Molecular Targets Development Program

分子靶点开发计划

• 批准号: 8158321
• 负责人: james mcmahon
• 金额: $ 548.03万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158321
• 关键词: Molecular Target; Program Development

Englerin: Investigation of this kidney cancer-specific compound advanced. It has excellent activity in mouse xenograft experiments. The mechanism of action became clearer, with GLUT-1 mRNA and protein levels reduced in cells prior to a decrease in HIF-2. This appears to fit our evolving appreciation of the importance of glycolytic pathways in kidney cancer. Large-scale recollection of the plant (Tanzania) and bulk isolation of several grams of englerin A was accomplished. New analogs were prepared and are now being evaluated for testing in kidney cancer cell lines. Results show that cells with 1 of 3 genetic lesions are sensitive to englerin A, indicating that it may be useful in therapy. An assay for englerin A in mouse serum is being developed to support pharmacokinetic (PK) studies prior to further xenograft experiments. HDAC: The HDAC/DNMT inhibitor screen identified 12 compounds with activity from the 4,363 compounds screened. These compounds are undergoing secondary/tertiary analysis of HDAC/DNMT activity at our collaborator's lab. Four hit compounds (whose structure varies from known compounds in this class) were identified that show activity in secondary assays. Each small molecule hit exhibits unique properties in its ability to block cell cycle progression, inhibit HDACs, alter global histone acetylation and induce cancer cell death. Chemical modulators will be explored to define specific targets and identify applications alone or in combination with existing anti-cancer drugs (manuscript submitted). MDM2: Screening of 148,000 natural product extracts is complete. Isolation and structural elucidation of 20 inhibitory products is complete. Fifteen pure compounds were identified and are now under evaluation (3 manuscripts have been published). Three compounds derived from this screen (and from similar work with LPDS) have been approved for preclinical evaluation as potential drug candidates. ABCG2: This collaborative effort between our lab and researchers at Ritsumeikan University (Shiga, Japan) resulted in the first synthesis of the botryllamides. Botryllamides A and G are now in preclinical development in CCR and synthetic supplies of these compounds will allow the required in vivo studies to proceed. Currently, we are working with the CBL to scale up synthesis of botryllamides A and G and generate selected analogs for further testing and characterization. HIF2: More than 130,000 natural product extracts were screened, identifying 153 active extracts. Candidaspongiolides and its core macrocycle have been identified as potent inhibitors. Work on isolating and identifying additional compounds from the active natural product extracts continues. More than 50 other compounds were isolated from marine and plant extracts, including stilbenes, flavanoids and pyrrolopyrimidines. Thirty-nine of these were provided to UOB for further evaluation. TRAIL: Withanolide E was identified as a potent and effective compound that synergistically increases the tumor cell killing effects of TRAIL. We verified the purity, identity and activity of a resupply of withanolide E from the DTP Repository and we are now testing withanolide E, both individually and in combination with TRAIL, in the NCI-60 cell line. Results from these studies will inform the planned in vivo xenograph studies designed to assess synergistic tumor cell killing in a mouse model system. Pdcd4: Thirteen pure compounds with activity in a primary Pdcd4 assay were isolated from plant and marine natural product extracts. These compounds appear to stabilize the tumor suppressor Pdcd4 and have been sent to our collaborators for additional testing. The results will help identify the potential targets of these compounds and how they prevent the proteosomal degradation of Pdcd4. Natural Products Chemistry: Ongoing isolation and structural studies are focused primarily on extracts with confirmed activity in Tdp1 and gp78 screening assays. Compounds active against the phosphodiesterase Tdp1 include crambescidin 800 and various structural analogs. Extract from the plant Minquartia guianensis (Olacaceae) provided the linear poly acetylenic compound minquartinoic acid as a potent Tdp1 inhibitor. Tdp1 inhibitory extracts are being fractionated to identify active agents. Gp78 is an E3 ligase involved with ER-associated degradation (ERAD) of proteins. We performed a screening assay that identified inhibitors of gp78. All extracts with confirmed activity were of fungal origin. Fungal cultures were re-grown on a larger scale and extracts of cultures that retained inhibitory activity were investigated. Pure compounds have been obtained and structural studies are ongoing. Large-scale chromatographic prefractionation of natural extracts continues. More than 3000 extracts were fractionated to produce more than 15,000 samples. Materials were added to our screening library for increased high-throughput screening. Installation of a new 3-mm cryoprobe for 600 MHz NMR and improvements to NMR infrastructure were completed. The addition of a cutting-edge NMR probe expanded our capacity to support isolation and structural studies of new bioactive natural products. Plk1: Development of the Plk1 ELISA-based assay is almost complete. Conversion of the 96-well assay to a 384-well format was successful. Signal-to-noise/background and reproducibility have been optimized. A resupply of GST-Plk1 full-length protein and phosphorylated pT78 13-mer peptide has been scheduled while we finalize robot handling procedures prior to high-throughput screening. ER: A high-content imaging system was used to screen for ER agonists and antagonists by quantitation of ER nuclear translocation. Primary screening is complete, and 160,000 synthetic compounds have been screened. Six pure compounds are being evaluated in secondary assays. Recently, we discovered 3 natural product extracts that exhibit activity. Purification and further testing of these extracts has begun (manuscript submitted). Schweinfurthin: The CGAP project showed that the tumor suppressor NF1 is altered in 20% of glioblastoma multiforme (GBM) cases. We have shown schweinfurthin A selectivity towards central nervous system (CNS) tumor cell lines and phenocopies of neurofibromatosis type 1 (NF1) in NF1-deficient cells. Both GBM and malignant peripheral nerve sheath tumor cell lines are sensitive to schweinfurthins. Our collaborators have synthesized a large number of analogs to help elucidate schweinfurthin mechanisms. Medicinal chemistry studies are aimed at improving these analogs so they can be viable drug candidates. An LC-MS assay for 3 candidate schweinfurthins was developed to support head-to-head PK studies in mice. Gp78: We used a cellular assay to screen for inhibitors of gp78 function and ERAD, which involves ubiquitination of proteins by gp78. Fungal extracts were fractionated to isolate active principles for further characterization. NF1 in CNS Tumors Screen: We ran a screen using cell lines from a Nf1-/+;Trp53-/+ NPcis mouse model of astrocytoma to find agents with activity in CNS tumors. The dual luciferase readout is a novel format that extends our repertoire of screening platforms. This project is in the HTS phase. p300: A biochemical screen has been developed using the CAD domain of HIF-1 and the CH1 domain of p300. Screening of natural product extract and pre-fractionated extract libraries is complete. Confirmation of active extracts has been initiated and will be followed by isolation and structure determination of the active constituents from these extracts.

ENGLERIN：对这种肾脏癌特异性化合物的研究。它在小鼠异种移植实验中具有出色的活性。作用机理变得更清晰，在HIF-2降低之前，细胞中的GLUT-1 mRNA和蛋白质水平降低。这似乎符合我们对糖酵解在肾癌中的重要性的不断发展的欣赏。完成了几克Englerin A的大规模回忆（坦桑尼亚）和大量隔离。准备了新的类似物，现在正在评估在肾脏癌细胞系中进行测试。结果表明，具有3个遗传病变中有1个的细胞对Englerin A敏感，表明它可能在治疗中有用。在进一步的异种移植实验之前，正在开发针对小鼠血清中的Englerin A的测定方法来支持药代动力学（PK）研究。 HDAC：HDAC/DNMT抑制剂屏幕鉴定出了12种化合物，其活性来自4,363种筛选的化合物。这些化合物正在我们的合作者实验室中进行HDAC/DNMT活动的次级/第三级分析。确定了四个命中化合物（其结构与此类的已知化合物不同），这些化合物显示了次级测定中的活性。每个小分子命中均具有阻断细胞周期进展，抑制HDAC，改变全局组蛋白乙酰化并诱导癌细胞死亡的能力，具有独特的特性。将探索化学调节剂，以定义特定靶标，并单独识别应用或与现有的抗癌药物（手稿）结合使用。 MDM2：完成148,000种天然产品提取物的筛选已完成。 20种抑制产物的隔离和结构阐明是完整的。确定了15种纯化合物，现在正在评估中（已发表了3个手稿）。该屏幕得出的三种化合物（来自与LPD的类似工作）已被批准作为潜在药物的临床前评估。 ABCG2：Ritsumeikan University（日本Shiga）的实验室与研究人员之间的合作努力导致了Botrylllamides的第一个合成。现在，Botryllamides A和G正在CCR的临床前开发中，这些化合物的合成供应将使所需的体内研究可以进行。当前，我们正在与CBL合作以扩展杂交酰胺A和G的合成，并生成所选的类似物，以进一步测试和表征。 HIF2：筛选了130,000多种天然产品提取物，识别153种活跃提取物。念珠菌及其核心大环已被确定为有效的抑制剂。从活性天然产物提取物中隔离和识别其他化合物的工作仍在继续。从海洋和植物提取物中分离出50多种其他化合物，包括stim虫，黄酮和吡咯吡汀。其中39个已向UOB提供了进一步的评估。 TRAIL：Wistanolide E被确定为一种有效且有效的化合物，从而协同增加了TRAIL的肿瘤细胞杀伤作用。我们验证了DTP存储库中withanolide e的补给的纯度，身份和活动，现在我们正在NCI-60细胞系中单独并与TRAIL结合使用withanolide e。这些研究的结果将为旨在评估小鼠模型系统中的协同肿瘤细胞杀死计划的计划内Xenograph学研究提供信息。 PDCD4：从植物和海洋天然产物提取物中分离出一级PDCD4分析中具有活性的13种纯化合物。这些化合物似乎可以稳定肿瘤抑制剂PDCD4，并已发送给我们的合作者进行其他测试。结果将有助于确定这些化合物的潜在靶标，以及它们如何防止PDCD4的蛋白体降解。 天然产品化学：持续的隔离和结构研究主要集中在TDP1和GP78筛选测定中证实活性的提取物上。针对磷酸二酯酶TDP1的活性化合物包括Crambescidin 800和各种结构类似物。从植物中的Minquartia Guianensis（Olacaceae）提取的提取物提供了线性的聚乙酰基化合物Minquartinicaic作为有效的TDP1抑制剂。 TDP1抑制提取物被分级以鉴定活性剂。 GP78是与蛋白质相关降解（ERAD）有关的E3连接酶。我们进行了筛选测定，该测定法确定了GP78的抑制剂。所有具有确认活性的提取物都是真菌起源的。真菌培养物在更大范围内重生，并研究了保留抑制活性的培养物。已经获得了纯化合物，并且正在进行结构研究。天然提取物的大规模色谱预分级继续。将3000多种提取物分馏出来生产15,000多个样品。将材料添加到我们的筛选文库中，以增加高通量筛选。为600 MHz NMR安装了新的3毫米冷冻探针，并完成了NMR基础架构的改进。尖端的NMR探针的添加扩大了我们支持新生物活性天然产物的隔离和结构研究的能力。 PLK1：基于PLK1 ELISA测定的开发几乎是完整的。将96孔测定法转换为384孔格式。信号到噪声/背景和可重复性已被优化。 GST-PLK1全长蛋白和磷酸化的PT78 13-MER肽的补充已安排在我们在高通量筛选之前最终确定机器人处理程序的同时。 ER：通过定量ER核易位，使用了高内感成像系统来筛选ER激动剂和拮抗剂。主要筛选完成，并筛选了160,000种合成化合物。在次级测定中评估了六种纯化合物。最近，我们发现了3种具有活性的天然产品提取物。这些提取物的纯化和进一步测试已经开始（手稿提交）。 Schweinfurthin：CGAP项目表明，抑制肿瘤的NF1在20％的多形胶质瘤（GBM）病例中改变了。我们已经显示了schweinfurthin对中枢神经系统（CNS）肿瘤细胞系和NF1缺陷细胞中1型神经纤维瘤病（NF1）的表型的选择性。 GBM和恶性周围神经鞘细胞系都对schweinfurthins敏感。我们的合作者综合了大量的类似物，以帮助阐明schweinfurthin机制。药物化学研究旨在改善这些类似物，因此它们可以成为可行的候选药物。开发了针对3种候选schweinfurthins的LC-MS测定法，以支持小鼠头对头的PK研究。 GP78：我们使用了一个细胞测定法来筛选GP78功能和ERAD的抑制剂，涉及GP78对蛋白质的泛素化。将真菌提取物分离以分离活性原理以进一步表征。 CNS肿瘤筛查中的NF1：我们使用来自星形胶质细胞瘤的NF1 - /+; TRP53 - /+ NPCIS小鼠模型的细胞系进行了筛选，以找到CNS肿瘤活性的剂。双荧光素酶读数是一种新颖的格式，可扩展我们的筛选平台曲目。该项目处于HTS阶段。 P300：使用HIF-1的CAD域和p300的CH1域开发了生化屏幕。筛选天然产物提取物和预分级提取物库的筛选已完成。已经启动了主动提取物的确认，然后将从这些提取物中隔离和结构确定活性成分。