Solid tumors require a constant supply of nutrients through blood vessels. When simple diffusion is no longer sufficient, tumors begin to continuously stimulate blood vessel growth to allow their rapid metabolic growth. The ability to stimulate angiogenesis comes largely from Hypoxia Inducible Factor-1 (HIF-1), a master transcription factor involved in cellular adaptation to hypoxia. HIF-1α, a subunit of the HIF transcriptional complex, is activated by low levels of oxygen and mediates the transcriptional responses to hypoxia, thus enabling the cell to survive in an oxygen depleted environment, as well as stimulate blood vessel growth. If the hypoxic response could be prevented in cancerous cells, new forms of cancer treatment would emerge. One particular target is the interaction between HIF-1α and p300, an essential transcriptional coactivator. Experimental evidence has found that specific blockade of this interaction leads to attenuation of HIF gene expression and a decrease in tumor growth2. A small molecule, chetomin, has been found prevent p300/HIF-1α binding. Chetomin abrogates the normal interaction and binding of p300 with both HIF-1α and HIF-2α by disrupting the structure and function of the domain of p300 that binds to HIF-1α and 2α. Chetomin significantly reduced the amount of p300 bound to HIF-1α in cells, as well as decreasing HIF mediated gene expression. Systemic administration of chetomin attenuates HIF-1 mediated gene expression within mice and possesses significant anti-tumor effects1. Chetomin is unlikely to be pursued clinically though, due to local coagulative necrosis, anemia and leukocytosis1, yet it still provides a lead compound. These results lead us to examine the structural activity relationship of chetomin and the class of compounds which chetomin belongs to, known as ETPs (epidithiodioxopiperazines). By elucidating the chemical mechanism of action, we hope to design a specific inhibitor with less toxicity. A fluorescent protein-protein interaction assay was setup for HIF-1α and p300, which allowed us to test a small library of natural and synthetic ETPs. Compounds possessing the core dithiodioxopiperazine moiety were active, but alterations to the core lead to a loss of activity. Through structural modifications we were able to narrow the activity down to the disulfide bridge. ETPs modified to contain reduced thiols were also active, but compounds with a single thiol group, methylated thiols, and no thiol groups were inactive. Peptidic compounds containing a disulfide without the surrounding dioxopiperazine moiety were inactive, indicating the dioxopiperazine moiety with two thiols in reduced or oxidized form are required for activity. We have now narrowed down the potential sites of action for the ETPs on the HIF-1α/p300, and through a combination of molecular modeling, in-silico design and combinatorial chemistry are designing a less toxic, potent, and highly specific inhibitor.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA