天然药物+临床样本+化合物库:三大找药物靶思路_MedChemExpress (MCE)

{"type":"doc","content":[{"type":"paragraph","attrs":{"id":"e93809d2-0b26-40ed-aedb-f90a25bf17f7","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"Section.01"}]},{"type":"paragraph","attrs":{"id":"0fb4a9d6-6647-448b-b7b0-b8c16a197901","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"思路 1：以药找靶"}]},{"type":"paragraph","attrs":{"id":"336ba484-057c-4d6e-88fd-f702ffcc1bc0","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"——天然药物的靶点确定"}]},{"type":"paragraph","attrs":{"id":"4b85489c-779d-4089-ba28-6d25d63f3b2c","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"以药找靶，即"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"已知药物具有活性，寻找药物作用靶点，"},{"type":"text","text":"其核心思路是利用已知的药物或活性分子作为探针，从复杂的生物体系中钓取其直接作用的蛋白质靶点。这种方法在阐明老药新用机制、探索天然产物作用靶点等研究领域中至关重要。"}]},{"type":"paragraph","attrs":{"id":"4d996f70-ab9b-4570-8f1e-7ec2fd749427","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"计算机预测"}]},{"type":"paragraph","attrs":{"id":"59f0cdf3-cbc8-478d-9e6d-d69915c0f7e1","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"计算机预测通常利用数据挖掘、药效团模型匹配、正反分子对接技术和药物-靶标相似性算法等，识别小分子可能作用的靶点。下面是一些常用的靶标预测网站，有需可参考喔~"}]},{"type":"paragraph","attrs":{"id":"2b17df93-ae1e-4d2d-9286-b24d82b634d7","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"表 1. 常用的靶标预测网站。"}]},{"type":"image","attrs":{"id":"d2945925-186b-495e-adc4-7380fd5678a9","src":"https://developer.qcloudimg.com/http-save/audit-10281355/f932f7756944f763fd9b405075b8b888.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"234e4708-6a55-4488-a8e8-51bb9636a07d","src":"https://developer.qcloudimg.com/http-save/audit-10281355/3b9da8f845015b6050c63ee1630a8f1f.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"d0dae8b1-79d8-44a1-94a9-819fd09f90a0","src":"https://developer.qcloudimg.com/http-save/audit-10281355/c4822498538165300400d2c2549d959b.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":true}},{"type":"image","attrs":{"id":"a86b87cd-4f77-464a-8e35-969d992ac66d","src":"https://developer.qcloudimg.com/http-save/audit-10281355/31e37f8418301fcb86d7b9ba01d55b0d.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"af0f8cd8-9119-4d7d-9015-7a28dc2a6ec8","src":"https://developer.qcloudimg.com/http-save/audit-10281355/355b2867c946a2ab79a0fa876398f509.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"234e4708-6a55-4488-a8e8-51bb9636a07d","src":"https://developer.qcloudimg.com/http-save/audit-10281355/3b9da8f845015b6050c63ee1630a8f1f.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"d0dae8b1-79d8-44a1-94a9-819fd09f90a0","src":"https://developer.qcloudimg.com/http-save/audit-10281355/c4822498538165300400d2c2549d959b.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"bab93fcf-c24d-48fd-86cd-70d15ad0b024","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"分子对接 药-靶相似性"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"5c31cb3e-c10c-440c-af98-819cc1d597cd","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"反向分子对接"},{"type":"text","text":" (Reverse Docking)：将小分子药物对一个包含数千甚至数万个蛋白质结构的数据库进行全局“对接”，根据计算出的结合亲和力排序，预测最可能的结合靶点。"}]},{"type":"paragraph","attrs":{"id":"f2fbf1b9-6abe-4596-93de-1ed47d4a01fd","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"• 早期的方法主要依靠结构相似性匹配，而最新的进展是利用如 HelixFold3 或 AlphaFold3 等结构预测模型，在统一的框架内同时模拟蛋白折叠和小分子对接，显著提升了预测的准确性。"}]},{"type":"paragraph","attrs":{"id":"3a3c4eaf-1d67-4772-9d64-3f0f0e6638aa","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"例如，研究者通过设置对接及相似性比较两条平行路径共同实现 "},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"italic"}],"text":"B. pinnatum"},{"type":"text","text":" 提取物化合物 1 (槲皮素糖苷) 的潜在靶点确定[1]。"}]},{"type":"paragraph","attrs":{"id":"bb506da5-6457-4db8-82d7-625316dd051b","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"在对接路径上，研究者通过 PDB 数据库构建了一个超过 9K 个蛋白的靶点配体数据库，使用 MarvinSketch 构建槲皮素糖苷的三维结构。为简化筛选流程，首先使用槲皮素糖苷的母核槲皮素与超过 9000 个蛋白靶点的配体数据库进行粗筛，以快速得到能足够容纳槲皮素糖苷口的潜在蛋白。随后选取粗筛 Top50 的潜在作用靶点与槲皮素糖苷进行逐一对接筛选，发现其中 TOP5 的靶点中有两个与抗炎抗氧化相关的关键 PDE 家族成员 PDE10A2 和 PDE4B，其中 PDE4B 抑制剂因其抗炎作用而广为人知。"}]},{"type":"paragraph","attrs":{"id":"33a8e5a9-b173-4daa-90d4-7225b38a3341","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"另一种路径是基于 SwissSimilality 的配体相似性比较，通过将槲皮素糖苷与筛选药物和生物活性使用 2D 分子指纹、超位置和快速非超位置等六种不同的方法进行预测潜在的 3D 相似性分子，发现其与同为 PDE 家族成员的 PDE5A1 酶内的共结晶配体的相似度为 0.952。推测槲皮素糖苷与 PDE5A1 具有活性。"}]},{"type":"paragraph","attrs":{"id":"889f3165-7b91-41dc-809a-dd59789f037a","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"表 2. 计算机反向找靶排名前五的靶点信息[1]。"}]},{"type":"image","attrs":{"id":"de9f5f64-676b-40ef-96d6-9681f063e208","src":"https://developer.qcloudimg.com/http-save/audit-10281355/b73f364ea0d8bfba598386b48de3b9a0.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"f215c4d3-ebb8-40fc-8aaf-f20cf1eff3fc","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"实验找靶"}]},{"type":"paragraph","attrs":{"id":"04ba4a55-6c6c-42ec-9474-864366305c52","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"实验找靶常用的方法主要有 DARTS、SPR 和 Pulldown 技术等，二者结合使用往往能取得最佳效果。"}]},{"type":"paragraph","attrs":{"id":"b265f386-0540-4d5f-ad9a-f3d961a2aedb","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"Section.02"}]},{"type":"paragraph","attrs":{"id":"bb435ace-ac5a-4053-863a-c6ce372864ba","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"思路 2：基于疾病临床样本"}]},{"type":"paragraph","attrs":{"id":"fa1aa15b-ed46-4e73-925f-2b8bab5ff3b4","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"——BACE1 靶点的发现"}]},{"type":"paragraph","attrs":{"id":"b88cdc04-414f-492c-9d43-241013a7f176","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"道理很简单，对比疾病人群的临床样本与正常对照，筛选出疾病发生发展过程中表达水平或功能发生显著改变的关键生物分子，如蛋白质、差异基因等。这些生物分子可以作为后续药物研究的潜在疾病靶点。"}]},{"type":"paragraph","attrs":{"id":"e49b06cd-32bb-4290-803d-aef491b9f5ec","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"下面我们结合发表于 "},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"Science Translational Medicine，"},{"type":"text","text":"题为 "},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"},{"type":"underline"}],"text":"“A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis”"},{"type":"text","text":" 的文献，一起来感受下该方法在科研中的实际应用[2]。"}]},{"type":"paragraph","attrs":{"id":"d872a3de-3e4f-49eb-83b0-d4501cbd7be2","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"Step 1. 样本处理：疾病模型的建立"}]},{"type":"paragraph","attrs":{"id":"d53659d4-58a1-499d-8a07-45b4f8107b0b","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"临床样本并不是拿过来就能直接用的！"}]},{"type":"paragraph","attrs":{"id":"589d3444-a5e2-4e51-bab0-1e562304b090","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"拿癌症举例，通常是采集患者的原代瘤体样本，也就是从患者体内直接切除下来的新鲜组织，接着提取出肿瘤细胞，种植到免疫缺陷的小鼠体内，让肿瘤在小鼠体内繁衍生长。"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"——即：患者来源异种移植瘤 (PDX) 。"},{"type":"text","text":"然后再从小鼠体内的肿瘤中提取细胞，在实验室的器皿中培养，最终形成了用来做科研实验的稳定细胞系。该细胞系能够高度还原患者原本的肿瘤特征，是科研研究和测试新药的宝贵材料[3]"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"。"}]},{"type":"paragraph","attrs":{"id":"d4908966-9521-4449-afb8-60311296c262","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"该文献案例中，作者使用的源自原代 LUAD 患者细胞系 CRUK0748-XCL，就是来源于 TRACERx 研究中的患者来源异种移植瘤。"}]},{"type":"image","attrs":{"id":"824a7e0f-7af0-4a6a-aee4-712415e98394","src":"https://developer.qcloudimg.com/http-save/audit-10281355/cc1609ea76604775fcb235c134da8aae.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"042d8bd7-3349-47ad-8ce5-054ddb39fb01","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"图 1. 肺癌 TRACERx 研究中 PDX 模型构建和扩增的研究方案示意图[4]。"}]},{"type":"paragraph","attrs":{"id":"136e3583-22b8-4a9c-aee9-561649c32907","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"Step 2. CRISPR 全基因组筛选"}]},{"type":"paragraph","attrs":{"id":"5943ad74-85e4-4943-9fb4-fbb91d5df5bb","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"CRISPR 全基因组筛选"},{"type":"text","text":"是一种高通量功能基因组学技术，利用 CRISPR/Cas 系统 (通常为 CRISPR-Cas9) 的基因编辑功能实现基因敲除、敲入、激活和干扰。"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"将CRISPR系统应用于构建基因组文库——构建全基因组 CRISPR 敲除 "},{"type":"text","text":"(CRISPRko)"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"、干扰 "},{"type":"text","text":"(CRISPRi) "},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"和激活 "},{"type":"text","text":"(CRISPRa) "},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"文库——进行高通量筛选"},{"type":"text","text":"，是疾病 (尤其是肿瘤) 靶基因研究的主要策略，广泛应用于包括细胞系、小鼠和斑马鱼在内的多种模型中。"}]},{"type":"paragraph","attrs":{"id":"ca56bfd0-bf82-4b5a-b404-affbc4805f23","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"CRISPR 文库多使用混合文库形式进行高通量筛选。其操作流程主要包括"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"设计合成 sgRNA 文库、Cas9 细胞系的构建、表型选择、高通量测序、生物信息学分析、候选基因和脱靶效应的验证"},{"type":"text","text":"等 (图 3)[4]。"}]},{"type":"image","attrs":{"id":"4515292b-fb78-4609-92c9-f6a107697a67","src":"https://developer.qcloudimg.com/http-save/audit-10281355/d53b15392a6dbe899b3a823b357228c2.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"511c090b-0345-4e96-821d-be53f9d1d83e","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"图 2. CRISPR/Cas9 高通量筛选流程[4]。"}]},{"type":"paragraph","attrs":{"id":"686bfef8-e385-42db-8ff3-0027ec66d555","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"CRISPR/Cas9 技术可实现全基因组范围的高通量筛选，其针对每个基因设计 3~10 条 sgRNA，利用芯片一次合成数万条覆盖整个基因组的 sgRNA 库，经慢病毒包装后以低感染复数感染宿主细胞，在适当的筛选条件下测试筛选前后 sgRNA 的丰度变化，进而找出候选基因。"}]},{"type":"paragraph","attrs":{"id":"1728b4f6-a7dd-4f18-92ae-bdc8452ce517","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"『"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"sgRNA 文库的设计与合成"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"47c51265-6327-4e1f-a165-58b18e0f7ac9","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"该文献案例中，作者使用的是"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"underline"}],"text":"“人 Calabrese 全基因组 CRISPRa 文库”"},{"type":"text","text":"，筛选能够增强原位肺腺癌 (LUAD) 肿瘤脑转移的基因。"}]},{"type":"paragraph","attrs":{"id":"d843ba8d-208c-42c1-a069-91a85447432f","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"——肿瘤转移通常不是因为某个基因丢失了，而是因为"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"某些原本不该表达的基因被异常激活、高表达，从而赋予了癌细胞侵袭和跨越屏障的能力"},{"type":"text","text":"。"}]},{"type":"paragraph","attrs":{"id":"27d3dac1-ffee-4d65-bcd8-6439e299f889","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"• Calabrese 文库通过 CRISPRa 技术，强行上调人体 18,000 多个编码基因的表达。赋予癌细胞“新的超能力”： 比如，某个肺癌细胞平时不具备穿透血脑屏障的能力，但文库恰好激活了它体内的某个基质金属蛋白酶基因，该细胞就突然获得了“通关”能力。"}]},{"type":"paragraph","attrs":{"id":"7e4b7c08-9a5b-4c83-bc49-4b030cbe8f96","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"• CRISPRa 系统的运作包含两部分：sgRNA (“导航地图”) 和 dCas9-转录激活因子 (“执行机器”) ； Calabrese 文库只有针对全基因组的 sgRNA 序列 (当然，也可以抗性筛选标记如 Puromycin，常用于寻找潜在的“耐药基因”)。"}]},{"type":"paragraph","attrs":{"id":"688f335b-4ea4-4789-b1d6-4956e11ead31","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"『"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"Cas9 细胞系的构建"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"f64800fa-0da3-44ce-aaea-8ecf919f54b7","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"dCas9-VP64 融合蛋白，需要细胞系预表达。该文献案例中，作者将 CRUK0748-XCL 细胞系改造为 CRUK0748-XCL-GLD 细胞，使其表达催化失活的 dCas9 与 VP64 融合的蛋白 (用于激活基因转录) 、GFP 和荧光素酶 (用于体内追踪)。"}]},{"type":"paragraph","attrs":{"id":"cb921da0-1f58-4020-a920-99d53a808777","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"简单来说：使用了一种“没有剪切活性”的突变 Cas9 蛋白 (称为 dCas9) 。在 dCas9 上连了一些“转录激活因子” (如 VP64、p65、HSF1) 。当它被向导 RNA (sgRNA) 带到基因的启动子区域时，它不剪断 DNA，而是像一个放大器一样，疯狂招募细胞自身的转录机器，从而强烈提高目标基因的表达量。"}]},{"type":"paragraph","attrs":{"id":"3f93185f-4597-4e34-be5f-96d31e04677e","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"『"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"慢病毒载体的构建及感染"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"a94b286e-fe94-418f-9044-22d4abac3235","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"作者将 CRUK0748-XCL-GLD 细胞以 0.3 的 MOI 进行转染，并筛选至最终文库覆盖度为 500 倍。随后经开胸手术，将转染文库的细胞直接接种到 30 只 NSG 小鼠的肺部 (图 3) 。"}]},{"type":"image","attrs":{"id":"74139382-0559-4b8a-9376-54008ffdbab1","src":"https://developer.qcloudimg.com/http-save/audit-10281355/57b330cd328efd2da20e60f8fc0126af.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"2bf58e3e-c080-4c14-9022-64c447288d76","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"图 3. 体内 CRISPR 激活筛选示意图[5]。"}]},{"type":"paragraph","attrs":{"id":"bf2e9b73-7861-4094-bbc4-55e4db35821f","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"『"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"高通量测序及分析"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"1b7c6836-f912-4019-9e25-6ed273356931","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"通过生物发光成像追踪肿瘤负荷，终点对肺和脑组织进行 sgRNA 测序并比较其富集程度，结合 TCGA 与 DepMap 数据库筛选出 12 个候选基因，其中 BACE1 的 sgRNA 在大脑中富集超过 150 倍。"}]},{"type":"paragraph","attrs":{"id":"7f477cb5-965e-444a-abb8-ee7f9a98b37b","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"『"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"验证候选基因"},{"type":"text","text":"』"}]},{"type":"paragraph","attrs":{"id":"2176c58d-08e7-42c1-bf51-22180af53044","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"随后通过独立 sgRNA 验证、组织微阵列及临床样本分析发现，BACE1 表达增加会增强 LUAD 的骨髓转移 (图 4) 。此外，在 BACE1 过表达的肺肿瘤小鼠的肝脏和骨骼中，转移负荷均有所增加。"}]},{"type":"image","attrs":{"id":"84591005-1a75-4ba1-8f08-c25e67f703ea","src":"https://developer.qcloudimg.com/http-save/audit-10281355/46514fca937cd76ede8eb4c035c09f15.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"787870db-5fa1-4e03-a47d-d52b20ed7c30","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"图 4. 使用两种独立的 sgRNA 激活 BACE1 表达可增加脑转移肿瘤负荷[5]。"}]},{"type":"paragraph","attrs":{"id":"33545dfe-2e47-4221-b51a-ecbc4a260413","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"Section.03"}]},{"type":"paragraph","attrs":{"id":"e6716505-cd38-4989-ba91-d9cbf3519094","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"思路 3：老药新用"}]},{"type":"paragraph","attrs":{"id":"6323a207-81f7-4a56-8510-6de3dc28366b","textAlign":"center","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"——基于化合物库筛选"}]},{"type":"paragraph","attrs":{"id":"2e728f11-c18d-4a94-bf88-87cbfb55e217","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"【文献案例】"},{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"underline"}],"text":"Targeting ATR signaling in sarcoma with homologous recombination deficiency"}]},{"type":"paragraph","attrs":{"id":"912d2bed-3197-4739-9990-7af2cfaae487","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","marks":[{"type":"textStyle","attrs":{"color":"","background":""}},{"type":"bold"}],"text":"【使用文库】MCE 定制库，"},{"type":"text","text":"包含 71 种药物，涵盖用于探索性研究和临床批准的靶向药物，以及临床上用于肉瘤患者治疗的一线和二线化疗药物[6]。"}]},{"type":"paragraph","attrs":{"id":"416ca2be-873e-44b3-bb38-5b50d7b38b19","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"通过构建包含 71 种靶向及化疗药物的化合物库，在患者来源的肉瘤 3D 细胞模型中开展中通量药物筛选 (基于 AUC 与 IC50 综合评估) ，成功鉴定出以 ATR、CHK1、WEE1 抑制剂为代表的 DNA 损伤应答 (DDR) 通路关键活性化合物。进一步通过联合用药筛选发现 ATR 抑制剂 (如 Camonsertib) 与 PARP 抑制剂或化疗药物 (如 Doxorubicin) 具有显著协同效应。"}]},{"type":"paragraph","attrs":{"id":"9aad4639-0dd4-4348-b797-3b924327eb71","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"机制研究表明，这类化合物通过抑制 ATR-CHK1 信号轴，导致 DNA 复制叉减速、复制压力累积及 DNA 损伤增加，进而引发有丝分裂异常和细胞凋亡；同时，同源重组缺陷 (HRD) 高表达肿瘤中 ATR 信号通路本身显著上调，揭示了“药物筛选命中化合物—作用于关键靶点—靶点依赖驱动疾病”的完整作用逻辑链。"}]},{"type":"image","attrs":{"id":"8b198b17-d83f-4899-bd58-7625a462245b","src":"https://developer.qcloudimg.com/http-save/audit-10281355/663c892a88c833fca118da9ae59130de.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"e4a071e5-0e08-4f38-aca5-5f355a13d51f","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false}},{"type":"image","attrs":{"id":"216dfe1c-eacd-410a-b7a3-70c4c5f0739d","src":"https://developer.qcloudimg.com/http-save/audit-10281355/a0b05f84799fc8ac849ccd3867c0ed26.png","extension":"","align":"center","alt":"","showAlt":false,"href":"","boxShadow":"","width":766,"aspectRatio":0,"status":"success","showText":true,"isPercentage":false,"percentage":0,"isHoverDragHandle":false}},{"type":"paragraph","attrs":{"id":"622e5e1d-643d-45da-b676-5db1cb0456e1","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"参考文献"}]},{"type":"paragraph","attrs":{"id":"13814131-ae10-45f7-b7ed-9361f931fbea","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[1] Lourenço E M G, et al. Identification of a selective PDE4B inhibitor from Bryophyllum pinnatum by target fishing study and in vitro evaluation of quercetin 3-O-α-L-arabinopyranosyl-(1→ 2)-O-α-L-rhamnopyranoside[J]. Frontiers in pharmacology, 2020, 10: 1582."}]},{"type":"paragraph","attrs":{"id":"dab680f8-56ce-4e3e-84b8-3a13a7b1921c","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[2] Chafe S C, et al. A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis[J]. Science translational medicine, 2025, 17(805): eadu2459."}]},{"type":"paragraph","attrs":{"id":"9a423f85-d7c5-4740-875b-2513e9d35a26","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[3] Olson B, et al. Mouse Models for Cancer Immunotherapy Research. Cancer Discov. 2018 Nov;8(11):1358-1365."}]},{"type":"paragraph","attrs":{"id":"97215bb6-a1a5-4060-b2f2-5ba923c23c12","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[4] Hynds RE, et al. Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models. Nat Commun. 2024 May 31;15(1):4653."}]},{"type":"paragraph","attrs":{"id":"542d5f9a-7ebc-4bd3-905f-3ed7781d220d","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[5] Lei T, et al. [Development and applications of CRISPR/Cas9 library screening technology in cancer research]. Nan Fang Yi Ke Da Xue Xue Bao. 2019 Nov 30;39(11):1381-1386. Chinese. "}]},{"type":"paragraph","attrs":{"id":"889f2f1e-1c8b-4d69-854a-c9c50b9c490b","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false},"content":[{"type":"text","text":"[6] Planas-Paz L, et al. Targeting ATR signaling in sarcoma with homologous recombination deficiency. Cancer Lett. 2026 Apr 1;642:218300. "}]},{"type":"paragraph","attrs":{"id":"87dd379c-08bd-4472-9392-0d96c15243bc","textAlign":"inherit","indent":0,"color":null,"background":null,"isHoverDragHandle":false}}]}","createTime":1783324231,"ext":{"closeTextLink":0,"comment_ban":0,"description":"","focusRead":0},"favNum":0,"html":"","isOriginal":0,"likeNum":0,