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 conduct 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. Immunomodulatory imide drugs (IMiDs) play a crucial role in the treatment landscape across various stages of multiple myeloma. Despite their evident efficacy, some patients may exhibit primary resistance to IMiD therapy, and acquired resistance commonly arises over time leading to inevitable relapse. It is critical to develop novel therapeutic options to add to the treatment arsenal to overcome IMiD resistance. We designed, synthesized, and screened a new class of polyfluorinated thalidomide analogs and investigated their anti-cancer, anti-angiogenic, and anti-inflammatory activity using in vitro and ex vivo biological assays. We identified four lead compounds that exhibit potent anti-myeloma, anti-angiogenic, anti-inflammatory properties using three-dimensional tumor spheroid models, in vitro tube formation, and ex vivo human saphenous vein angiogenesis assays, as well as the THP-1 inflammatory assay. Western blot analyses investigating the expression of proteins downstream of cereblon (CRBN) reveal that Gu1215, our primary lead candidate, exerts its activity through a CRBN-independent mechanism. Our findings demonstrate that the lead compound Gu1215 is a promising candidate for further preclinical development to overcome intrinsic and acquired IMiD resistance in multiple myeloma. Introduction of fluorine into bioactive molecules has attracted much attention in drug development. For example, tetrafluorination of the phthalimide moiety of immunomodulatory drugs (IMiDs) has a strong beneficial effect on the ability to inhibit angiogenesis. The neomorphic activity of E3 ligase complexes is induced by the binding of IMiDs to cereblon. In collaboration with Dr. Gutschow, we investigated that a set of eight thalidomide analogs, comprising non- and tetrafluorinated counterparts, did not induce the degradation of neomorphic substrates (IKZF3, GSPT1, CK1a, SALL4). Hence, the antiangiogenic activity of fluorinated IMiDs was not triggered by neosubstrate degradation features. A fluorine scanning of non-traditional IMiDs of the benzamido glutarimide chemotype was performed. By measuring the endothelial cell tube formation, no angiogenesis inhibitors were identified, confirming the narrow structure-activity window of IMiD-induced antiangiogenesis. Natural Products Drug Screening. 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-1alpha 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-1alpha through inhibiting the binding interaction between the C-terminal transactivation domain (CTAD) of HIF-1alpha and the cysteine/histidine-rich 1 (CH1) domain of p300. Using our HIF-1alpha/p300 assay, we performed high-throughput screen of NCI's Natural Products Repository in collaboration with the 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-1alpha/p300 inhibitors. Efforts are ongoing to extract more discorhabdins from New Zealand sponges in order to continue preclinical studies to further understand the mechanisms of these novel compounds. Epidithiodiketopiperazine (ETPs) possess diverse biological activities including anticancer, antifungal, antibacterial and antiviral properties. ETPs are known as a class of compounds that have been shown to inhibit HIF-1alpha with the ETP core itself being sufficient to block the HIF-1alpha and p300 interaction in vitro. There is considerable interest in synthetic chemistry of these natural products and preparation of analogs is actively pursued; however, they are structurally challenging to synthesize. This study is undertaken to screen synthetic ETP analogs rationally designed and synthesized by our collaborator Dr. Tom Snaddon and to determine the activity of these novel compounds in biological assays. We have recently patented several lead compounds with promising activity and will further evaluate them in preclinical models.