Emricasan

撰文 | 咸姐 细胞死亡是多细胞生物发育、稳态维持和病原防御的核心环节，其中程序性细胞死亡主要分为细胞凋亡（apoptosis）和细胞焦亡（pyroptosis）两条通路，二者均由天冬氨酸特异性半胱氨酸蛋白酶（caspase）家族介导。炎性caspase（包括人源的caspase-1、-4、-5以及小鼠的caspase-1和-11）在炎症小体复合物中被激活，进而切割成孔蛋白Gasdermin D（GSDMD），释放其N端成孔结构域，在细胞膜上形成孔洞，引发细胞焦亡——一种裂解性、促炎性的细胞死亡方式，并伴随IL-1β和IL-18等炎症因子的大量释放。与此相对，凋亡则由caspase-8和-9启动，继而激活执行型caspase-3和-7，以非裂解方式清除细胞。尽管凋亡和焦亡在生理条件下发挥重要功能，但二者的过度激活均已被证明与多种炎症性疾病的发生发展密切相关【1】。因此，靶向caspase活性以抑制病理性细胞死亡，成为抗炎治疗的重要策略。然而，既往开发的细胞可渗透性广谱caspase抑制剂在临床试验中屡遭挫败，主要归因于毒性反应或疗效不足，部分原因在于广谱抑制会干扰caspase-8的功能，从而在某些细胞中诱发替代性坏死性凋亡（necroptosis）【2】。此外，由于caspase家族成员之间存在高度的结构同源性，开发高选择性的caspase抑制剂一直面临巨大挑战。近年来，有研究发现靶向GSDMD的细胞外纳米抗体在某些条件下可进入早期GSDMD孔并减少后续孔的形成，从而减轻焦亡性死亡【3】。这一现象提示，GSDMD孔可能在孔形成早期阶段允许某些大分子或通常无法穿透细胞膜的分子通过，这为设计新型选择性caspase抑制剂提供了新的思路。 基于以上，来自美国基因泰克公司的Vishva M. Dixit团队提出假设：若能开发出对炎性caspase具有选择性、且正常状态下无法穿透细胞膜的小分子抑制剂，那么这些抑制剂将仅在细胞启动焦亡、形成GSDMD孔后才进入靶细胞，从而实现“按需”抑制，避免广谱抑制正常细胞所带来的毒副作用。在这一背景下，该团队于近日在Nature杂志在线发表题为Gasdermin D-mediated delivery of caspase inhibitors to suppress pyroptosis的文章，系统设计并筛选了一系列四肽类共价抑制剂，旨在利用GSDMD孔作为药物进入的“通道”，实现对焦亡途径的选择性阻断，并通过体外和体内模型验证其疗效和机制。 本文研究人员首先利用杂交组合底物文库（HyCoSuL）系统筛查了人caspase-1、-4和-5的底物偏好，发现与小鼠caspase-1和-11类似，它们均偏好P2位组氨酸和P3位谷氨酸，但在P4位存在差异（caspase-11偏好小脂肪族残基，而caspase-1/4/5偏好大体积残基），且caspase-4与小鼠caspase-11虽功能相似但底物偏好不同。鉴于人caspase-1/4/5具有相似的切割偏好，研究人员又测试了先前报道的一组四肽caspase底物，发现含有人pro-IL-1β中Trp-Val-His-Asp序列的底物最易被人caspase-1切割，而含有GSDMD中Phe-Leu-Thr-Asp序列的底物效率约低三倍，且caspase-1的催化效率较其他炎性caspase高出一个数量级。此外，在四肽序列中引入非天然氨基酸既能提高切割效率，也能增强对促炎性caspase相对于促凋亡caspase的选择性。 根据caspase的切割位点偏好，研究人员合成了约100种含酰氧基甲基酮（AOMK）反应基团的四肽共价抑制剂，在重组人caspase上筛选出KGR-3、KGR-23和KGR-72为人caspase-1的有效选择性抑制剂，且能抑制小鼠caspase-1，但这些化合物在缺乏外排泵MDR1的gMDCKI细胞中的渗透性极低。为验证其能否经GSDMD孔进入细胞，研究人员又测试了它们对THP-1细胞中尼日利亚菌素诱导的NLRP3/caspase-1依赖性焦亡的抑制，发现多种四肽可显著减少细胞裂解。随后，利用表达FKBP-caspase-4或-11融合蛋白的THP-1细胞，通过可透过细胞的B/B配体二聚化引发焦亡，研究人员鉴定出若干种与广谱caspase抑制剂emricasan相当的化合物，例如KGR-3可阻断caspase-4诱导的死亡但对caspase-11效果较弱，而KGR-23、KGR-53和KGR-72则对二者均有抑制。为排除非GSDMD依赖的进入方式，研究人员还测试了这些化合物对星形孢菌素诱导的凋亡的影响，结果显示与emricasan不同，所选四肽均不能抑制凋亡。 进一步地，KGR-3作为有效的caspase-1和-4抑制剂被深入表征。实验结果显示，KGR-3在多种细胞体系中均能阻断焦亡和IL-1β分泌，包括THP-1 FKBP-caspase-4细胞、ATP刺激的PMA分化THP-1细胞以及VBP激活NLRP1炎症小体的Ker-CT细胞（后者依赖GSDMD和GSDME）。在EA.hy926细胞中，VBP通过CARD8-caspase-1炎症小体触发细胞死亡，KGR-3抑制GSDMD依赖性焦亡，但对caspase-1驱动的凋亡小泡和caspase-3切割无效，表明其需经GSDMD孔进入细胞；即使在VBP处理后数小时加入KGR-3，仍能阻止后续裂解和IL-1β释放，且细胞可继续生长，提示GSDMD孔形成后存在干预窗口。在表达FKBP-caspase-4的EA.hy926细胞中，KGR-3同样有效，而ESCRT蛋白VPS4A和CHMP3的显性负突变可抵消其保护。此外，由于在凋亡细胞中具有低渗透性，KGR-3不影响TRAIL诱导的外源性凋亡、MCL-1抑制剂S63845诱导的内源性凋亡以及李斯特菌溶血素O诱导的坏死。与此同时，通过检测膜不通透的荧光小分子PI和YOYO-1在GSDMD孔形成后的细胞摄取，研究人员证实了在caspase-1被KGR-3抑制的情况下，初始GSDMD孔仍会短暂开放，允许小分子进入活细胞胞质，且该过程严格依赖caspase-1和GSDMD；这为KGR-3等低通透性抑制剂通过GSDMD孔进入细胞提供了直接证据。不过，由于荧光标记KGR-3会损害其活性或选择性，直接观察抑制剂通过内源性Gasdermin孔的动态过程仍有待未来实现。 最后，鉴于caspase-11依赖性焦亡在LPS诱导的内毒素休克中起重要作用，研究人员对KGR-53进行了深入表征——该化合物对caspase-11的抑制效力强于KGR-3，但在LPS转染的小鼠骨髓来源巨噬细胞（BMDMs）中仅能部分抑制焦亡。考虑到GSDMD孔道可能对带负电荷化合物的转运效率较低，研究人员去除KGR-53 P3位谷氨酸的负电荷，得到KGR-53P。优化后的KGR-53P作为低细胞通透性的caspase-1/11抑制剂，在体外能有效阻断LPS诱导的巨噬细胞焦亡、GSDMD切割及IL-1β/IL-18分泌，延迟给药仍有效，且不影响凋亡。在小鼠内毒素休克模型中，KGR-53P可显著降低血清IL-18和IL-1β水平，表明低通透性caspase抑制剂可通过GSDMD孔进入细胞并限制炎性因子的产生。不过，研究人员也观察到KGR-53P在无LPS情况下会升高血清肝酶水平，这可能属于工具化合物的脱靶效应，提示其仍需进一步优化。 上述结果综合表明，传统上因通透性差而被认为不适合药物开发的caspase抑制剂，可利用焦亡细胞特有的GSDMD孔作为“入口”实现靶向递送，从而在体内有效抑制炎症反应。本研究开发的低通透性但高选择性的caspase-1/4/11抑制剂（KGR-3和KGR-53P），实现了对焦亡和炎症因子释放的选择性阻断，而不影响正常细胞的凋亡。毫无疑问，这一策略为脓毒症、急性呼吸窘迫综合征和COVID-19等以过度炎症小体激活为特征的疾病提供了新的治疗思路。 原文链接： https://doi.org/10.1038/s41586-026-10957-y 制版人： 十一 参考文献 1. Newton, K., Strasser, A., Kayagaki, N. & Dixit, V. M. Cell death. Cell 187, 235–256 (2024). 2. Dhani, S., Zhao, Y. & Zhivotovsky, B. A long way to go: caspase inhibitors in clinical use. Cell Death Dis. 12, 949 (2021). 3. Schiffelers, L. D. J. et al. Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application. Nat. Commun. 15, 8266 (2024). 学术合作组织 （*排名不分先后） 战略合作伙伴 （*排名不分先后） · 推荐直播 · 转载须知 【原创文章】BioArt原创文章，欢迎个人转发分享，未经允许禁止转载，所刊登的所有作品的著作权均为BioArt所拥有。BioArt保留所有法定权利，违者必究。 BioArt Med Plants 人才招聘 点击主页推荐活动 关注更多最新活动！

Not content with navigating Rezdiffra’s trajectory to blockbuster territory, Madrigal Pharmaceuticals has spent 2026 shopping for a range of drugs that the company hopes could work alongside its metabolic dysfunction-associated steatohepatitis (MASH) trailblazer. Madrigal kicked off the year paying $50 million to Pfizer for the rights to a DGAT2i inhibitor, before spending $60 million the following month on six preclinical siRNA programs from China’s Ribo Life Science and its Swedish subsidiary Ribocure Pharmaceuticals that Madrigal wants to use to silence genes implicated in MASH. The company explained at the time that both deals were aimed at securing therapies that could be combined with Rezdiffra. The biopharma was back at the dealmaking table in May, paying $25 million upfront to Arrowhead Pharmaceuticals for a phase 1-stage RNA interference therapeutic that had previously been rejected by Johnson & Johnson. Early clinical data had suggested the asset could reduce liver fat in a mutation found in 30% of MASH patients, including high prevalence in Hispanic populations. Combined with a preclinical GLP-1 agonist licensed from China’s CSPC Pharmaceutical for $120 million upfront last year, Madrigal now has a diverse array of MASH assets coming up behind Rezdiffra. So what’s the strategy behind this rapid pipeline expansion? “The question early on was what mechanisms we wanted,” Madrigal CEO Bill Sibold told Fierce in an interview at the company’s office in Waltham, Massachusetts. “My answer was: we want all of them,” Sibold continued. “We want anything that makes sense mechanistically or has proven already to have an effect on MASH.” It’s a sign of just how much Rezdiffra has changed the game since being approved as the first-ever treatment for MASH in 2024. Before that point, Sibold said he had counted 23 drugs across the industry that had failed to treat the fatty liver disease previously known as nonalcoholic steatohepatitis (NASH). This rollcall of failure meant MASH had become “the graveyard of drug development,” lumped in with other difficult-to-treat conditions like sepsis and lupus, he remembered. The disease occurs when fat builds up in the liver, causing inflammation and eventually cell death. It is the leading cause of liver transplants for women and the second biggest cause for men in the U.S. “Industry had been really interested in this area for a long time, but nobody could crack the nut,” Sibold said. Many companies tried and failed to find a breakthrough. Gilead’s ASK1 inhibitor selonsertib and its LOXL2 enzyme-targeting simtuzumab both flopped in the clinic, as did Genfit’s dual PPAR alpha/delta agonist elafibranor and Conatus Pharmaceuticals' pan-caspase inhibitor emricasan. Rezdiffra’s origins go back to Roche. Madrigal founder Rebecca Taub, Ph.D., left the Swiss pharma’s metabolic unit to work for VIA Pharmaceuticals, which licensed the thyroid hormone receptor (THR)-β agonist. Madrigal assumed the rights to that agreement when it was founded in 2011 and continued to develop the therapy. Sibold remembers getting a call from a recruiter about the Madrigal CEO position in 2023 while he was president for North America at Sanofi, having helped launch immunology blockbuster Dupixent. “There's no way I'm going to leave this to go to that,” he remembers of his initial reaction to the offer. But as Sibold dug into the company, educating himself on the trials and the science behind its lead asset, he became convinced that this was a unique opportunity to make a special impact. The year after Sibold joined up, Madrigal had secured Rezdiffra’s historic FDA approval. As well as the company's headquarters in West Conshohocken, Pennsylvania, the company now also has an office in Massachusetts—which Fierce visited—as well as an international outpost in Switzerland. “There were so many Big Pharma companies trying to solve [MASH], and this little Pennsylvania company of very few people had the tenacity, the guts and the grit to get there,” Sibold said. Madrigal reported $958.4 million in annual net sales for the drug in 2025. By the second quarter of this year, Rezdiffra was bringing in $364.3 million, equivalent to year-over-year growth of 71%. Sibold told Fierce that it wasn’t always this way. Early on, a MASH advocate told him: “We didn't know whether to believe you at first because it seemed like such a huge hill to climb that you guys didn't even have an approved product. But you are doing exactly what you said you would do.” After establishing itself as the market leader in MASH, what’s next? Does the company stay in its lane, expand into immunology targets or look at new indications? For Sibold, the answer lies in Madrigal’s five-word purpose: leading the fight against MASH. The biopharma wants to continue working with the MASH community it has come to know so well to build out its pipeline with more effective and comprehensive treatments for different subpopulations. Rather than generate data and hope to get bought by a bigger fish, Sibold said he is planning for an independent and sustainable company 10 to 20 years down the line. His vision is for MASH treatments to grow like other specialty markets, such as rheumatoid arthritis, psoriasis or inflammatory bowel disease, with dozens of products that generate tens of billions of dollars in annual sales. “We feel like MASH will emerge like that, and as a result, we want to have the portfolio of products that can best help all or subpopulations of patients,” Sibold said. Unlike a Big Pharma, where MASH may be one of a myriad of indications being focused on, Sibold suggested Madrigal's strength comes from the fact it is committed to the disease. This approach is reflected in trial enrollment and feedback, he said. “The community values expertise, and I think we've made a conscious decision to be experts in MASH,” he said. “Through expertise comes insight into what might be the next best mechanism, or the next best mechanism to combine with your current mechanism, or the best way to design the next trial.” Because reducing liver fat is so important to preventing or treating MASH, GLP-1s are moving into the space and posing a potential threat. Last year, Novo Nordisk’s Wegovy was approved to treat MASH, and some people have warned Sibold that Madrigal could be crushed by GLP-1s in the future. Instead, the CEO insisted more activity in the space is a good thing for Madrigal. “It's great to have another company who's putting resources into diagnosis, awareness and treatment,” he said. “There are some patients that are getting Wegovy, but it is helping to grow the market. It's not to our detriment.” But other Big Pharmas are circling. Last year saw GSK acquire the FGF21 analog efimosfermin from Boston Pharmaceuticals for $1.2 billion upfront, while Roche picked up a similar drug called pegozafermin as part of its $3.5 billion buyout of 89bio . Madrigal may be looking to become the MASH version of what Vertex Pharmaceuticals has achieved in cystic fibrosis, but that doesn’t mean Sibold and his team couldn’t eventually broaden their focus. “Would we look at other diseases? Yes. We would in time, or if the biology takes us there,” he told Fierce. But for now, the company is leaning into MASH specialization, developing relationships with patient groups, clinicians and researchers while raising Rezdiffra’s profile and growing a specialized portfolio of complementary therapies. “It's been fun. It's making a difference. It's helping patients,” Sibold said. “This is as good as it gets.