Desflurane

本文结合文献与专利介绍一下近期E3新配体的开发现状。这次首先讲一下DCAF1。 DCAF1生物学背景 DCAF1（也称为VprBP）是一个多功能的E3泛素连接酶底物受体，参与调控多种细胞过程。它是Cullin-RING连接酶（CRL）家族的一部分，特别是CRL4连接酶复合物的一部分。DCAF1在细胞周期、细胞生长和细胞分裂中起着重要作用，并且与某些癌症的发展有关。此外，DCAF1还在葡萄糖剥夺条件下通过失活Rheb-mTORC1途径促进癌细胞存活。在低葡萄糖条件下，DCAF1增强了Rheb的K48链多聚泛素化和蛋白酶体依赖性降解，从而抑制mTORC1活性，诱导自噬，并促进癌细胞在葡萄糖剥夺条件下的存活。DCAF1 也是一些病原体（如 HIV 和 SIV）的靶点，这些病原体通过劫持 DCAF1 的功能来抑制宿主免疫反应。 由于 DCAF1 在细胞生理和病理过程中的重要作用，它被认为是靶向蛋白质降解 (TPD) 领域的一个新兴的连接酶靶点。 对DCAF1的表达分析也可以发现，其存在一定的组织特异性与肿瘤特异性，是个高价值的E3连接酶，进一步证明了其在TPD领域的开发潜力。 然而，缺乏有效针对 DCAF1 的已知配体限制了对其功能和治疗潜力的研究。 不过近期一些DCAF1小分子配体与相关PROTAC的报道， 似乎打开了DCAF1应用的大门。 文献报道 MY-11B The Scripps Research Institute于2022年在JACS上报道了利用化学蛋白质组学方法，发现了一类可以立体选择性和位点特异性地与 DCAF1 蛋白上的半胱氨酸 C1113 共价反应的氮杂环丁烷丙烯酰胺化合物MY-11B（J. Am. Chem. Soc. 2022, 144, 40, 18688–18699）。当然他们还对此申请了专利（WO2023225625A2）。 文章中展示了 DCAF1 蛋白的结构域组成，以及 C1113 位于 DCAF1-Vpx 复合物中与 HIV 病毒蛋白相互作用的界面。 基于MY-11B开发了靶向降解 FKBP12 和 BRD4 蛋白的亲电 PROTACs，表现出良好的降解活性（DC50 < 0.5 μM），并证明其有立体选择性和 DCAF1 依赖性。 至于MY-11B与DCAF1的亲和力，文中提到了用凝胶 ABPP 方法测定出 MY-11B 对 DCAF1 的体外靶点参与度 (TE50) 大约是 25 μM。 CYCA-117-70 AIDD明星公司Recursion Pharmaceuticals在Journal of Chemical Information and Modeling上报道了基于AI筛选发现的新型DCAF1配体(J. Chem. Inf. Model. 2023, 63, 4070−4078)。 本文采用了一种基于人工智能的药物-靶点相互作用 (DTI) 预测模型 MatchMaker，结合化学信息学筛选和分子对接技术，来识别 DCAF1 WD40 结构域 (WDR) 的配体。通过虚拟筛选与体外实验验证，发现了活性化合物 CYCA-117-70。通过X 射线晶体学研究，解析其与 DCAF1 WDR 结构域的复合物结构，确定其结合模式。 不过其活性一般，对DCAF1 WDR 结构域结合活性KD仅为70 μM。 OICR-8268 多伦多大学发表在Journal of Medicinal Chemistry上的文章(J. Med. Chem. 2023, 66, 5041−5060)，该研究通过使用 X-Chem 公司专有的 1140 亿个化合物的 DEL 库对 DCAF1 的 WDR 结构域进行筛选，并结合基于结构的药物设计，成功发现了第一个纳摩尔级的DCAF1配体OICR-8268，其 SPR Kd 值为 38 ± 1.5 nM，ITC Kd 值为 278 nM，并在细胞内具有良好的靶点结合能力 (CETSA EC50 = 10.5 μM)，为开发基于DCAF1的PROTACs提供了新的可能性。 研究人员解析了 OICR-8268 与 DCAF1 WDR 结构域的共晶结构，确认了其结合模式，并为进一步优化提供了结构基础。 Cpd.13 诺华团队也在ACS Medicinal Chemistry Letters上报道了其基于口袋鉴定、苗头化合物发现。共晶结构解析与基于结构的药物设计等策略发现了化合物13，SPR 测定的 Kd 值为 31 nM，可以作为开发 DCAF1 基于的 PROTACs 的起点（ACS Med. Chem. Lett. 2023, 14, 949−954）。 有趣的是，文章中还总结了上图中Cpd.8与前文提到的OICR-8268、CYCA-117-70的共晶结构。，可知这三个骨架各异的化合物具有相近的结合模式。通过比较这些化合物的结合模式，可以为进一步优化 DCAF1 配体提供指导。 故事并没有结束。在今年的nature communications上，诺华进一步报道了基于Cpd.13的PROTAC设计，表明DCAF1 PROTACs是一种很有潜力的克服 PROTACs 耐药性的策略。DCAF1 作为一个必需基因，以及其与 CRBN 和 VHL 不同的细胞定位，使其成为开发新一代 PROTAC 分子的理想靶点（Nat Commun 15, 275 (2024).）。 首先文章中做了DCAF1 与其他 E3 连接酶受体的基因依赖性评分，表明 DCAF1 是一个必需基因，而 CRBN与 VHL不是。目前大多数 PROTAC 分子依赖于 CRBN 和 VHL 这两种 E3 连接酶，而这两种连接酶在临床治疗中都面临着耐药性问题。 接着文章中利用非共价 DCAF1配体Cpd.13，设计了DCAF1-BRD9 PROTAC (DBr-1)、基于酪氨酸激酶抑制剂达沙替尼的PROTAC (DDa-1)、DCAF1-BTK PROTAC (DBt-10),均表现出良好的降解活性。 进一步的，研究人员使用 CRBN-BTK PROTAC 耐药的细胞系，证明了 DCAF1-BTK PROTAC 可以有效地降解 BTK 并抑制细胞增殖，而 CRBN-BTK PROTAC 则失去了活性。这表明 DCAF1 PROTACs 可以克服 CRBN 介导的 PROTACs 耐药性。 总而言之，这项研究表明，虽然配体结构与PROTAC活性仍有待优化，DCAF1 PROTACs 是一种很有潜力的克服 PROTACs 耐药性的策略。DCAF1 作为一个必需基因，以及其与 CRBN 和 VHL 不同的细胞定位，使其成为开发新一代 PROTAC 分子的理想靶点。 专利信息 Cullgen，睿跃生物 睿跃生物的主要研发方向便是利用其独有的uSMITE™技术平台，扩展公司的E3泛素连接酶工具箱。 早在2022年，Cullgen便报道具有新颖结构骨架的DCAF1小分子配体，专利WO2022194087A1。 不过本专利的活性却并不尽如人意。代表性化合物与DCAF1的亲和活性仅为Kd ≤ 40 μM（下图）。 专利中也给出了某个化合物与DCAF1的共晶结构，可见与上文三个化合物的共晶结合同一个口袋位点。 WO2024056077A1，Cullgen的第二篇专利。很明显的可以看到专利化合物设计上参考了上文中Recursion发现的CYCA-117-70。 代表性化合物的亲和活性为Kd ≤ 25 μM，相比于上一篇专利有所提升。与第一篇专利一样，也有共晶结构。 同时，专利中还存在一类DCAF1的共价配体，但并未给出其与DCAF1的亲和活性。 进一步的，专利基于CYCA-117-70与多激酶抑制剂TL13-87设计了一系列PROTAC，活性尚可。 自然也少不了BRD4 PROTAC，活性尚可。 还基于CDK2/4/6抑制剂PF-06873600，设计了新的CDK4 PROTAC，活性有一定的提升。 专利中还基于上述提到的新设计的DCAF1配体B-072，设计了TYK2 PROTAC（未给出活性）与ERα PROTAC（活性一般）。 本专利很好的实现了将CYCA-117-70转化成PROTAC的概念验证，但专利中这类PROTAC分子的亲和力、降解活性、以及linker设计上，距离达到成药性预期明显还有一段距离。后续Cullgen在此的发展值得观望。 Kymera WO2024092009A1，Kymera的第一篇DCAF1相关专利。专利中设计了近千个DCAF1配体分子。这类分子分为两类：基于CYCA-117-70的类似物，以及基于OICR-8268的类似物（部分为共价配体设计，但亲和力一般）。专利中部分实施例具有良好的DCAF1亲和活性（Kd < 10 μM）。 进一步的，基于配体分子I-653设计了一系列BRD4-DCAF1 PROTAC分子。不过专利中仅给出了两个实施例的活性数据。可以看到，最优实施例I-907具有不错的降解活性。DC50达到了10~100 nM，Dmax = 70~90%。 WO2024092011A1，Kymera的第二篇DCAF1相关专利。专利中设计了11个IRAK4-DCAF1 PROTAC。可以明显的发现，专利中的DCAF1配体为第一篇专利中的I-886。不过专利中仅给出PROTAC与DCAF1的亲和力，并未给出这类PROTAC分子。 不过Kymera现在在专利撰写中对活性数据有所隐藏。鉴于OICR-8268的亲和力达到了38 nM，I-653这一系列基于此改造得到的化合物，不至于比OICR-8268亲和力差。 可以明显看到，第二篇专利中的I-7采用了与Kymera临床IRAK4 PROTAC分子KT-474完全相同的warhead与linker，区别仅在E3 ligand。想必其成药性，有一定的保证。 后话 总体而言，鉴于DCAF1的必需性，以及其特异性的肿瘤/组织分布，其有望成为TPD领域新的开发方向。同时，MY-11B、CYCA-117-70、OICR-8268、Cpd.13等骨架各异的DCAF1配体也为开发基于DCAF1的PROTAC奠定了基础，也为后人开发新型DCAF1配体提供了良好的参考方向。 同时，Kymera的DCAF1 PROTAC似乎展现出了良好的降解活性与成药性。其后续是否会有新的进展，值得期待。 至于其他新的E3，下一篇文章再作详细介绍吧。 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↓

Each puff of a metered-dose inhaler expels a potent hydrofluorocarbon gas with upwards of 3,000 times the greenhouse warming potential of carbon dioxide. It is becoming more widely known that the healthcare sector is a significant contributor to greenhouse gas emissions, accounting for nearly 9% of the total in the U.S. But perhaps less recognized is that common substances routinely used in care—anesthetic gasses, even inhalers—are part of the problem.  Digging into the science, the irony becomes obvious: The mechanisms for keeping patients safe during a procedure or an asthma attack are also poisoning the environment. Desflurane, a common inhaled anesthetic, has heat-trapping properties hundreds to thousands of times greater than carbon dioxide. Meanwhile, nitrous oxide, a sedative used in surgery, has a warming potential 273 times that of carbon dioxide and greatly damages the ozone layer, which is crucial to absorbing harmful radiation from the sun. Even metered-dose inhalers that contain a seemingly small amount of hydrofluorocarbon gas have been shown to contribute meaningfully to emissions. At the end of last month, a small group of safety net hospitals and municipal health departments joined a 10-month collaborative in an effort to test decarbonization interventions and measure their results. The cohort, led by the Institute for Healthcare Improvement, includes sustainability professionals, clinicians and other healthcare leaders across six states. Each organization is expected to pick an improvement project and will receive support from experts and peers to carry it out. “For safety net hospitals who deliver care on tight margins and serve patient populations disproportionately impacted by the effects of climate change, investing in lowering carbon emissions holds the potential to advance their mission, strategy, and financial health,” the IHI said in its request for applications. The last IHI decarbonization cohort wrapped up in December 2023. More than a dozen organizations participated in 16 quality improvement projects. More than half measured reductions either in waste or purchases of anesthetic gas. Several sites decommissioned central nitrous oxide and eliminated desflurane from their operating rooms. Fierce Healthcare spoke to some of the participants about what they were able to achieve.   Thinking big by going small   Nitrous oxide, commonly used in dentistry, can be stored in one of two ways: in large centralized tanks that deliver the gas to operating rooms through piping, or in portable cylinder tanks. Choosing between the two might seem inconsequential. Indeed, one benefit of a centralized system is having to change the tanks less often. However, several hospitals around the world recently discovered that between 75% and 95% of their nitrous oxide leaks out through the central piping before it ever reaches the patient, wasting resources and polluting the atmosphere.  Addressing this waste was a goal for Northwell, which joined the last IHI decarbonization cohort. But to do so, the health system would need to figure out how the gas was being procured and used at its facilities. With more than 800 ambulatory sites and 20-odd hospitals, this would prove no small task.  “When you’re as big as we are, that was quite a challenge,” Teresa Murray Amato, M.D., VP of Clinical Sustainability and Resource Stewardship and director of geriatric emergency medicine at Northwell Health, told Fierce Healthcare. Amato started by looking for allies—“probably the best way to start,” she said—such as the head of anesthesia at her own hospital, who was really interested in sustainability. “Try to go for the easiest first,” Amato said.  As part of its emergency management work, Northwell works to ensure it has a three-day supply of essential resources. Keeping that in mind, Amato’s team decided to test if anesthesiologists would have enough nitrous oxide if they were to only use the portable tanks for three days. The clinicians recorded how long they used the gas and at what flow rates.  That information and anecdotal conversations revealed the gas was being used very infrequently, and Amato’s team extrapolated the use and determined that decommissioning centralized nitrous oxide systems would not negatively impact clinical practice. Spreading awareness of the environmental harms also reduced nonessential uses of nitrous oxide. For Northwell, eliminating the large tanks didn't mean getting rid of nitrous oxide altogether, it just consolidates resources in a more practical way. There are some cost savings too, though not as great as the enormous benefits to the environment. Even if someone left a portable tank open by accident and it leaked entirely, the impact would be a fraction of that of a centralized tank, according to Amato. “It’s such a simple solution, but it’s a challenge in a very large, highly matrixed organization,” Amato said. Armed with the success from this project, the proof of concept is now helping fuel other similar projects forward. “Now, everybody wants to be the next one to go,” Amato noted. Northwell’s new goal is to have almost no central tanks by the end of 2024.   The dark side of inhalers    To expel the medication, each puff of a metered-dose inhaler relies on a hydrofluorocarbon gas, a potent greenhouse gas with upwards of 3,000 times the warming potential of carbon dioxide. This gas contributes to climate change, which can lead to events that worsen asthma and in turn further drive up the use of inhalers. Though each inhaler seems small, 144 million were prescribed in the U.S. alone in 2020. And three-quarters of those contained the gas. In England, metered-dose inhalers have been found to represent 3% of total National Health Service carbon emissions, equal to the carbon emissions from all the electricity used by the NHS. “They actually have a pretty substantial contribution to healthcare’s overall environmental impact,” Gregg Furie, M.D., medical director for climate and sustainability at Brigham and Women's Hospital, told Fierce Healthcare.  Alternatives, like dry powder and soft mist inhalers, exist. This was an area Mass General Brigham wanted to tackle through the IHI collaborative. “We saw it as an opportunity to provide some structure to a project that we initially wanted to focus on,” Furie said.  The health system leaned on the institute's staff to learn how to bring a range of diverse stakeholders to the table. “There are way more stakeholders than you think there are,” Jonathan Slutzman, medical director for environmental sustainability at Mass General Hospital, told Fierce Healthcare. The collaboration also offered regular check-ins and a framework for accountability, which forced momentum internally, Slutzman added. Their goal was to reduce the overall share of metered-dose inhalers that are prescribed and to shift to alternatives where possible. They also hoped to raise awareness of the environmental impact of certain inhalers within the organization. They don't yet have concrete data on the project's outcomes to share. “This is not something that many practitioners are aware of,” Furie noted. “We are the ones who ultimately prescribe these devices… this is something that we feel we have real agency to do something about.” While the shift to alternative inhalers across the U.S. has been slow, Furie expects that to begin to change. That shift is being partly driven by increased scrutiny of the patent process and high prescription costs at the federal level. Apart from provider-driven efforts to move away from metered-dose inhalers, other solutions need to come from industry innovations and policy reform, per Furie. The FDA is studying the challenges of developing inhalers with a lower warming potential—but Slutzman believes the agency can be doing more to protect sustainability. One could argue that environmental impact should be a part of evaluating the safety of a drug, he said. “FDA should be able to encourage or make it easier for drug delivery devices that we know are environmentally better,” Slutzman said. “That would enable us to prescribe better inhalers.”   Centering climate as a broader priority    The idea for the collaboration began when the Agency for Healthcare Research and Quality commissioned IHI to develop a primer on decarbonization. IHI sought input from an expert panel, collaborating with the National Academy of Medicine and Health Care Without Harm to co-design resources along the way. “There are more and more organizations engaging in this area, which is very exciting and it is very important to ensure there is no duplication of efforts,” IHI Director Bhargavi Sampath, MPH, told Fierce Healthcare. Afterwards, the organization wanted to put the primer into practice by way of a collaborative, intended to inspire leader engagement, boost teams’ confidence and skills and build will across departments. “It really takes a very big group of people to do this work,” said IHI Project Director Becka DeSmidt.  Anyone looking for organizational buy-in on sustainability initiatives is advised to stress the potential for cost savings, the moral obligation to address the climate for patient health and that prioritizing such initiatives can help retain current or recruit future staff. “We’ve heard a lot from clinicians directly about how meaningful and engaging this work can be and clinician advocacy can be really powerful in helping an organization make these commitments,” DeSmidt told Fierce Healthcare. Two organizations participating in the collaborative successfully made the case for additional sustainability hires by leveraging the progress they were making on their projects as well as the cost savings they were seeing. Two others integrated clinical decarbonization metrics into their key performance indicators amid “the growing acceptance of integrating climate priorities into broader system strategies,” DeSmidt said. “Something we were really excited about was the promise of bringing climate more centrally into health system priorities,” Sampath echoed, “to advance some of the climate work that really needs to take place more locally at the frontline and with clinician leadership.”