Development of Angiogenesis Inhibitors

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Development of immunomodulatory drugs. The antiangiogenic properties of thalidomide reported by D'Amato and colleagues prompted its clinical evaluation in various solid tumors, including prostate cancer. 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 collaborate with Drs. Nigel Greig (NIA/NIH) and Michael Gutschow to synthesize novel thalidomide analogs, evaluate them using in vitro and in vivo models to assess activity, and characterize their structure-activity-relationships for further rational drug design. We have synthesized over 315 novel analogs of thalidomide and screened them for inhibition of inflammation and angiogenesis using various in vitro, ex vivo, and in vivo drug development models (e.g., rat aorta ring model, human saphenous vein model, cultured endothelial cells, migration and tube formation assays). In collaboration with Dr. Neil Vargesson, we conducted an in vivo screen of a library of new analogs to determine which agents demonstrate activity using the in vivo zebrafish and chicken embryo model systems. We identified the most potent of these agents and have patented them. We continue to develop these compounds, which appear to have minimal side effects in initial preclinical toxicology studies and may have improved pharmacology over the two FDA approved thalidomide analogs. We have optimized for both antiangiogenic and anti-inflammatory properties of these immunomodulatory drugs (IMiDS), which means the clinical indication can go beyond hematological malignancies and could have activity in solid tumors. This work in antiangiogenic/anti-cancer drug development serves not only to advance the field of antiangiogenic therapy but also to discover new treatment paradigms that focus on immunomodulation for advanced, metastatic disease. We have recently completed characterization of the antiangiogenic activity of polyfluorinated benzamides as well as adamantyl and noradamantyl phthalimidines. Efforts are ongoing to identify potential leads for in vivo toxicology and pharmacology studies in xenograft models. Cereblon (CRBN) is a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex, and a binding target of IMiDs. CRBN is responsible for the pleiotropic effects of IMiDs, yet its function in angiogenesis and in mediating the antiangiogenic effects of IMiDs remains unclear. We investigated the role of CRBN in the angiogenic process and in propagating the antiangiogenic effects of IMiDs in vitro. siRNA-mediated CRBN knock down in human endothelial cells (HUVEC and HMVEC-L), did not affect endothelial cell proliferation, migration, or tube formation. Using CRBN-deficient mice, we further demonstrated that microvessal formation can occur independently of cereblon in the ex vivo mouse aortic ring model. The cereblon E3 ubiquitin ligase complex can recruit endothelial cell-specific factors, AGO2 (associated with angiogenesis), and SALL4 (associated with embryogenesis/angiogenesis), for ubiquitin-mediated degradation. Knockdown of CRBN caused a corresponding increase in AGO2 and SALL4 protein expression and IMiD treatment was able to rescue the siCRBN effect to increase the CRBN expression. These findings suggest one potential mechanism of action that likely involves a tightly coordinated regulation of CRBN with endothelial cell targets and highlight the need to further elucidate the mechanism(s), which could include cereblon-independent pathways, through which IMiDs exert their antiangiogenic effects. Carfilzomib-lenalidomide-dexamethasone (KRd) therapy has yielded promising results in patients with newly diagnosed multiple myeloma (NDMM). CRBN is the direct molecular target of lenalidomide and genetic polymorphisms in CRBN have been associated with lenalidomide efficacy. In this study, we assessed the correlation of five single nucleotide variants (SNVs) in the CRBN gene with clinical response and outcomes in patients with NDMM administered KRd therapy with lenalidomide maintenance, achieving favorable trial endpoints in a prospective Phase II study (NCT01402284). Of the observed SNVs, no associations with KRd therapy response were found in this patient cohort, although strong trends in hypoalbuminemia grade and hyperbilirubinemia grade emerged across the CRBN rs1672753 genotype (P = 0.0008) and the rs1714327 genotype (P = 0.0010), respectively. Our results do not provide conclusive support for the predictive utility of CRBN gene polymorphisms as potential biomarkers of clinical response to lenalidomide-based therapy in our patient population. However, these findings remain to be validated in prospective studies using larger patient populations. Development of HIF-1alpha inhibitors. The hypoxia-inducible factor (HIF) is fundamentally involved in tumor angiogenesis, invasion, and energy metabolism. Inhibition of HIF-1 represents an attractive therapeutic strategy for targeting hypoxia, a hallmark of many solid tumors, and tumor angiogenesis. One promising approach for directly inhibiting HIF-1 activity is by disrupting the tight binding between HIF-1a and p300. Previously, our laboratory developed an in vitro fluorescence binding assay that can be used in a high-throughput screen to identify small-molecule inhibitors of HIF-1a through inhibiting the binding interaction between the C-terminal transactivation domain (CTAD) of HIF-1a and the cysteine/histidine-rich 1 (CH1) domain of p300. Using our HIF-1a/p300 assay, we performed high-throughput screen of NCI's Natural Products Repository in collaboration with Dr. Kirk Gustafson (Molecular Targets Laboratory/NCI). This effort led to the discovery of a series of pyrroloiminoquinone alkaloids including discorhabdin and makaluvamine alkaloids, originating from a Latrunculia sp. of marine sponge, as potential HIF-1a/p300 inhibitors. Preclinical studies are ongoing to understand the mechanisms of these novel compounds.