HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

It has been shown that oxygen deprivation results in apoptotic cell death, and that hypoxia inducible factor 1 (HIF1) and the tumor suppressor p53 play key roles in this process. However, the molecular mechanism through which hypoxia and HIF1 induce apoptosis is not clear. Here we show that the expression of pro-apoptotic gene BNIP3 is dramatically induced by hypoxia in various cell types, including primary rat neonatal cardiomyocytes. Overexpression of HIF1a, but not p53, induces the expression of BNIP3. Overexpression of BNIP3 leads to a rather unusual type of apoptosis, as no cytochrome c leakage from mitochondria was detected and inhibitors of caspases were unable to prevent cell death. Taken together, these data suggest that HIF1-dependent induction of BNIP3 may play a significant role during hypoxiainduced cell death. Cell Death and Differentiation (2001) 8, 367 ± 376.

The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC 50 s of 14 and 0.7 Mmol/L, respectively. These results correlated with the direct

There is increasing evidence that tumor-associated macrophages promote the malignancy of some cancers. Colonystimulating factor-1 (CSF-1) is expressed by many tumors and is a growth factor for macrophages and mediates osteoclast differentiation. Herein, we report the efficacy of a novel orally active CSF-1 receptor (CSF-1R) kinase inhibitor, JNJ-28312141, in proof of concept studies of solid tumor growth and tumor-induced bone erosion. H460 lung adenocarcinoma cells did not express CSF-1R and were not growth inhibited by JNJ-28312141 in vitro. Nevertheless, daily p.o. administration of JNJ-28312141 caused dose-dependent suppression of H460 tumor growth in nude mice that correlated with marked reductions in F4/ 80 + tumor-associated macrophages and with increased plasma CSF-1, a possible biomarker of CSF-1R inhibition. Furthermore, the tumor microvasculature was reduced in JNJ-28312141-treated mice, consistent with a role for macrophages in tumor angiogenesis. In separate studies, JNJ-28312141 was compared with zoledronate in a model in which MRMT-1 mammary carcinoma cells inoculated into the tibias of rats led to severe cortical and trabecular bone lesions. Both agents reduced tumor growth and preserved bone. However, JNJ-28312141 reduced the number of tumor-associated osteoclasts superior to zoledronate. JNJ-28312141 exhibited additional activity against FMS-related receptor tyrosine kinase-3 (FLT3). To more fully define the therapeutic potential of this new agent, JNJ-28312141 was evaluated in a FLT3-dependent acute myeloid leukemia tumor xenograft model and caused tumor regression. In summary, this novel CSF-1R/FLT3 inhibitor represents a new agent with potential therapeutic activity in acute myeloid leukemia and in settings where CSF-1-dependent macrophages and osteoclasts contribute to tumor growth and skeletal events.

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