撰文/@Jie Jack Li
翻译/新阳@意药同萌
杰克说药是著名药史专家Jie Jack Li（李杰）教授专为同写意打造的药林外史精品专栏，将讲述一个个药物发现背后的故事。李杰教授现为上海睿智的副总裁，先后出版了30本有机和药物化学方面的书籍以及药物发现史，其中10本与诺奖得主E. J. Corey合作完成。其《Blockbuster Drugs》一书获 2015 Alpha Sigma Nu Science Book 奖，并被翻译成中文出版，深受欢迎。 开设写意专栏，请联系同写意秘书处（微信号tongxieyimishuchu） 2020年10月25日，圣地亚哥的一家叫Mirati的小型生物技术公司Mirati宣布了其抗癌药物adagrasib（1）在I/1B期和II期临床试验的初步结果。结果表明，adagrasib对非小细胞肺癌（NSCLC）、结直肠癌（CRC）和其他实体肿瘤具有良好和持久的抗肿瘤活性，为KRAS G12C突变的患者提供了新的希望。在adagrasib（1） 作为单一疗法晚期非小细胞癌的治疗中，其客观缓解率可达45%。这一消息振奋了包括制药界以及癌症患者、医生、研究人员和投资者在内的所有人：什么是KRAS？ 什么是 G12C？ 为什么KRAS-G12C抑制剂会引起公众的关注？
RAS 是鼠肉瘤的缩写，而RAS肿瘤基因是Robert Weinberg在1982年发现的第一个人类肿瘤基因。具有讽刺意味的是，当Michael Bishop和Harold Varmus在1989年因发现“逆转录病毒肿瘤基因的细胞起源”而获得诺贝尔奖时，人们普遍认为Robert Weinberg应该与他们分享这个奖项。因为Robert不仅发现了第一个人类肿瘤基因RAS，他还为肿瘤抑制基因Rb.1的发现做出了重大贡献。KRAS-G12C中G12C的突变表明，位于KRAS蛋白密码子-12的糖氨酸氨基酸（Gly，G）已经变异为半胱氨酸氨基酸（Cys，C）。这一突变是是必然会发生的，因为半胱氨酸的活性嗜核硫醇（+SH）会与众多KRAS-G12C抑制剂形成共价键，Mirati的adagrasib （1） 就是众多抑制剂之一。RAS基因家族是人类癌症中最常见的突变基因。在90%的胰腺癌、45%的结肠癌和35%的肺癌中都存在RAS突变。在三个Ras基因中，Kirsten-RAS（KRAS）是最常发生突变的亚型，比例高达86%，因此获得了最多的关注。其他两个亚型神经母细胞瘤-RAS（NRAS）和哈维-RAS （HRAS）因突变率较低（11%和3%），学术界对此则不太感兴趣。KRAS蛋白，有以下几个突变。KRAS-G12C突变在NSCLC（突变KRAS-G12C约占45-50%）中占主导地位。另一方面，KRAS-G12D（其中在密码子-12位点的甘氨酸突变为天冬氨酸），这一突变在胰腺癌（61%）、结肠癌（42%）和NSCLC（22%）中显得非常重要。由于RAS蛋白在肿瘤学中的重要性，自从Robert Weinberg四十年前发现RAS肿瘤基因以来，科学家们投入了大量精力来寻找作用于这个有价值的靶点的药物。但是，由于RAS缺乏与小分子抑制剂结合的位点，该领域在此前三十年的药物发现工作中并未取得显著的成果。Amgen化学家甚至将RAS蛋白的表面比作电影《星球大战》中的死星表面。因此，RAS虽与众多癌症密切相关，但依然被认为是一个不可成药的药物靶点也就不奇怪了。最近以KRAS-G12C抑制剂为代表的众多直接的RAS抑制剂的研究取得一定成果，证明了众多科学家的创造性和坚持不懈的努力。2013年，加州大学旧金山分校的Kevan Shokat教授发表了他的里程碑式论文，在文中描述了这一突破，此论文发表在《自然》杂志上。在论文中，Shokat描述了众多KRAS-G12C抑制剂，这些抑制剂通过引起变构控制鸟苷三磷酸（GTP）亲和力和与其他因子的相互作用。他的团队通过使用共价键抑制剂来达到这个艰难的目标。所使用的化合物包括二硫化物、硫化物、氯丙烯酰胺（例如丙烯酰胺2）等，它们与在10位的半胱氨酸发生反应，形成共价S-C键。现在，在药物发现方面，我们已经对共价键抑制剂有足够多的了解，其优势包括：适用于一些“不可成药”的靶点，KRAS-G12C抑制剂的研发中尤为明显;更高效;起效的持续期更长;能够直接与目标靶点匹配。Shokat找到了一家叫Wellspring Biosciences公司，希望合作实现这一革命性发现。他们使用了一种特异性共价结合KRAS-G12C的抑制剂ARS-853（3），这是第一个显示具有细胞活性的化合物。2018年披露的一种KRAS-G12C的特异性抑制剂含有类似喹啉刚性结构的化合物ARS-1620 （4） 在生物体内也具有活性。最终，Wellspring 和 Janssen 合作于2019年7月将 ARS-3248（JNJ-74699157，结构至今未披露）带到临床试验的阶段。但是，这已是此类药物中第三种用于临床试验的药物。尽管先发，Wellspring还是落后于Amgen and Mirati。2019年4月，在奥兰多举行的ACS会议上，Amgen宣布了他们的候选药物AMG 510（Sotorasib，5），一种特异性共价结合KRAS-G12C抑制剂开始在晚期KRAS-G12C突变实体肿瘤患者中进行首次人体试验。震惊了整个制药界。为什么Amgen比任何人都早地拥有第一个临床候选化合物？事实证明，位于加利福尼亚州Thousand Oaks的 Amgen 团队在研究表皮生长因子受体 （EGFR） 抑制剂之前，在靶向共价抑制剂方面已有丰富的经验。通过与伯克利生物技术公司 Carmot 公司的合作，使Amgen 在两两年半时间筛选了 100,000 种未纯化的化合物。更令人兴奋的是，2019年 7 月，Amgen 报告了其的首创的共价性 KRAS-G12C 特异性抑制剂 AMG 510（sotorasib， 5）的I期临床试验结果：13 名可评估的 NSCLC 患者中，在正在进行的第 1 阶段研究中，54%的患者在使用了960mg的目标剂量后出现了部分应答。同时，46%的患者病情稳定，在目标剂量下疾病控制率为100%。FDA授予AMG 510（sotorasib，5）快速评审通道，以治疗的KRAS-G12C突变的转移性NSCLC。。KRAS-G12C药物靶点，这个以前被视为不可成药的靶点，现在已成为最热门的的癌症靶点之一。AMG 510（sotorasib，5）热度不减，，Mirati和Array生物制药也宣布他们的共价KRAS-G12C特异性抑制剂MRTX849（1）6在2019年夏天进入I期临床试验。事实上， Mirati 和 Array 在一年前已经有一个相当不错的候选药物 Mrtx1257 （6） 。然而，他们决定进一步优化该化合物以求得到一个更理想的药物用于临床试验。现在，随着 MRTX849 （1） 的临床试验逐渐披露，他们的决定被证明是明智的。在药物发现中，一些里程碑式的结果比另一些更为重要。当使用某些作用机制 （MoA） 的药物在第II期临床试验中被证明有效时，我们认为该特定的 MOA 已实现成药性的概念验证 （POC）。从历史上看，与没有成药性概念验证的药物机理相比，具有成药性概念验证的药物机理成功进入市场的可能性是非常高的。这个结论对病人，医生和投资者都是好的。我们只触及到了KRAS-G12C抑制剂的浅显的表面，但发现它们的这一过程，对药物发现者来说，给我们上了价值连城的一课。在未来，这将是药物发现历史上的另一个故事。
Drug the Undruggable Target
—KRAS-G12C Inhibitors to Treat CancerJie Jack Li, Vice President of Discovery Chemistry at Shanghai ChemPartnerOn October 25, 2020, Mirati Therapeutics, a small biotech company in San Diego, announced their preliminary phase I/1b and II results of their cancer drug adagrasib (1). It showed deep and durable anti-tumor activity in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and other solid tumors, providing renewed hope for patients that harbor a KRAS G12C mutation. It is compelling for a 45% confirmed objective response rate for adagrasib (1) as a monotherapy in advanced NSCLC. The news electrifying the drug industry and everyone, including the cancer patients, doctors, researchers, and investors alike, is getting excited:Why a KRAS-G12C inhibitor is generating so much publicity?RAS is an abbreviation for Rat Sarcoma. RAS oncogene, on the other hand, was actually the first human oncogene discovered by Robert Weinberg in the 1982. Quite ironically, when Michael Bishop and Harold Varmus received the Nobel Prize in 1989 for their “discovery of cellular origin of retroviral oncogenes”, it was widely believed that Robert Weinberg should have shared the Prize with them. Not only did Weinberg discover RAS, the first human oncogene, he also made significant contributions to the discovery of tumor suppressor geneRb.1 G12C in KRAS-G12C mutations indicates that the glycine amino acid (Gly, G) at codon-12 of the KRAS protein has mutated to a cysteine amino acid (Cys, C). This is consequential because cysteine will be employed as the reactive nucleophile thiol (–SH) to form covalent bonds with the warheads of targeted covalent KRAS-G12C inhibitors. Mirati’s adagrasib (1) is one of them.RAS gene family are the most frequently mutated oncogenes in human cancers. Mutant RAS appears in 90% of pancreatic, 45% of colon, and 35% of lung cancers. Among the three Ras genes, Kirsten-RAS (KRAS) is the most frequently mutated isoform (86%), therefore, of the greatest interest. The other two isoforms neuroblastoma-RAS (NRAS, 11%) and Harvey-RAS (HRAS, 3%) are of less interest. With regard to the KRAS proteins, there are several mutations. The KRAS-G12C mutation, in particular, predominates in NSCLC (45–50% of mutant KRAS-G12C). On the other hand, KRAS-G12D (where glycine at codon-12 has mutated to aspartate), is important in pancreatic cancer (61%), colon cancer (42%), and NSCLC (22%).2 Because the importance of the RAS protein in oncology, much effort has been devoted to find inhibitors of this valuable drug targets ever since Weinberg discovered the RAS oncogene forty years ago. But first thirty years of drug discovery effort in this field was completely fruitless because RAS’s lack of deep pockets for binding of small molecule inhibitors. Amgen chemists compared the surface of the RAS protein to the surface of the Death Star in the movie Star Wars. It is not that surprising that RAS was considered a “undruggable” target although it is implicated in many cancers.Recent successes with RAS direct inhibitors, KRAS-G12C inhibitors in particular, are a testimony of scientistsʹ ingenuity and perseverance. The breakthrough came when Prof. Kevan Shokat at the University of California at San Francisco published his landmark paper in 2013. In his Nature paper, Shokat described a series of KRAS-G12C inhibitors that allosterically control guanosine triphosphate (GTP) affinity and effector interactions.3 His team chose to use covalent inhibitors to tackle this tough target, specifically the switch-II pocket (S-IIP). The warheads that they used included disulfides, vinyl sulfonamides, and acrylamides (e.g., acrylamide 2). All of these warheads reacted with the cysteine amino acid at the 10 position to form a covalent S–C bond.Today, we have learned enough to understand that targeted covalent inhibitors have many advantages in drug discovery. They include:· Applicability to some “undruggable” targets, obviously it is the case for KRAS-G12C inhibitors;· Extended duration of action; and· Ability to directly measure target.A biotech company Wellspring Biosciences that Shokat helped to found capitalized his revolutionary discovery. They came up with a covalent KRAS-G12C-specific inhibitor ARS-853 (3), which was the first compound to show cellular activities. A more rigid quinoline-containing analogue ARS-1620 (4), disclosed in 2018, was one of the first covalent KRAS-G12C-specific inhibitors that were active in vivo.4 Eventually, Wellspring and Janssen collaborated to bring ARS-3248 (JNJ-74699157, structure not disclosed thus far) to clinical trials in July 2019. But that was the third of such drugs that went to clinical trials. Despite starting first, Wellspring lagged behind both Amgen and Mirati.In April 2019 at the ACS meeting in Orlando, Amgen announced their drug candidate AMG 510 (sotorasib, 5), a covalent KRAS-G12C-specific inhibitor started its first-in-human trials in patients with advanced KRAS-G12C mutant solid tumors.5a That was the first of this class of drugs that went to clinical trials. The whole drug industry was stunned.Where did they come from?Why did Amgen have the first clinical candidate before everyone else?It turned out that the Amgen team in Thousand Oaks, California already had abundant experience with targeted covalent inhibitors previously when they worked on epidermal growth factor receptor (EGFR) inhibitors. Collaboration with a Berkeley biotech company Carmot Therapeutics helped Amgen screen 100,000 unpurified compounds in a matter of two and half years. More excitingly, in July, 20195b Amgen reported their Phase I clinical trial outcome of their first-in-class covalent KRAS-G12C-specific inhibitor AMG 510 (sotorasib, 5): 54% of 13 evaluable NSCLC patients experienced a partial response (PR) at the target dose of 960 mg in the ongoing Phase 1 study. Meanwhile, 46% of patients had stable disease for a disease control rate of 100% at the target dose. The FDA granted AMG 510 (sotorasib, 5) fast track designation for previously treated metastatic NSCLC with KRAS-G12C mutation. The KRAS-G12C drug target, the previously “undruggable” target has now become one of the most popular cancer targets.Hot on the heel of AMG 510 (sotorasib, 5), Mirati and Array Biopharma also announced their covalent KRAS-G12C-specific inhibitor MRTX849 (1)6 went to phase I clinical trials in the summer of 2019. As a matter of fact, Mirati and Array already had a pretty good drug candidate MRTX1257 (6) one year ago already. However, they decided to optimize a bit further to have a more ideal drug to go to clinical trials. Now with the clinical readings out for MRTX849 (1), their decision has been proven wise. Here is the answer to the question:In drug discovery, some milestones are more important than others. When a drug using certain mechanism of action (MoA) has been proven efficacious in phase II clinical trials, we consider that particular MoA has achieved proof-of-concept (PoC). Historically, mechanism of action with proof-of-concept has an exponentially highly probability of succeeding in going to the market than MoA without PoC. That, is good for the patients, doctors, and investors.We only have scratched the surface of the KRAS-G12C inhibitors. Their discovery is a treasure trove from which we can learn valuable lessons. But that is for another blog in the future.1. Weinberg. Robert A. Racing to the Beginning of the Road, The Search for the Origin of Cancer, W. H. Freeman and Company: New York, NY, 1998.2. Stephen, A. G.; Esposito, D.; Bagni, R. K.; McCormick, F. Cancer Cell 2014, 25, 272–281.3. Ostrem, J. M.; Peters, U.; Sos, M. L; Wells, J. A.; Shokat, K. M. Nature2013, 503, 548–551.4. (a) Janes, M. R.; Zhang, J.; Li, L.-S.; Hansen, R.; Peters, U.; Guo, X.; Chen, Y.; Babbar, A.; Firdaus, S. J.; Darjania, L.; et al. Cell 2018, 172, 578–589. (b)5. (a) Fell, J. B.; Fischer, J. P.; Baer, B. R.; Ballard, J.; Blake, J. F.; Bouhana, K.; Brandhuber, B. J.; Briere, D. M.; Burgess, L. E.; Burkard, M. R.; et al.ACS Med. Chem. Lett. 2018, 9, 1230–1234. (b) Anon, Cancer Discov.2019, 9, 988–989.6. Fell, J. B.; Fischer, J. P.; Baer, B. R.; Blake, J. F.; Bouhana, K.; Briere, D. M.; Brown, K. D.; Burgess, L. E.; Burns, A. C.; Burkard, M. R.; et al. 2020,63, 6679–6693.
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