Development of Anticancer Agents

Division Of Clinical Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Development of immunomodulatory drugs. Immunomodulatory drugs (IMiDs) are thalidomide analogs, which possess pleiotropic anti-myeloma properties including immune-modulation, anti-angiogenic, anti-inflammatory and anti-proliferative effects. Thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. The development of novel IMiDs with improved efficacy and decreased toxicity is an ongoing research effort in our laboratory. In collaboration with Drs. Nigel Greig (NIA, NIH) and Michael Gutschow (University of Bonn, Germany) to we have synthesized novel IMiDs, evaluated them using various preclinical models to assess activity, and characterize their structure-activity-relationships for further rational drug design. We have synthesized over 315 350 novel analogs and screened them using in vitro, ex vivo, and in vivo drug development models (e.g., rat aorta ring model, human saphenous vein model, migration and tube formation assays, anti-inflammatory assays). We identified the most potent of these analogs and have patented them. We use in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the novel analogs. Studies are ongoing for SAR analysis of rationally designed and synthesized IMiD analogs. 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, CK1alpha, 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. Introduction of a monoterpenoid group into the thalidomide backbone to replace the classic glutarimide that is involved in cereblon binding resulted in IMiD/monoterpenoid analogues that lack human cereblon binding. Polyfuorination of the phthalimide ring resulted in agents that possessed significant anti-proliferative action against lenalidomide sensitive (MM.1S) and resistant MM (U266 R10R) cells, as well as anti-angiogenesis and anti-inflammatory activities via cereblon-independent mechanisms. From our cellular studies in lenalidomide sensitive and resistant MM cell lines (anti-proliferative assay), human umbilical vein endothelial cells (anti-angiogenesis assay), and RAW 264.7 mouse macrophage cells challenged with lipopolysaccharide (anti-inflammatory assay), we describe four novel lead compounds that warrant further investigation as cereblon-independent IMiDs in cancer and inflammatory disorders. 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-1 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 Program (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. New discorhabdin D derivatives have been isolated from Latrunculia kaakaariki and Latrunculia brevis and tested for their growth inhibitory activity against the NCI-60 human tumor cell line panel. Efforts are ongoing to extract more novel discorhabdins from marine sponges in order to characterize their biological activity and 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. In collaboration with Dr. Tom Snaddon (Indiana University), a series of 24 novel ETP analogs were rationally designed, synthesized and screened for biological activity. anti-angiogenic activity in the rat aortic ring ex vivo assay. We further evaluated the lead compounds using rat aortic ring ex vivo assay, endothelial tube formation and cytotoxicity assays, as well as three-dimensional colon cancer and multiple myeloma spheroid models. Results indicate that several compounds have anticancer activity in both colon cancer and multiple myeloma models and are worthy of further drug development efforts. We have recently patented several lead compounds with promising activity and will further evaluate them for preclinical development.