The angiogenic property of thalidomide reported by D'Amato and colleagues has prompted its clinical evaluation in various solid tumors, including prostate cancer. As a follow-up to our previous phase II study combining docetaxel with two antiangiogenic (bevacizumab and thalidomide) drugs, Dr. Bill Dahut (GMB, CCR, NCI) and I conducted a phase II trial of docetaxel, prednisone, bevacizumab, and lenalidomide in mCRPC patients. Lenalidomide was substituted for thalidomide in this regimen because of the reduced side effect profile (fatigue and neuropathy). Patients were also given broad supportive measures (growth factor and anticoagulation) in an attempt to mitigate the morbidity and treatment-limiting toxicities. Toxicities were manageable with most common adverse events (AEs) being hematological. Twenty-nine patients (46%) had grade 4 neutropenia, 20 (32%) had grade 3 anaemia and seven (11%) had grade 3 thrombocytopenia. Of 61 evaluable patients, 57 (93%), 55 (90%) and 33 (54%) had PSA declines of 30, 50 and 90%, respectively. Of the 29 evaluable patients, 24 (86%) had a confirmed radiographic partial response. The median times to progression and overall survival were 18.2 and 24.6 months, respectively. With appropriate supportive measures, combination angiogenesis inhibition can be safely administered and potentially provide clinical benefit. These hypothesis-generating data would require randomized trials to confirm the findings. Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenesis pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort in our laboratory. 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 collaborated with Drs. Nigel Greig (NIA, NIH) and Michael Gutschow to synthesize and characterize novel thalidomide analogs and evaluated them using in vitro and in vivo models to assess activity. In collaboration with Dr. Neil Vargesson, we conduct an in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both using the in vivo zebrafish and chicken embryo model systems. Additionally, 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 were seen to reduce microvessel outgrowth in rat aortic rings as well as inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The anti-angiogenic properties of the compounds was also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos. The compounds anti-tumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogs as a promising immunomodulatory class with anti-cancer effects that warrant further development to characterize their mechanisms of action. Angiogenesis inhibitors are now widely used in the clinic; however, there are relatively few published studies on the mechanism of their presumed teratogenic effects. To address this issue, we screened a variety of angiogenesis inhibitors in developing zebrafish and chicken embryo models to assess for developmental defects and potential teratogenic effects. Weconfirmed previous reports that sunitinib, sorafenib and TNP-470 are teratogenic and demonstrate that axitinib, pazopanib, vandetanib, and everolimus are also teratogens in these models. A dose response study identified the drugs inhibit HUVEC cell proliferation in vitro, and also target the developing blood vessels of embryos in vivo. This provides further evidence for the potential risk of fetal toxicity when using these drugs in a clinical setting, and emphasizes the importance of the development and maintenance of the vasculature in the embryo. We conclude that angiogenesis inhibitors, regardless of the molecular target, are teratogenic when exposed to chicken embryos. 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. Development of HIF-1a/p300 inhibitors has been hampered by preclinical toxicity; therefore, we aimed to identify novel HIF-1a/p300 inhibitors with 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 of 170,298 crude natural product extracts and prefractionated samples and identified 25 active extracts. One of these extracts, originating from the marine sponge Latrunculia sp., afforded six pyrroloiminoquinone alkaloids that were identified as positive hits (IC50 values: 1-35 uM). Luciferase assays confirmed inhibition of HIF-1a transcriptional activity by discorhabdin B (1) and its dimer (2), 3-dihydrodiscorhabdin C (3), makaluvamine F (5), discorhabdin H (8), discorhabdin L (9), and discorhabdin W (11) in HCT 116 colon cancer cells (0.1-10 uM, p0.05). Except for 11, all of these compounds also reduced HIF-1a transcriptional activity in LNCaP prostate cancer cells (0.1-10 uM, p0.05). These effects occurred at noncytotoxic concentrations (50% cell death) under hypoxic conditions. At the downstream HIF-1a target level, compound 8 (0.5 uM) significantly decreased VEGF secretion in LNCaP cells (p0.05). Pyrroloiminoquinone alkaloids are a novel class of HIF-1a inhibitors, which interrupt the protein-protein interaction between HIF-1a and p300 and consequently reduce HIF-related transcription. Preclinical studies of these compounds are ongoing. Continuing our collaboration with Dr. Kirk Gustafson (Molecular Targets Laboratory, CCR, NCI), the natural product screen also identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A and B were isolated from the extract, and their structures assigned by spectroscopic analyses. We recently synthesized the novel heptacyclic structure of eudistine C. These compounds inhibited CH1/C-TAD binding, albeit only at relatively high concentrations. Initial biological characterization of eudistidine C suggests potential applications in the context of cancer and malaria. Compounds based on the eudistidine framework represent possible chemical probes into the role of HIF-1a and p300 in the adaptation of cancer cells to low oxygen environments and are leads for the development of cancer therapeutics.
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