Achalasia Addisonianism Alacrimia Syndrome

侃言 蛋白水解靶向嵌合体（PROTACs）是一种异双功能分子，通过诱导泛素-蛋白酶体系统(UPS)对靶标蛋白质进行泛素化和降解。这是一种事件驱动的作用机制，相比于小分子抑制剂有诸多优势，比如：催化性质的低浓度低剂量需求、减少给药量和给药频率、减少毒副作用、更加长效持久、有效降低药物耐药、靶点适用自由度高等，具有非常高价值的治疗应用前景。 然而，由于可利用的E3配体的开发和应用有限，高频过度的使用仅有的如VHL(60.1%)、CRBN(30.1%)配体（数据来自iScience)，导致PROTACs耐药性问题成为困扰当前PROTACs治疗的一大难题。因此，开发肿瘤组织特异性E3配体就变得非常重要，依靠肿瘤自身癌蛋白(oncoprotein)，将极大地降低突变耐药的机率。 近日，PROTACs奠基人、Arvians创始人、耶鲁大学著名科学家Craig Crews教授在JACS上刊载了其团队最新研究成果，突出强调基于肿瘤组织，特异性E3连接酶底物衔接蛋白配体的开发，在解决PROTACs耐药性突变问题上的重要性。 文章的第一作者李科博士为我多年好友。许多关键性问题，也和李博士进行了探讨。 文章于2024年8月27日在线，地址如下： https://doi.org/10.1021/jacs.4c05393 1 PROTACs耐药性 与     肿瘤特异性E3复合体蛋白   PROTACs作为一种异双功能分子，有效地诱导靶蛋白和E3连接酶邻近，形成三元复合物，使靶蛋白泛素化，进而被蛋白酶体识别后降解。然而，由于过度依赖和高频使用VHL、CRBN等E3连接酶配体，也出现了PROTACs耐药性问题。 依赖肿瘤自身癌蛋白，开发肿瘤组织特异性E3连接酶配体，是解决当前PROTACs耐药性问题的关键。 E3连接酶底物衔接蛋白：MAGE-A3 黑色素瘤抗原A3型蛋白(MAGE-A3)，是一种组织特异性表达的蛋白，也是一种E3连接酶的底物衔接蛋白，与E3连接酶结合，通过改变亚细胞位置或靶向泛素化的独特底物，来调节E3连接酶的活性。 MAGE蛋白家族包括I型(AMGE-A/B/C)和II型(AMGE-D/E/F/G/H/L/Necdin)两种抗原类型、以及众多亚族。其中，I型为MAGE睾丸癌抗原，主要在生殖组织中表达，若启动子去甲基化异常表达则易导致癌症；而II型MAGE蛋白可在多种组织中表达。 MAGE同源结构域(MHD)在I/II型蛋白中都是高度保守的。然而，尽管结构相似，MHD底物结合间隙中的独特残基，被赋予了与不同蛋白伴侣特异性结合的能力。 MAGE-A3就是这种具有与E3连接酶结合能力的组织特异性衔接蛋白，因此，极具潜力作为开发肿瘤特异性PROTACs的靶点： 能够作为E3连接酶底物招募模块 在体细胞组织中极少表达 能够在多种肿瘤类型中发现 最近的研究发现，MAGE-A3与三重基序蛋白28(TRIM28)相互作用，并调节TRIM28。而TRIM28的表达无处不在，具有多种功能，包括E3连接酶激活。 因此，由MAGE-A3配体组成的PROTACs，可以在表达MAGE-A3的肿瘤组织中，特异性地招募由TRIM28介导的靶蛋白泛素化。 MAGE-A3在免疫治疗中的应用 此外，诱导MAGE-A3降解，可能对癌细胞具有抗增殖作用。而MAGE蛋白家族本身就是癌症免疫治疗的热门靶点，已有多项临床试验针对MAGE-A3抗原。 PROTACs介导的MAGE-A3降解有可能增加抗原在同源MHC复合物上的呈递，进而改善免疫治疗的结果。 2 DNA编码化合物库技术（DEL) 助力     E3连接酶衔接蛋白MAGE-A3配体发现    研究人员利用DEL技术，针对MAGE-A3的MHD进行亲和力筛选，如上图A，包含约109个DEL化合物。 DEL具有广泛的靶点适用性，也非常适合缺乏酶功能的靶标、以及非活性位点抑制剂类配体的发现。并以发现的苗头化合物为起点，指导靶标特异性异双功能小分子的设计。 DEL苗头化合物发现和类药性优化 从这个包含约十亿多种化合物的超级化合物库中，对MAGE-A3同源结构域进行筛选后数据分析，最终发现了两个来自不同文库的相似的“化学型”家族：苯甲醛支架、氨基嘧啶支架，如上图B。 在这两个相似的“化学型”富集中，通过对全长分子的富集度、物化特性、组成合成砌块、以及起始原料的综合考虑分析对比，最终选择了一些列分子进行off-DNA合成、验证。化合物KL346就是其中用于后续验证的代表，如下图。 通过对KL346和MAGE-A3 MHD进行ITC生物物理表征，发现了低微摩尔结合亲和力，如上图B，KD=8.1±2.3 μM。同时也发现了KL346:MAGE-A3复合体构成的化学计量比例为1：2（N~0.5)。 配体“成药性”也是PROTACs成功的关键，出于此考虑，研究人员根据采用常见的策略来截断DEL苗头化合物，获得化合物MJBIA9836(如上图A)：去除组成苗头化合物中靠近寡核苷酸标签的合成砌块，一般认为其多作为间隔而不是药效团，对配体亲和结合力贡献较小。 如上图B，结果也证实，截取后的分子保留了相当的结合亲和力，但其类药性特性却获得了极大的改善。 如上图C，通过细胞热移试验(CETSA)，MJBIA9836相比于DMSO对照组，稳定了两个MAGE-A3阳性细胞系内源性MAGE-A3或增加了蛋白水平。这表明，改善后的化合物具有细胞渗透性。 靶标:配体相互作用的结构表征 如上图，研究人员利用分子对接技术，通过MAGE-A3晶体结构，评估了MJBIA9836的潜在结合位点，并对观察到的1:2结合化学计量进行了合理化。 单体与二聚体模型对接结果揭示了一个相似的结合位点。在没有变构的情况下，二聚体模型中的结合界面是位于预测的显示1：2结合化学计量的结合位点。因此，研究人员进行了进一步的结构表征以验证配体结合位点和辅助基于结构的配体优化。 然而，MJBIA9836的溶解度有限，研究人员获得了其吗啉类似物KL861，其结合亲和力与MJBIA9836相当，但溶解度提高，并用于单晶结构分析，如下图。 单晶衍射与分子对接结果一致，疏水作用是配体结合的驱动力。同时，也再次印证了单个配体分子与MAGE-A3二聚体结合。 为了确定MAGE-A3与配体的生物活性位点，研究人员利用靶标突变体进行进一步的研究。观察到R211A侧链靠近二聚体界面，并且位于配体羰基的氢键距离内，研究人员获得了R211A突变的MAGE-3A MHD，如上图B。 3    MAGE-A3配体驱动的PROTACs    在获得了MJBIA9836在体外、结构、以及细胞内均能够结合MAGE-A3的证实后，研究人员以此进行PROTACs设计，并验证MAGE-A3配体在靶向降解剂中的实用性。 PROTACs设计及验证 如上图，研究人员首先基于VHL和CRBN设计PROTACs，进行概念性验证（POC）。然而，仅基于VHL招募的PROTACs以剂量依赖性的方式降低MAGE-A3的水平，并观察到VHL蛋白水平升高，如下图B。 最有效的PROTAC为KL465，如上图A，装配有（rh)-VHL。有效降低HeLa细胞MAGE-A3水平，Dmax~60%，DC50~2μM。 VHL的重要性 为了进一步验证VHL在靶标降解中的重要性，研究人员设计了KL465的无活性版本——KL633，如下图B。在KL633组成中，对于VHL结合至关重要的羟基脯氨酸部分，其立体化学倒置，因此在保持了相当的物化特性的同时，不具有VHL招募活性。 在上图A中可以看出，两种浓度下，KL465能够诱导靶标有效降解；但KL633、MJBIA9836并不能显著降低MAGE-A3的蛋白水平。这表明，KL465降解靶标是VHL依赖性的。 如图C，KL465在MAGE-A3阳性细胞中，其对细胞增殖的抑制作用更大。这表明，与单独结合配体相比，MAGE-A3的降解显著降低了细胞活力。 总之，这些数据表明，使用KL465降解MAGE-A3可以降低癌细胞活力，并且这种观察到的效果不是由于脱靶毒性导致。 KL465不影响MAGE-A3:TRIM28相互作用 利用MAGE-A3配体作为肿瘤特异性TPD的E3招募元件，还需要小分子配体不破坏MAGE-A3:TRIM28的相互作用，验证如下图。 利用TR-FRET试验，最终表明，基于MAGE-A3的配体或基于配体设计的PROTAC都不能与TRIM28竞争性结合MAGE-A3，不影响其相互作用。 4       总结与展望     当前的PROTACs设计和应用，依然依赖于VHL和CRBN，两者都是Cullin-E3复合物的E3连接酶衔接蛋白。然而，它们的广泛表达限制了其组织和疾病特异性降解。而且，高频过度的使用，也出现了耐药性问题。这主要归因于E3连接酶机制的突变，而不是靶蛋白，因此，发现更多E3连接酶配体将势在必行。 而compartment-特异性降解在这个形势下将具有远大的前景。PROTACs开发的下一步逻辑必将是在特定组织或疾病状态中，利用E连接酶靶向降解。 然而，针对E3连接酶底物结合位点可能需要占据较浅的口袋或内源性蛋白质底物的竞争，这两种情况都为E3连接酶结合带来挑战。幸运的是DEL技术为此提供了一种快速高效的组合方法。DEL技术已经成功发现多种E3连接酶伙伴配体，如DCAF1、GID4、MAGE-A3等，助力PROTACs开发。 整合DEL筛选数据、与机器学习（ML）强大能力，可以增强可扩展性、结构多样性、总体命中率、效力和类药性。 本文中KL465对靶标适度降解，可能有以下几个因素： 在组织培养中观察到的MAGE-A3半衰期短，重合成速度快，这限制了观察到的最大降解； 开发PROTACs使用的配体仅表现出中等结合亲和力，减少了低效三元复合物的形成； PROTACs的连接子还没有进一步探索优化。 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↓

Hamilton, Bermuda, July 19, 2024 (GLOBE NEWSWIRE) -- Study shows significant reduction in aorta dilation, delayed rupture and lower mortality in established animal model of abdominal aortic aneurysmAltamira’s SemaPhore™ nanoparticles delivering SOD2 mRNA successfully to mitochondria in aorta wall Positive outcomes suggest potential use of treatment in management of small abdominal aortic aneurysm and prevention of ruptures Altamira Therapeutics Ltd. (“Altamira” or the “Company”) (Nasdaq:CYTO), a company dedicated to developing and commercializing RNA delivery technology for targets beyond the liver, today announced the preprint publication of a study demonstrating effective treatment of abdominal aortic aneurysm (AAA) in an animal model.1 The study was conducted by a research group from Washington University, St. Louis MO, and the University of South Florida, Tampa FL. It showed that treatment with SOD2 mRNA delivered systemically with peptide-based nanoparticles (SemaPhore™ by Altamira) to AAA mice resulted in a significant reduction in aorta dilation (p<0.05), delayed rupture and a highly significant improvement in survival rates (p<0.01) compared to untreated controls. AAA is a localized abnormal enlargement (bulge) of the abdominal aorta, i.e. the part of the main artery which runs through the belly. The rupture of an AAA may be life-threatening; more than 50% of patients die before they reach the emergency room, and those who survive have very high morbidity.2 The prevalence of AAAs increases with age and is 4-6 times more common in men than in women; it develops in approximately 1% of men between 55 and 64 years of age, and increases by 2 to 4% per decade thereafter.3 Surgery is the main treatment for large AAAs or those that are growing rapidly. AAA is an inflammatory disease involving oxidative stress caused by excessive levels of reactive oxygen species (ROS). Although the use of antioxidants would appear a promising treatment strategy, clinical efficacy has turned out to be mostly unsatisfactory. By targeting SOD2 (superoxide dismutase 2), an enzyme known for its capacity to eliminate ROS, the researchers used a different approach. They delivered SOD2 mRNA through systemic injections of Altamira’s peptide-based SemaPhore nanoparticles in an established murine AAA model and were thus able to boost mitochondrial SOD2 expression, reduce levels of oxidative stress and in turn mitigate the expansion of small AAA and largely prevent rupture. The research group concluded: “This nanotherapeutic mRNA delivery approach may find translational application in the medical management of small AAA and the prevention of AAA rupture.” “Using SOD2 mRNA to modulate oxidative stress appears a very promising approach in various challenging cardiovascular disorders such as abdominal aortic aneurysm or atherosclerosis and in other inflammatory or degenerative disease where ROS is a critical disease driver”, commented Samuel Wickline, M.D., Chief Scientific Adviser of Altamira and one of the co-authors of the study. “Importantly, the SemaPhore nanoparticles allowed for systemic delivery of the mRNA payload with efficient uptake and SOD2 expression in the aortic wall. Moreover, there was a good safety profile with no sustained accumulation or SOD2 expression in major organs, and no change in hematologic parameters or liver / kidney function. Last, but not least, the nanoparticles showed good stability over time.” About Altamira Therapeutics Altamira Therapeutics (Nasdaq: CYTO) is developing and supplying peptide-based nanoparticle technologies for efficient RNA delivery to extrahepatic tissues (OligoPhore™ / SemaPhore™ platforms). The Company currently has two flagship siRNA programs using its proprietary delivery technology: AM-401 for KRAS driven cancer and AM-411 for rheumatoid arthritis, both in preclinical development beyond in vivo proof of concept. The versatile delivery platform is also suited for mRNA and other RNA modalities and made available to pharma or biotech companies through out-licensing. In addition, Altamira holds a 49% stake (with additional economic rights) in Altamira Medica AG, its commercial-stage legacy asset Bentrio®, an OTC nasal spray for allergic rhinitis. Further, the Company is in the process of partnering / divesting its inner ear legacy assets. Founded in 2003, Altamira is headquartered in Hamilton, Bermuda, with its main operations in Basel, Switzerland. For more information, visit: https://altamiratherapeutics.com/ Forward-Looking Statements This press release may contain statements that constitute "forward-looking statements" within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended. Forward-looking statements are statements other than historical facts and may include statements that address future operating, financial or business performance or Altamira’s strategies or expectations. In some cases, you can identify these statements by forward-looking words such as "may", "might", "will", "should", "expects", "plans", "anticipates", "believes", "estimates", "predicts", "projects", "potential", "outlook" or "continue", or the negative of these terms or other comparable terminology. Forward-looking statements are based on management's current expectations and beliefs and involve significant risks and uncertainties that could cause actual results, developments and business decisions to differ materially from those contemplated by these statements. These risks and uncertainties include, but are not limited to, the success of strategic transactions, including licensing or partnering, with respect to Altamira’s legacy assets, Altamira’s need for and ability to raise substantial additional funding to continue the development of its product candidates, the clinical utility of Altamira’s product candidates, the timing or likelihood of regulatory filings and approvals, Altamira’s intellectual property position and Altamira’s financial position, including the impact of any future acquisitions, dispositions, partnerships, license transactions or changes to Altamira’s capital structure, including future securities offerings. These risks and uncertainties also include, but are not limited to, those described under the caption "Risk Factors" in Altamira’s Annual Report on Form 20-F for the year ended December 31, 2023, and in Altamira’s other filings with the Securities Exchange Commission (“SEC”), which are available free of charge on the SEC’s website at: www.sec.gov. Should one or more of these risks or uncertainties materialize, or should underlying assumptions prove incorrect, actual results may vary materially from those indicated. All forward-looking statements and all subsequent written and oral forward-looking statements attributable to Altamira or to persons acting on behalf of Altamira are expressly qualified in their entirety by reference to these risks and uncertainties. You should not place undue reliance on forward-looking statements. Forward-looking statements speak only as of the date they are made, and Altamira does not undertake any obligation to update them in light of new information, future developments or otherwise, except as may be required under applicable law. Investor Contact: Hear@altamiratherapeutics.com 1 Yan et al. (2024), Systemic delivery of murine SOD2 mRNA to experimental abdominal aortic aneurysm mitigates expansion and rupture, bioRxiv: 2024.06.17.599454. 10.1101/2024.06.17.599454 2 Shaw et al. (2024), Abdominal aortic aneurysm, StatPearls. https://www.ncbi.nlm.nih.gov/books/NBK470237/ 3 Aggarwal et al. (2011), Abdominal aortic aneurysm: A comprehensive review, Exp Clin Cardiol 16(1): 11–15.

Phase II/III clinical trial to initiate enrollment in Q4 2023 TEMPE, Ariz.--(BUSINESS WIRE)-- Nectero Medical, a clinical-stage biotechnology company pioneering novel therapies to treat aneurysmal disease, today announced that the U.S. Food and Drug Administration (FDA) has granted Breakthrough Therapy designation for the Nectero Endovascular Aneurysm Stabilization Treatment (Nectero EAST®) System to treat patients with infrarenal AAAs, maximum diameter 3.5 – 5.0cm. Nectero Medical is initiating a randomized, controlled Phase II/III clinical trial (stAAAble) to evaluate the product’s safety and effectiveness with first subject enrollment targeted in Q4 2023. Breakthrough Therapy designation is an FDA program intended to facilitate design and conduct of an efficient product development program. It is a challenging status to obtain. Designation requires that the novel drug treats a serious condition, and preliminary clinical evidence indicates that the drug may provide a substantial improvement over available therapy on a clinically significant endpoint(s). Nectero Medical’s Breakthrough Therapy designation is supported by results from the first-in-human (FIH) study (NCT05133492) of the Nectero EAST System. Early clinical results of the FIH study were recently published in the Journal of Vascular Surgery and showed that a single, localized PGG administration to patients with small- to medium-sized infrarenal AAAs was safe and demonstrated the potential to slow the growth of AAAs. Additional follow-up data will be presented at the VEITH Symposium in New York later this year. In addition to the benefits of Fast Track (received in August 2023), which allows for more frequent FDA interactions and eligibility for priority and rolling review, Breakthrough Designation adds FDA’s organizational commitment to involve senior managers. Nectero Medical will meet with the Agency near term to review the development program and establish a plan for ongoing communications. AAA is a serious condition associated with the potential for rupture and sudden death. The Nectero EAST System is intended to treat early, in small to mid-size aneurysms, and potentially avoid the need for a future, more invasive procedure that carries greater risks of morbidity and mortality. “This designation reinforces Nectero Medical’s belief that the Nectero EAST System has the potential to have a significant impact for patients with AAA disease,” said Jack Springer, President and Chief Executive Officer of Nectero Medical. “We look forward to working closely with the FDA to advance this product to patients as quickly and efficiently as possible.” About the Nectero EAST® System The Nectero EAST System is a single-use, endovascular system for the treatment of infrarenal abdominal aortic aneurysms (AAA). It is estimated that >1M Americans are living with AAA and ~60,000 undergo treatment each year; complications of AAA account for approximately 10,000 deaths in the U.S. annually. Available treatments are primarily reserved for AAAs larger than 5.0/5.5cm (female/male) in diameter, symptomatic patients, or rapidly expanding aneurysms. Smaller AAAs are traditionally monitored with serial ultrasound or CT surveillance. Patients with small- and mid-sized aneurysms carry a .5-5% annual risk of rupture. The Nectero EAST System is comprised of a dual-balloon delivery catheter and stabilizer mixture containing pentagalloyl glucose (PGG). The system delivers PGG locally into the aneurysmal wall where it binds to elastin and collagen to strengthen the aortic vessel wall and to potentially reduce the risk of further degradation. The procedure does not require any specialized tools, takes less than an hour to complete, leaves no permanent implant behind and does not preclude any future interventions. If successful, the Nectero EAST System may offer patients with smaller AAAs a first approved therapeutic option beyond surveillance. For more information, please visit necteromedical.com. CAUTION: There is no assurance as to the results of the study described in this press release or the efficacy of the Nectero EAST System.