作者: Zhou, Guizhen ; Zheng, Mingyue ; Li, Yupeng ; Huo, Ruifeng ; Gu, Yuejiao ; Zhou, Chuan ; Ge, Zhiming ; Fan, Zisheng ; Xu, Tianfeng ; He, Wei ; Tao, Zhaofan ; Wang, Dan ; Gao, Mingshan ; Zhang, Sulin ; Cui, Rongrong

KRAS^G12D, the most frequent KRAS oncogenic mutation, is a promising target for cancer therapy. Herein, we report the design, synthesis, and biological evaluation of a series of KRAS^G12D PROTACs by connecting the analogues of MRTX1133 and the VHL ligand. Structural modifications of the linker moiety and KRAS inhibitor part suggested a critical role of membrane permeability in the degradation activity of the KRAS^G12D PROTACs. Mechanism studies with the representative compound 8o demonstrated that the potent, rapid, and selective degradation of KRAS^G12D induced by 8o was via a VHL- and proteasome-dependent manner. This compound selectively and potently suppressed the growth of multiple KRAS^G12D mutant cancer cells, displayed favorable pharmacokinetic and pharmacodynamic properties in mice, and showed significant antitumor efficacy in the AsPC-1 xenograft mouse model. Further optimization of 8o appears to be promising for the development of a new chemotherapy for KRAS^G12D-driven cancers as the complementary therapeutic strategy to KRAS inhibition.

2023-12-01·Journal of Proteome Research

Metabolomic, Proteomic, and Single-Cell Proteomic Analysis of Cancer Cells Treated with the KRAS^G12D Inhibitor MRTX1133

Article

作者: Orsburn, Benjamin C

Mutations in KRAS are common drivers of human cancers and are often those with the poorest overall prognosis for patients. A recently developed compound, MRTX1133, has shown promise in inhibiting the activity of KRAS^G12D mutant proteins, which is one of the main drivers of pancreatic cancer. To better understand the mechanism of action of this compound, I performed both proteomics and metabolomics on four KRAS^G12D mutant pancreatic cancer cell lines. To obtain increased granularity in the proteomic observations, single-cell proteomics was successfully performed on two of these lines. Following quality filtering, a total of 1498 single cells were analyzed. From these cells, 3140 total proteins were identified with approximately 953 proteins quantified per cell. At 48 h of treatment, two distinct populations of cells can be observed based on the level of effectiveness of the drug in decreasing the total abundance of the KRAS protein in each respective cell, with results that are effectively masked in the bulk cell analysis. All mass spectrometry data and processed results are publicly available at www.massive.ucsd.edu at accessions PXD039597, PXD039601, and PXD039600.

2023-11-23·Journal of medicinal chemistry1区 · 医学

Design, Synthesis, and Pharmacological Evaluation of Multisubstituted Pyrido[4,3-d]pyrimidine Analogues Bearing Deuterated Methylene Linkers as Potent KRAS^G12D Inhibitors

1区 · 医学

Article

作者: Liu, Xiaogu ; Feng, Juanjuan ; Xiao, Xuanzheng ; Zhang, Ao ; Xia, Xinting ; Zhang, Jian ; Ding, Chunyong ; Ma, Jing ; Pang, Xiufeng

The breakthrough in drug development of KRAS^G12C inhibitors provides inspiration for targeting alternative KRAS mutations, especially the most prevalent KRAS^G12D variant. Based on the structural analysis of MRTX1133 in complex with KRAS^G12D, a comprehensive structure-activity study was conducted, which led to the discovery of several compounds (22, 28, and 31) that showed higher potency in suppressing the clonogenic growth of KRAS^G12D-dependent cancer cells. These new compounds markedly and selectively inhibited the binding of RBD peptide to GTP-bound KRAS^G12D with IC₅₀ values between 0.48 and 1.21 nM. These new inhibitors were found to have dose-dependent anti-tumor efficacy in the AsPC-1 xenograft mouse models with a tumor growth inhibition of approximately 70% at a dose of 20 mg/kg twice daily (i.p.). Despite the non-optimal pharmacokinetic properties similar to those of MRTX1133, the high in vitro and in vivo potency of these new inhibitors call for further profiling.

113

项与 MRTX-1133 相关的新闻（医药）

2024-04-12

·CPHI制药在线

KRAS G12D打破“不可成药”魔咒，多款进入临床，国内外药企积极布局

关注并星标CPHI制药在线----扫码进入天然提取物线上主题展展厅----RAS是肿瘤中突变最为广泛的癌基因，占所有肿瘤突变的20%~30%。RAS家族包括KRAS、HRAS和NRAS三种亚型，其中KRAS基因突变是最常见的RAS突变亚型，约占RAS基因家族突变的85%，且比RAS家族其他基因突变更易导致肿瘤的发生。关于KRAS及其相关突变 KRAS基因编码的KRAS蛋白是一种小型膜结合GTP酶（GTP水解酶），当KRAS蛋白与GTP结合呈激活状态，与GDP结合呈关闭状态。活化后的KRAS蛋白可以激活多条下游信号通路，如MAPK信号通路、PI3K信号通路和Ral-GEFs信号通路。这些信号通路在促进细胞生存、增殖和细胞因子释放方面具有重要作用。 KRAS在失活与激活状态之间的转换受鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAPs）的调控，其中GEF催化KRAS与GTP的结合，从而促进KRAS的激活，其中包括SOS蛋白（属GEFs/鸟苷释放因子/鸟苷酸交换因子)。GAPs能够促进与KRAS结合的GTP水解成为GDP终止活性状态，从而抑制KRAS的活性。在人类癌症中，KRAS基因突变出现在接近90%的胰腺癌、30-40%的结肠癌、17%的子宫内膜癌和15-20%的肺癌（大多为NSCLC）中。此外，KRAS基因突变还会出现在胆管癌、宫颈癌、膀胱癌、肝癌和乳腺癌等癌症中。研究发现，KRAS突变位置主要是4个热点密码子（12、13、61和146）。密码子12是所有4个热点密码子中突变频率最高的，其中G12D突变最常见，其次是G12V、G12C等。根据不同的癌症类型，G12D突变占KRAS突变癌症的20-50%，包括50%的壶腹癌、48%的阑尾腺癌和44%的胆管癌。而且，与其他KRAS突变相比，G12D突变肿瘤细胞上PD-L1表达减少，细胞毒性CD8+ T细胞浸润肿瘤减少，这也导致G12D突变肿瘤对抗PD1治疗的耐药性增加。 KRAS G12D抑制剂进展 KRAS G12D突变虽然更常见，但目前全球还没有针对KRAS G12D突变的靶向药获批上市，反而有两款针对KRAS G12C突变的靶向药Krazati（adagrasib）和Lumakras（sotorasib）已获批上市，这两款药物均被批准用于先前至少接受过一种全身性疗法、携带KRAS G12C突变的NSCLC。由于突变型KRAS对GTP有较高的亲和力，又存在催化位点小、蛋白表面光滑等难以靶向的因素，KRAS长期以来被认为是“不可成药”靶点。不过凭借KRAS G12C突变新引入的半胱氨酸易形成共价键的特性，科学家设计出了能够不可逆地靶向结合KRAS 12号密码子上半胱氨酸残基的共价小分子抑制剂。与KRAS G12C相比，KRAS G12D是同一个密码子上氨基酸突变种类不同。KRAS G12D是KRAS 12号密码子突变引入天冬氨酸，而不是半胱氨酸。这导致其流行病学上的差异：KRAS G12C主要集中在肺癌领域，KRAS G12D主要集中在胰腺癌领域。而这也进一步导致KRAS G12C抑制剂无法直接用于治疗KRAS G12D突变肿瘤，毕竟天冬氨酸无法与KRAS G12C抑制剂形成共价结合。借鉴KRAS G12C抑制剂的开发策略，目前全球药企已研发出多款KRAS G12D抑制剂，详见下表。这些在研KRAS G12D抑制剂大多处于早期临床，最快处于1/2期临床。而且，在研KRAS G12D抑制剂药物类型除了小分子化药，还涉及降解剂，这也导致其给药方式有所不同，包括静脉给药、口服给药。资料来源：公开资料具体品种上，MRTX1133进展最快，处于1/2期临床。该药是一款选择性非共价KRAS G12D抑制剂，能够与处于失活状态和激活状态的KRAS G12D突变体结合，并表现出对KRAS G12D突变体的特异性。在针对多种癌症类型的临床前动物研究中，MRTX1133显著降低肿瘤大小，尤其是在胰腺癌肿瘤模型中，MRTX1133在8/11（73%）的胰腺癌模型中观察到应答。而且，MRTX1133联合西妥昔单抗相比于单药能够抑制肿瘤细胞pERK和pS6的表达，显著提高抗肿瘤效应。 ASP-3082是安斯泰来开发的一款高选择性新型KRAS G12D降解剂，临床前数据显示其能够实现KRAS G12D蛋白的有效降解，对细胞外信号调节激酶磷酸化及其下游基因具有显著的抑制作用，并有效诱导半胱天冬酶-3的切割。而且，ASP-3082通过静脉给药能对KRAS G12D突变肿瘤显示生长抑制作用，并在多种异种移植小鼠模型中得到证实。 RMC-9805是KRAS G12D的GTP结合型和活性RAS(ON)形式的首个口服突变选择性共价抑制剂。在体外和体内的肿瘤模型中，RMC-9805处理引起KRASG12D选择性和持续性修饰，导致RAS通路活性受到深度和持久的抑制，细胞增殖受到抑制，并诱导细胞凋亡。我国药企也积极布局KRAS G12D抑制剂，如恒瑞医药、齐鲁制药等。恒瑞医药的HRS-4642进展较快，已进入1期临床。该药是一种新型、长效、选择性KRAS G12D抑制剂，能够特异性结合KRAS G12D，进而抑制下游MEK、ERK蛋白的磷酸化，发挥抗肿瘤作用。HRS-4642采用脂质体包裹制成，具有靶向给药、可控且持续进行药物释放等优势。 2023年ESMO上公布的HRS-4642治疗KRAS G12D突变晚期实体瘤的1期临床研究数据显示：截止2023年8月4日，共入组18例患者（肺腺癌n=10，结直肠腺癌n=5，阑尾粘液腺癌、卵巢癌和胰腺癌各1例），这些患者接受既往治疗中位数为3线（范围为2-7）。其中13名患者至少进行了一次基线评估，11名患者（61.1%）处于疾病稳定状态，6名（33.3%）出现了靶病变缩小，包括肺癌和结直肠癌。HRS-4642的半衰期约为40小时。安全性方面，研究中未观察到任何剂量限制性毒性（DLTs），且最大耐受剂量（MTD）尚未确定。9例患者（50.0%）出现了≥3级的不良事件（AEs）。6例患者（33.3%）出现了≥3级的与治疗相关不良事件（TRAEs），包括高胆固醇血症（16.7%）、脂肪酶增高（11.1%）和贫血（11.1%）。而且，AE的发生率没有观察到剂量依赖性趋势。祐森健恒也布局了KRAS G12D抑制剂。2023年11月，祐森健恒就其KRAS G12D抑制剂UA022项目与阿斯利康达成全球独家授权协议。据协议，祐森健恒将有资格获得2400万美元的首付款，最高达3.95亿美元的潜在开发和商业化里程碑付款，以及净销售额的分级特许权使用费。此外，今年2月围绕KRAS抑制剂也达成了一项交易，Jazz Pharmaceuticals斥资8.8亿美元收购Redx Pharma的KRAS抑制剂研发项目，而其KRAS G12D项目处于临床前研究。总结目前来看，KRASG12D抑制剂进展较慢，但已取得不错进展，已从“不可成药”实现“成药”。但新药研发失败风险非常高，能否通过临床试验、监管批准顺利上市，KRAS G12D抑制剂还有很长的路要走。我国药企在KRAS G12D抑制剂领域也积极布局，虽较国外慢些，但也有实现弯道超车的可能。期待KRAS G12D抑制剂领域早日实现重大突破。【智药研习社近期直播预告】来源：CPHI制药在线声明：本文仅代表作者观点，并不代表制药在线立场。本网站内容仅出于传递更多信息之目的。如需转载，请务必注明文章来源和作者。投稿邮箱：Kelly.Xiao@imsinoexpo.com▼更多制药资讯，请关注CPHI制药在线▼点击阅读原文，进入智药研习社~

突破性疗法上市批准申请上市

2024-04-05

·BioSpace

Novocure Announces 20 Presentations On Tumor Treating Fields, Highlighting Preclinical Effects in Pancreatic Cancer and Immune Effects, at American Association for Cancer Research (AACR) Annual Meeting 2024

ROOT, Switzerland--(BUSINESS WIRE)-- Novocure (NASDAQ: NVCR) today announced 20 presentations on Tumor Treating Fields (TTFields) therapy will be delivered at the American Association for Cancer Research (AACR) Annual Meeting 2024, to be held April 5 to 10 in San Diego. The presentations, describing preclinical investigations, include new insights on how TTFields therapy can potentially enhance the immune system’s ability to combat cancer cells and on TTFields therapy’s effects and mechanisms in pancreatic cancer. “The research we and our collaborators are presenting at the AACR Annual Meeting underscores the potential of Tumor Treating Fields therapy in treating various solid tumor types,” said Moshe Giladi, Ph.D., Novocure’s Chief Science Officer. “We continually explore the science of TTFields therapy to improve our product and make a difference in the lives of patients with cancer, and we are eager to discuss some of our latest insights with leading cancer investigators.” Highlights include: A preclinical study in a murine model of pancreatic cancer showing that TTFields applied concurrently with standard chemotherapy led to significantly enhanced therapeutic effects. When used alongside gemcitabine and nab-paclitaxel (Gem/NabP), TTFields significantly reduced tumor growth compared to the control group, and a complete regression of tumors was observed in a number of animals. This is a treatment regimen similar to the PANOVA-3 clinical trial examining the efficacy of TTFields in patients with locally advanced pancreatic cancer. A preclinical study demonstrating a novel immunomodulatory role for TTFields by promoting in vitro pro-inflammatory polarization of macrophages. Macrophages exposed to TTFields showed an increase in markers and chemicals that indicate a shift towards a pro-inflammatory, tumor-fighting mode. The study suggests TTFields can potentially modulate macrophages to better attack cancer cells by pushing them toward a state more hostile to tumors. An in vitro study suggesting concomitant application of TTFields and PARP inhibitors in BRCA wild type pancreatic cancer cells leads to improvement over monotherapy. Applied individually, both PARP inhibitors and TTFields killed cancer cells, stopped them from multiplying, and triggered apoptosis. These effects were amplified when TTFields and PARP inhibitors were applied together. These findings contribute to existing data suggesting that TTFields induce a state of BRCAness in cancer cells. Presentations from Novocure-sponsored and partner programs include: Tumor Treating Fields (TTFields) targeted self assembling nanoparticles for pancreatic cancer treatment: In vitro and in vivo assessment. Presenter: P.P. Desai. 1:30 p.m. PDT on Sunday, April 7. Tumor Treating Fields induce the integrated stress response, alter the transcriptional signatures of cellular metabolism, and modulate immune-related cytokines dependent and independent of p53. Presenter: P.R. Srinivasan. 1:30 p.m. PDT on Sunday, April 7. Tumor Treating Fields (TTFields) disrupt cancer cell invasion by impacting cell-ECM traction forces. Presenter: S.M. Short. 9 a.m. PDT on Monday, April 8. N-cadherin-mediated activation of PI3K/Akt pathway following application of Tumor Treating Fields (TTFields). Presenter: T. Voloshin. 9 a.m. PDT on Monday, April 8. Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure. Presenter: M. Mazzanti. 9 a.m. PDT on Monday, April 8. Co-treatment with KRAS G12D inhibitor MRTX1133 plus TTFields against human pancreatic and Colorectal cancer cell lines results in synergistic up-regulation of cleaved PARP in KRAS G12D & unexpectedly in KRAS G12V as well. Presenter: V. Tajiknia. 9 a.m. PDT on Monday, April 8. Tumor Treating Fields (TTFields) for spinal metastasis: Clinical trial concept for use of conductive implants as waveguides to enhance TTFields strength. Presenter: C.E. Tatsui. 9 a.m. PDT on Monday, April 8. Tumor Treating Fields (TTFields) increase cancer cell membrane permeability and improve sensitivity to doxorubicin in vitro and in vivo. Presenter: T. Voloshin. 9 a.m. PDT on Tuesday, April 9. Development of paclitaxel-loaded nanoparticles with high charge density. Presenter: K.Y. Bang. 1:30 p.m. PDT on Tuesday, April 9. Tumor Treating Fields alter PDGFR-β localization in immortalized human pericytes in an in vitro model of the blood-brain barrier. Presenter: C. Hagemann. 1:30 p.m. PDT on Tuesday, April 9. Designing arrays for delivering Tumor Treating Fields (TTFields) to the mouse head. Presenter: I Tzchori. 1:30 p.m. PDT on Tuesday, April 9. Tumor Treating Fields (TTFields) induce an anti-tumor immune response in a pancreatic cancer mouse model. Presenter: T. Voloshin. 9 a.m. PDT on Wednesday, April 10. Macrophage pro-inflammatory phenotype skewing by the application of Tumor Treating Fields (TTFields). Presenter: T. Voloshin. 9 a.m. PDT on Wednesday, April 10. Quantitative proteomics reveal the mechanism of TTFields therapy for glioblastoma. Presenter: Q. Mei. 9 a.m. PDT on Wednesday, April 10. Treatment of pancreatic cancer cells with Tumor Treating Fields (TTFields) and PARP inhibitors. Presenter: E. Dor-On. 9 a.m. PDT on Wednesday, April 10. Preclinical effects of Tumor Treating Fields (TTFields) with PARP inhibitors in ovarian cancer. Presenter: E. Dor-On. 9 a.m. PDT on Wednesday, April 10. Preclinical investigations of concomitant Tumor Treating Fields (TTFields) with cisplatin for treatment of cervical cancer. Presenter: I. Tzschori. 9 a.m. PDT on Wednesday, April 10. Tumor Treating Fields (TTFields) can induce immunogenic cell death in GBM resulting in enhanced immune modulation. Presenter: R.T. Nitta. 9 a.m. PDT on Wednesday, April 10. Tumor Treating Fields (TTFields) show efficacy in Triple-Negative breast cancer (TNBC) cells alone and in combination with PARP inhibitor Talazoparib. Presenter: M. Ghandali. 9 a.m. PDT on Wednesday, April 10. TTFields and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines. Presenter: V. Tajiknia. 9 a.m. PDT on Wednesday, April 10. ABOUT TUMOR TREATING FIELDS THERAPY Tumor Treating Fields (TTFields) are electric fields that exert physical forces to kill cancer cells via a variety of mechanisms. TTFields do not significantly affect healthy cells because they have different properties (including division rate, morphology, and electrical properties) than cancer cells. The multiple, distinct mechanisms of TTFields therapy work together to selectively target and kill cancer cells. Due to its multimechanistic actions, TTFields therapy can be added to cancer treatment modalities in approved indications and demonstrates enhanced effects across solid tumor types when used with chemotherapy, radiotherapy, immune checkpoint inhibition, or PARP inhibition in preclinical models. TTFields therapy provides clinical versatility that has the potential to help address treatment challenges across a range of solid tumors. To learn more about Tumor Treating Fields therapy and its multifaceted effect on cancer cells, visit tumortreatingfields.com. ABOUT NOVOCURE Novocure is a global oncology company working to extend survival in some of the most aggressive forms of cancer through the development and commercialization of its innovative therapy, Tumor Treating Fields. Novocure’s commercialized products are approved in certain countries for the treatment of adult patients with glioblastoma, malignant pleural mesothelioma and pleural mesothelioma. Novocure has ongoing or completed clinical studies investigating Tumor Treating Fields in brain metastases, gastric cancer, glioblastoma, liver cancer, non-small cell lung cancer, pancreatic cancer and ovarian cancer. Headquartered in Root, Switzerland and with a growing global footprint, Novocure has regional operating centers in Portsmouth, New Hampshire and Tokyo, as well as a research center in Haifa, Israel. For additional information about the company, please visit Novocure.com and follow @Novocure on LinkedIn and Twitter. FORWARD-LOOKING STATEMENTS In addition to historical facts or statements of current condition, this press release may contain forward-looking statements. Forward-looking statements provide Novocure’s current expectations or forecasts of future events. These may include statements regarding anticipated scientific progress on its research programs, clinical study progress, development of potential products, interpretation of clinical results, prospects for regulatory approval, manufacturing development and capabilities, market prospects for its products, coverage, collections from third-party payers and other statements regarding matters that are not historical facts. You may identify some of these forward-looking statements by the use of words in the statements such as “could” “anticipate,” “estimate,” “expect,” “project,” “intend,” “plan,” “believe” or other words and terms of similar meaning. Novocure’s performance and financial results could differ materially from those reflected in these forward-looking statements due to general financial, economic, environmental, regulatory and political conditions and other more specific risks and uncertainties facing Novocure such as those set forth in its Annual Report on Form 10-K filed on February 22, 2024, and subsequent filings with the U.S. Securities and Exchange Commission. Given these risks and uncertainties, any or all of these forward-looking statements may prove to be incorrect. Therefore, you should not rely on any such factors or forward-looking statements. Furthermore, Novocure does not intend to update publicly any forward-looking statement, except as required by law. Any forward-looking statements herein speak only as of the date hereof. The Private Securities Litigation Reform Act of 1995 permits this discussion. View source version on businesswire.com: Contacts INVESTORS & MEDIA: Ingrid Goldberg investorinfo@novocure.com media@novocure.com 610-723-7427 Source: Novocure View this news release online at:

免疫疗法

2024-03-18

·精准药物

【NCB】KRAS-G12D的选择性共价抑制剂设计

KRAS是人体内最常见的致癌突变基因，每年造成全球超过300万的新增患者负担。FDA近期批准的药物sotorasib与adagrasib作为突破性疗法，通过共价结合KRAS-G12C突变蛋白的12位半胱氨酸，将其锁定在信号失能状态，实现抑制肿瘤增殖。G12D突变是人类肿瘤中最常见的KRAS突变，尤其在胰腺癌中普遍存在。然而，由于缺乏靶向天冬氨酸的共价弹头，靶向KRAS-G12D的共价抑制剂开发仍然面临挑战。近日，加州大学旧金山分校的研究人员发现了一类基于β-丙内酯的共价亲电弹头，与MRTX1133的化学骨架结合后，能够与KRAS-G12D形成稳定的共价络合物，并展现出对KRAS-G12D驱动的癌细胞增殖和小鼠异种移植肿瘤生长的高度选择性抑制效果。由于羧基侧链的低亲核性，天冬氨酸是公认最难以亲核反应进行共价修饰的氨基酸之一。KRAS-G12D共价抑制剂开发的关键挑战在于设计一种能够在生理pH条件下与天冬氨酸羧基侧链高效结合的共价弹头，同时保证该弹头在水溶液中的稳定性，且不会与谷胱甘肽（GSH）等具有高亲核性的内源物质发生反应。为了寻找合适的共价弹头，研究人员探索了一系列三元和四元杂环共价基团，这些基团是反应活性很强的应变释放亲电试剂（strain-release electrophiles），已有报道能够与羧基发生烷基化或酰基化反应。共价修饰速率实验结果显示，基于β-丙内酯的四元杂环弹头可在多个位点（包括Asp12）以剂量依赖的方式高效共价修饰KRAS-G12D蛋白。而氮丙环和环氧乙烷类弹头则展现了中等程度的KRAS-G12D共价交联效率（40%-60%）。在成功发现靶向Asp12的潜在共价弹头后，研究团队将此弹头与MRTX1133的化学骨架结合，得到了外消旋体化合物G12Di-1。全蛋白质组质谱分析显示，G12Di-1能够与KRAS-G12D·GDP形式的蛋白发生快速的共价偶联反应（半衰期=99s），而对KRAS野生型、KRAS-G12S、KRAS-G13D以及另一种酸性氨基酸突变蛋白KRAS-G12E均未显示出共价反应性。值得注意的是，G12Di-1对GTP结合状态下的KRAS-G12D蛋白同样展现出一定的共价反应性，这一发现与先前报道的KRAS突变体抑制剂存在显著的区别。鉴于上述发现，研究人员进一步探究了这种共价修饰是否会影响KRAS在活性状态下与效应蛋白（如Raf）的相互作用。TR-FRET分析结果表明，G12Di-1能够以剂量依赖地抑制Raf-Ras结合域（Raf-RBD）和KRAS-G12D之间的相互作用，而对KRAS野生型与Raf的结合无显著影响。此外，生物膜干涉实验进一步证实，与KRAS-G12D·GppNHp相比，KRAS-G12D·GppNHp·G12Di-1复合物与Raf-RBD的结合能力显著降低。这样的共价修饰特性对G12D突变体可能尤为有利，因为该突变型的GTP水解酶活性受到严重影响，导致大多数KRAS-G12D分子持续处于GTP结合的激活状态。为了深入探究G12Di-1与KRAS蛋白的共价相互作用机制，研究团队对KRAS-G12D·GDP与G12Di-1的复合物晶体结构进行了解析。结果显示，G12Di-1的分子结合构象与MRTX1133及AMG510类似，其中G12Di-1的β-丙内酯环打开，通过其β-碳原子与Asp12发生烷基化反应，从而实现共价交联。有趣的是，绝大多数G12Di-1分子以S型构象与KRAS-G12D结合，这引导研究者们推测，这一现象很有可能来源于R型构象的β-丙内酯弹头在结合Asp12时发生的SN2构型翻转。因此，研究团队通过手性拆分G12Di-1，获得了(R)-G12Di-2和(S)-G12Di-2两种对映异构体，并对它们与KRAS-G12D的反应性进行了评估。实验结果表明，(R)-G12Di-2对KRAS-G12D·GDP的共价反应性明显更强，这进一步证实了研究人员的假设。尽管(R)-G12Di-2作为KRAS-G12D的有效共价配体显示出良好的活性，其β-丙内酯共价弹头的高反应性却导致其在生理条件下的水溶液中稳定性较低，这限制了其在细胞实验中的应用。为了克服这一挑战，研究人员在β-丙内酯环的α碳原子上引入取代基，以降低水解反应发生的可能。结果显示，通过异丙基取代得到的G12Di-4，虽然它的共价反应速率在空间位阻的影响下有所下降，但其在PBS溶液中的稳定性显著提高。为了进一步提高G12Di-4对KRAS-G12D的亲和力，研究小组继续对其结构进行优化，通过整合MRTX1133的完整化学结构，得到了G12Di-6。该分子在亲和力和共价反应速率方面均获得了显著提升，更值得注意的是，G12Di-6是第一个展示出对KRAS蛋白在GTP结合状态下选择性优于GDP结合状态的共价分子，这一特性与MRTX1133存在显著的区别。细胞水平的Western Blot实验也进一步验证了G12Di-6能够在细胞内高效共价结合KRAS-G12D蛋白，并显著抑制下游ERK的磷酸化水平。基于共价标记动力学的构效关系研究表明，左侧萘环3号位的羟基取代是引起G12Di-6对GTP结合状态下的KRAS-G12D高选择性的关键因素，这与该羟基和KRAS蛋白的Asp69侧链形成的氢键密切相关。然而，G12Di-6在抑制多种癌细胞系增殖的实验测试中，并未表现出显著的KRAS突变特异性。经过研究发现，由于异丙基单取代的β-丙内酯结构在真核生物的蛋白酶体抑制剂中较为常见（如belactosin C），G12Di-6因与癌细胞内蛋白酶体的高效结合而发生脱靶效应。为了解决这一问题，研究团队在丙内酯结构的β-碳原子上再次引入了甲基取代，从而得到了新的化合物G12Di-7。这一改进不仅有效阻止了G12Di-6与蛋白酶体的脱靶结合，而且进一步提高了化合物的稳定性。更为重要的是，G12Di-7不仅展现了与GDP及GTP结合状态下的KRAS-G12D蛋白的高效共价结合能力，还表现出对KRAS-G12D突变型细胞的高度选择性。此外，G12Di-7在KRAS-G12D基因转化的小鼠BA/F3癌细胞模型中同样表现出显著的浓度依赖性抑制效果。无论是在传统的2D单层细胞培养，还是更为复杂的3D细胞培养模型中，G12Di-7均展现出了对KRAS-G12D突变驱动的癌细胞株（SW1990、AsPC-1和AGS）生长的显著抑制作用。而对非KRAS-G12D突变驱动的癌细胞株则均无明显影响，且在10μM浓度下未表现出明显的毒性，强调了其高选择性和安全性。接着，研究人员通过在小鼠体内模型中评估G12Di-7对KRAS-G12D转化细胞系衍生的异种移植瘤组织的抗肿瘤活性，进一步评估G12Di-7的体内有效性。结果显示，G12Di-7通过静脉滴注给药的方式，剂量依赖性地抑制了SW1990异种移植瘤的生长。与此同时，在治疗期间两组小鼠的体重增加情况相似，表明了G12Di-7治疗方案未引起明显的分子系统性毒性。综上，通过将改良后的β-丙内酯衍生物共价弹头与MRTX1133的化学骨架结合，研究人员成功设计了首个针对KRAS-G12D的共价抑制剂。这类分子能够有效地共价修饰GDP和GTP结合状态下KRAS-G12D的12位突变天冬氨酸，且具有出色的KRAS-G12D突变型细胞选择性和体内肿瘤抑制效果，为KRAS-G12D突变驱动的癌症治疗提供了一种新的策略。参考文献Zheng Q, Zhang Z, Guiley KZ, Shokat KM. Strain-release alkylation of Asp12 enables mutant selective targeting of K-Ras-G12D. Nat Chem Biol. Published online March 5, 2024. doi:10.1038/s41589-024-01565-w声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号粉丝群/投稿/授权/广告等请联系公众号助手觉得本文好看，请点这里↓

突破性疗法

100 项与 MRTX-1133 相关的药物交易

登录后查看更多信息

研发状态

适应症	最高研发状态	国家/地区	公司
胰腺腺癌	临床2期	美国更多	Mirati Therapeutics, Inc. 更多
晚期恶性实体瘤	临床2期	美国更多	Mirati Therapeutics, Inc. 更多
非小细胞肺癌	临床2期	美国更多	Mirati Therapeutics, Inc. 更多
结直肠癌	临床2期	美国更多	Mirati Therapeutics, Inc. 更多

登录后查看更多信息

临床结果

适应症

分期

评价

查看全部结果

研究	分期	人群特征	评价人数	分组	结果	评价	发布日期


B032 (AACR_Targeting RAS2023)	N/A	胰腺癌	-	MRTX1133	艱廠顧鑰艱繭壓鹽壓鬱(蓋衊鏇繭糧鏇鑰觸鹹鬱) = 膚鹹醖蓋獵鏇網顧構蓋築壓鑰遞蓋齋簾壓網齋 (鹹顧願膚鬱廠衊夢繭鑰 )	积极	2023-05-01