 inhibits Notch processing in tumor cells as measured by the reduction of intracellular Notch expression by Western blot. This leads to reduced expression of the Notch transcriptional target gene Hes1. RO4929097 does not block tumor cell proliferation or induce apoptosis but instead produces a less transformed, flattened, slower-growing phenotype. RO4929097 is active following oral dosing. Antitumor activity was shown in 7 of 8 xenografts tested on an intermittent or daily schedule in the absence of body weight loss or Notch-related toxicities. Importantly, efficacy is maintained after dosing is terminated. Angiogenesis reverse transcription-PCR array data show reduced expression of several key angiogenic genes. In addition, comparative microarray analysis suggests tumor cell differentiation as an additional mode of action. These preclinical results support evaluation of RO4929097 in clinical studies using an intermittent dosing schedule. A multicenter phase I dose escalation study in oncology is under way.

The accumulation of ␤-amyloid peptides (A␤) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated A␤ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent A␤ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of A␤1-42 and the toxic fragment A␤25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic ␤-sheet conformation by A␤ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of A␤1-40 and A␤25-35 to negatively charged lipid vesicles and A␤ induced cell toxicity. These results suggest that A␤ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.␤-amyloid, the major constituent of senile plaques in Alzheimer disease patients (1) has been proposed to be the cause of the neurodegeneration that occurs in Alzheimer disease brains. A␤1-42, A␤1-40, and certain fragments, notably A␤25-35, are directly toxic to neuronal cell cultures at high micromolar concentrations (2-5). The observed cell death has been correlated with an effect of amyloid peptides on the membrane integrity as determined by lipid peroxidation (2). Furthermore, it has recently been shown that low nanomolar concentrations of A␤ peptides increase the susceptibility of the plasma membrane to additional insults (6).Substantial evidence has been provided suggesting that a crucial step for the formation of toxic A␤ is the transition of random coil to ␤-sheet conformation that is necessary for fibril aggregation. Those fibrils have been demonstrated to cause cell death (7-9). On the other hand studies with lipid vesicles demonstrated that formation of ␤-sheet structures is enhanced in the presence of negatively charged lipid vesicles (10-12). Decreasing the negative charge of a membrane may, therefore, result in a decrease in membrane association of A␤ peptides. Such a decrease in the negative charge of lipid membranes by a decrease in the membrane dipole potential has been demonstrated for phloretin, a lipophilic dipolar substance shown to decrease the membrane dipole potential (13-15).Here we demonstrate that phloretin and a structural analogue, exifone, not only reduce the association of toxic A␤ peptides with the membrane but also prevent A␤ toxicity to neuron-like PC12 cells. These results suggest that a physicochemical interaction of A␤ peptides with negatively charged membranes might be responsible for the toxic effect of A␤ to neuronal cells. MATERIALS AND METHODSMaterials. Rat PC12 pheochromocytoma cells were a gift from E. Sho...

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Supplementary Methods and Materials from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with <i>In vivo</i> Efficacy and Pharmacodynamic Properties

Supplementary Figure 2 from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with <i>In vivo</i> Efficacy and Pharmacodynamic Properties

Supplementary Figure 1 from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with <i>In vivo</i> Efficacy and Pharmacodynamic Properties

Supplementary Figure 1 from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with <i>In vivo</i> Efficacy and Pharmacodynamic Properties

Supplementary Figure 2 from Preclinical Profile of a Potent γ-Secretase Inhibitor Targeting Notch Signaling with <i>In vivo</i> Efficacy and Pharmacodynamic Properties