Development of Anticancer Agents

免疫调节药物的开发：达马托及其同事报道的沙利度胺的抗血管生成特性促使其在包括前列腺癌在内的各种实体肿瘤中进行临床评估。沙利度胺在影响免疫调节和血管生成途径的各种恶性肿瘤中显示出临床活性。开发疗效更好、毒性更低的新型沙利度胺类似物是我们实验室正在进行的研究工作。此前，我们已经证明了沙利度胺细胞色素P450 2C19同工酶生物转化的产物之一5‘-OH-沙利度胺是该药物抗血管生成活性的原因之一。基于这种代谢物的化学结构，我们与奈杰尔·格雷格博士(NIA，NIH)和迈克尔·古斯鲁博士合作合成了新型沙利度胺类似物，使用体外和体内模型评估它们的活性，并表征它们的结构-活性-关系，以进一步合理的药物设计。我们已经合成了超过315个新的沙利度胺类似物，并利用各种体外、体外和体内药物开发模型(例如，大鼠主动脉环模型、人隐静脉模型、培养内皮细胞、迁移和管形成试验)筛选了它们抑制炎症和血管生成的作用。与Neil Vargesson博士合作，我们对新的类似物库进行了体内筛选，以确定哪些制剂使用体内斑马鱼和鸡胚胎模型系统显示了活性。我们确定了这些药物中最有效的，并为它们申请了专利。我们继续开发这些化合物，它们在最初的临床前毒理学研究中似乎具有最小的副作用，并且可能已经改善了FDA批准的沙利度胺和免疫调节药物(IMiDS)来那度胺和泊马度胺的药理学。我们最近完成了多氟苯甲酰胺的细胞毒性、抗血管生成和抗炎特性的表征，并在耐药多发性骨髓瘤模型中进行了测试。目前正在进行研究，以确定三维(3D)骨髓瘤球体模型中的潜在线索，随后将评估该模型在异种移植模型中的体内毒理学和药理学研究，并了解其作用机制，因为这些化合物在结构上类似于沙利度胺，但缺乏与药物靶点Cereblon(CRBN)结合的戊二酰亚胺部分。这些IMiD与CRBN的结合改变了连接酶的底物专一性，从而介导了在癌症治疗中利用的多种效应。由于其抗血管生成和抗免疫调节活性，尽管沙利度胺具有致畸作用，但它仍然受到临床的关注，合成更安全、临床活性更高的沙利度胺类似物的努力仍在继续。我们进行了构效关系研究，以评价新型沙利度胺类似物的抗血管生成活性和电子CRBN结合分析。在电子药效团分析和分子对接与人脑蛋白的晶体结构被用来研究新的沙利度胺类似物的脑蛋白结合能力。对合理设计和合成的IMID类似物的SAR分析的研究正在进行中。缺氧诱导因子-1α抑制物的研究进展：缺氧诱导因子在肿瘤血管生成、侵袭和能量代谢中起重要作用。抑制HIF-1是针对缺氧和肿瘤血管生成的一种有吸引力的治疗策略，缺氧是许多实体肿瘤的标志。直接抑制HIF-1活性的一个有希望的方法是通过破坏HIF-1α和p300之间的紧密结合。此前，我们实验室建立了一种体外荧光结合试验，通过抑制HIF-1a的C末端反式激活结构域(CTAD)和p300的半胱氨酸/富含组氨酸的1(CH1)结构域之间的结合作用，从而识别HIF-1a的小分子抑制剂。使用我们的HIF-1α/p300检测，我们与分子靶标实验室(NCI)合作，对NCI的天然产物储存库进行了高通量筛选。这一努力导致了一系列的吡咯亚氨基醌生物碱的发现，包括来自一个Latrunculia sp.的discorhabdin和makaluvamine生物碱。作为潜在的HIF-1a/p300抑制剂。目前正在努力从新西兰海绵中提取更多的不协调素，以便继续进行临床前研究，以进一步了解这些新化合物的机制。表二硫代二酮基哌嗪(Epidithiodiketopiperazines，ETP)具有多种生物活性，包括抗癌、抗真菌、抗菌和抗病毒等。ETP是一类已被证明抑制HIF-1α的化合物，ETP核心本身在体外足以阻断HIF-1α和p300的相互作用。人们对这些天然产物的合成化学有相当大的兴趣，并积极进行类似物的制备；然而，它们的合成在结构上具有挑战性。这项研究旨在筛选由我们的合作者Tom Snaddon博士合理设计和合成的合成ETP类似物，并确定这些新化合物在生物检测中的活性。我们最近已经申请了几项具有良好活性的先导化合物的专利，并将在临床前模型中进一步评估它们。

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. 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 FDA approved thalidomide and immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide. We have recently completed characterization of the cytotoxicity, antiangiogenic and anti-inflammatory properties of polyfluorinated benzamides as well as tested them in IMiD-resistant multiple myeloma models. Studies are ongoing to identify potential leads in three dimensional (3D) myeloma spheroid models which will subsequently be evaluated for in vivo toxicology and pharmacology studies in xenograft models as well as to understand the mechanisms of action since the compounds are structurally similar to thalidomide but lacks the glutarimide moiety that binds to the drug's target,cereblon (CRBN). The binding of these IMiDs to CRBN alters the substrate specificity of the ligase, thereby mediating multiple effects that are exploited in cancer therapy. Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. We conducted a structure-activity relationship study to evaluate the antiangiogenic activity and in silico CRBN binding analysis of novel thalidomide analogs. 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 thalidomide analogs. Studies are ongoing for SAR analysis of rationally designed and synthesized IMiD analogs. 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-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-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-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-1a/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. Epidithiodiketopiperazines (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.