The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors, including prostate cancer. Previously, we showed that one of the products of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5'-OH-thalidomide, is responsible for the drug's antiangiogenic activity. Based on the chemical structure of this metabolite, we synthesized 118 novel analogs of thalidomide and evaluated them using 4 in vitro models to assess activity in the inhibition of angiogenesis (rat aorta model, human saphenous vein model, cultured endothelial cells, and tube formation assay). We continue to develop these compounds, which appear to have minimal side effects in initial preclinical toxicology studies. Clinical development included completion of a phase II trial of thalidomide with weekly docetaxel, which showed the 18 mo survival was 42.9% in the docetaxel alone group and 68.2% in the combined group. The median overall survival in the docetaxel alone group was 14 mo compared with 28 mo for the combination arm (p=0.11). Dr. Dahut and I conducted a Phase II trial of thalidomide, docetaxel, prednisone and bevacizumab in chemo-naive castration-resistant prostate cancer patients. In the mouse model, combination therapy of docetaxel, bevacizumab, and thalidomide inhibited tumor growth most effectively. In the clinical trial, 90% of patients receiving the combination therapy had PSA declines of greater than or equal to 50%, and 88% achieved a PSA decline of greater than or equal to 30% within the first 3 mo of treatment. The median time to progression was 18.3 mo and the median overall survival was 28.2 mo in this group with a Halabi predicted survival of 14 mo. While toxicities were manageable, all patients developed grade 3/4 neutropenia. The addition of bevacizumab and thalidomide to docetaxel is highly active with manageable toxicities. The estimated median survival is encouraging given the generally poor prognosis of this patient population. These results suggest that definitive clinical trials combining antiangiogenic agents with different mechanisms with docetaxel are warranted to improve treatment outcomes for patients with metastatic CRPC. To maintain the activity of this combination while reducing its associated side effects, we have replaced thalidomide with a structurally similar drug, lenalidomide. In this ongoing trial, patients with chemo-naive CRPC will be treated with docetaxel, prednisone, bevacizumab, and lenalidomide. In collaboration with Dr. Neil Vargesson, we further investigated the mechanism of action of thalidomide's teratogenic effect. We demonstrated that loss of immature blood vessels is the primary cause of thalidomide-induced teratogenesis and explained its action at the cell biological level. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined that pomalidomide, displays a high degree of cell specificity, and has antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations as compared to thalidomide or lenalidomide. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogs (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). All compounds inhibited the extent of outgrowth of newly developing blood vessels in vivo in fli1:EGFP zebrafish embryos and in rat aortic ring assays. Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, while Gu998 and Gu992 showed no difference. The compounds were also evaluated in vivo using the human prostate cancer PC3 xenograft model to evaluate for anti-tumor effects and screened in vivo in chicken embryos to investigate their teratogenic potential. These novel thalidomide analogs are promising immunomodulatory class compounds with anti-cancer effects that warrant further development to characterize their mechanisms of action. A principal mechanism by which cancer cells adapt to the hypoxic microenvironment is through the activity of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1a). HIF-1a expression under hypoxic conditions regulates genes that play key roles in metastasis, angiogenesis, cancer cell metabolism, and resistance. Therefore, the inhibition of transcription driven by HIF (via disrupting the complex that HIF forms with p300, an essential transcriptional coactivator) has the potential for cancer treatment. We have previously shown in our laboratory that several members of the epidithiodiketopiperazine (ETP) family of natural products are able to block the interaction between HIF-1a and p300 by a zinc ejection mechanism. Structure-activity studies using both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1a and p300. Studies are currently underway to identify additional compounds that inhibit the HIF-p300 interaction and involve a high throughput screen using the in vitro fluorescence binding assay developed in our laboratory (composed of a biotinylated synthetic peptide of the C-TAD domain of HIF-1a immobilized on 96-well streptavidin-coated plates and a recombinant GST-tagged protein containing the CH1 domain of p300). We screened a library consisting of 170,000 compounds from the NCI Natural Products Repository that include a collection of pre-fractionation compounds, a selection of compounds known to affect HIF transactivation through unknown mechanisms, and a library of natural compounds. This screen screen led to the identification of indandione and benzoquinone derivatives that reduce the tight interaction between a HIF-1a fragment and the CH1 domain of p300. The indandione derivatives were shown to fragment to give ninhydrin, which was identified as the active species. Both the naphthoquinones and ninhydrin were observed to induce Zn(II) ejection from p300 and the catalytic domain of the histone demethylase KDM4A. Together with previous reports on the effects of related compounds on HIF-1a and other systems, the results suggest that care should be taken in interpreting biological results obtained with highly electrophilic/thiol modifying compounds. The natural product screen also identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 uM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1a interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein-protein binding events. Molecular and mass spectrometry studies are being conducted to verify that these compounds can disrupt the HIF-1a/p300 complex and subsequent downstream HIF-mediated signaling in cell-based assays.
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