更新于：2024-05-01

ALOX12

12-脂氧合酶

更新于：2024-05-01

基本信息

别名

花生四烯酸12-脂氧合酶、12LO、12S-lipoxygenase

+ [13]

简介

Catalyzes the regio and stereo-specific incorporation of molecular oxygen into free and esterified polyunsaturated fatty acids generating lipid hydroperoxides that can be further reduced to the corresponding hydroxy species (PubMed:17493578, PubMed:1851637, PubMed:8319693, PubMed:8500694, PubMed:18311922, PubMed:32404334). Mainly converts arachidonate ((5Z,8Z,11Z,14Z)-eicosatetraenoate) to the specific bioactive lipid (12S)-hydroperoxyeicosatetraenoate/(12S)-HPETE (PubMed:17493578, PubMed:22984144, PubMed:24282679, PubMed:8319693, PubMed:8500694). Through the production of bioactive lipids like (12S)-HPETE it regulates different biological processes including platelet activation (PubMed:8319693, PubMed:8500694). It can also catalyze the epoxidation of double bonds of polyunsaturated fatty acids such as (14S)-hydroperoxy-docosahexaenoate/(14S)-HPDHA resulting in the formation of (13S,14S)-epoxy-DHA (PubMed:23504711). Furthermore, it may participate in the sequential oxidations of DHA ((4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoate) to generate specialized pro-resolving mediators (SPMs) like resolvin D5 ((7S,17S)-diHPDHA) and (7S,14S)-diHPDHA, that actively down-regulate the immune response and have anti-aggregation properties with platelets (PubMed:32404334). An additional function involves a multistep process by which it transforms leukotriene A4/LTA4 into the bioactive lipids lipoxin A4/LXA4 and lipoxin B4/LXB4, both are vasoactive and LXA4 may regulate neutrophil function via occupancy of specific recognition sites (PubMed:8250832). Can also peroxidize linoleate ((9Z,12Z)-octadecadienoate) to (13S)-hydroperoxyoctadecadienoate/ (13S-HPODE) (By similarity). Due to its role in regulating both the expression of the vascular endothelial growth factor (VEGF, an angiogenic factor involved in the survival and metastasis of solid tumors) and the expression of integrin beta-1 (known to affect tumor cell migration and proliferation), it can be regarded as protumorigenic (PubMed:9751607, PubMed:16638750, PubMed:22237009). Important for cell survival, as it may play a role not only in proliferation but also in the prevention of apoptosis in vascular smooth muscle cells (PubMed:23578768).

关联

项与 ALOX12 相关的药物

SRI-11226

靶点

5-LOX x ALOX12

作用机制

5-LOX抑制剂 [+1]

在研机构

SRI International

原研机构

SRI International

在研适应症

阿尔茨海默症

非在研适应症-

最高研发阶段临床前

首次获批国家/地区-

首次获批日期1800-01-20

SRI-31291

靶点

12/15-LOX x 5-LOX x ALOX12

作用机制

12/15-LOX抑制剂 [+2]

在研机构

Western University of Health Sciences [+1]

原研机构

Western University of Health Sciences

在研适应症

创伤性脑损伤

非在研适应症-

最高研发阶段临床前

首次获批国家/地区-

首次获批日期1800-01-20

SKL-11197

靶点

12/15-LOX x ALOX12 x HCN channels x VDCCs

作用机制

12/15-LOX抑制剂 [+3]

在研机构-

原研机构

SK Biopharmaceuticals Co., Ltd.

在研适应症-

非在研适应症

糖尿病性神经病变 [+2]

最高研发阶段终止

首次获批国家/地区-

首次获批日期1800-01-20

项与 ALOX12 相关的临床试验

NCT01521598 / Completed临床2期

A Phase 2, Multicenter, Randomized, Double-blind, Placebo Controlled Study Assessing the Efficacy and Tolerability of SKL11197 for the Pain of Diabetic Peripheral Neuropathy

Primary Objective: To evaluate the efficacy of SKL11197 for the treatment of diabetic peripheral neuropathy pain (DPN).
Secondary Objective: To evaluate the safety and tolerability of SKL11197 in subjects with painful diabetic peripheral neuropathy.
Primary Efficacy Endpoint: The primary efficacy outcome variable will be the time to exit from the double-blind phase because of inadequate pain relief.

开始日期2012-01-01

申办/合作机构

SK Life Science, Inc.

100 项与 ALOX12 相关的临床结果

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100 项与 ALOX12 相关的转化医学

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0 项与 ALOX12 相关的专利（医药）

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1,310

项与 ALOX12 相关的文献（医药）

2024-12-01·Renal failure

Elevated ALOX12 in renal tissue predicts progression in diabetic kidney disease.

Article

作者: Meixi Wang ; Jingjing Wang ; Jinni Wang ; Yonggui Wu ; Xiangming Qi

Diabetic kidney disease (DKD) is one of the major causes of end-stage renal disease and one of the significant complications of diabetes. This study aims to identify the main differentially expressed genes in DKD from transcriptome sequencing results and analyze their diagnostic value. The present study sequenced db/m mouse and db/db mouse to determine the ALOX12 genetic changes related to DKD. After preliminary validation, ALOX12 levels were significantly elevated in the blood of DKD patients, but not during disease progression. Moreover, urine ALOX12 was increased only in macroalbuminuria patients. Therefore, to visualize the diagnostic efficacy of ALOX12 on the onset and progression of renal injury in DKD, we collected kidney tissue from patients for immunohistochemical staining. ALOX12 was increased in the kidneys of patients with DKD and was more elevated in macroalbuminuria patients. Clinical chemical and pathological data analysis indicated a correlation between ALOX12 protein expression and renal tubule injury. Further immunofluorescence double staining showed that ALOX12 was expressed in both proximal tubules and distal tubules. Finally, the diagnostic value of the identified gene in the progression of DKD was assessed using receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) value for ALOX12 in the diagnosis of DKD entering the macroalbuminuria stage was 0.736, suggesting that ALOX12 has good diagnostic efficacy. During the development of DKD, the expression levels of ALOX12 in renal tubules were significantly increased and can be used as one of the predictors of the progression to macroalbuminuria in patients with DKD.

2024-02-15·International journal of molecular sciences

Loss of 15-Lipoxygenase in Retinodegenerative RCS Rats.

Article

作者: Andrew James Mead ; Kabir Ahluwalia ; Brandon Ebright ; Zeyu Zhang ; Priyal Dave ; Zeyang Li ; Eugene Zhou ; Aditya Anil Naik ; Rachael Ngu ; Catherine Chester ; Angela Lu ; Isaac Asante ; Dimitrios Pollalis ; Juan Carlos Martinez ; Mark Humayun ; Stan Louie

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

2024-01-05·Computers in biology and medicine

A novel PCDscore based on programmed cell death-related genes can effectively predict prognosis and therapy responses of colon adenocarcinoma.

Article

作者: Yangjie Peng ; Cheng Ouyang ; Yijun Wu ; Rui Ma ; Hao Li ; Yanke Li ; Jingjing Jing ; Liping Sun

Emerging evidence suggests a correlation between oncogenesis and programmed cell death (PCD). However, comprehensive studies that incorporate all identified PCD-related genes to guide colon adenocarcinoma (COAD) prognosis and precision treatment strategies are lacking. In this study, a series of bioinformatics analyses were comprehensively conducted using data from the TCGA-COAD, GSE17538, and GSE39582 cohorts. A total of 21 PCD-associated prognostic genes were identified through univariate Cox analysis. LASSO and multivariate Cox methods were employed to establish a prognostic gene signature (ALOX12, HSPA1A, IL13, MID2, RFFL, and SLC39A8) and the corresponding scoring system, termed PCDscore, which exhibited robust predictive ability. The ssGSEA and ESTIMATE algorithms were utilized to evaluate the tumor microenvironment of COAD. The high PCDscore group demonstrated a poorer prognosis, characterized by lower CD4⁺ T cell infiltration and a higher stromal score. In contrast, the low PCDscore group exhibited sensitivity to common chemotherapy drugs such as Cisplatin and 5-Fluorouracil. Single-cell sequencing analysis further revealed that the high-PCDscore group displayed a lower proportion of CD4⁺ T cells. Colorectal cancer samples from the years 2013-2017 were employed to validate the PCDscore, while those from 2018 to 2019 served as a temporal external validation set for the PCDscore. In vitro experimental results indicated that the overexpression of SLC39A8 inhibited the proliferation and invasion of colorectal cancer cells. The study developed a novel PCDscore system based on the analysis of genes related to all identified PCD types, providing valuable insights into clinical prognosis and drug sensitivity for patients with COAD.

项与 ALOX12 相关的新闻（医药）

2023-09-15

·CPHI制药在线

研发 | 简述慢性咳嗽治疗靶点研究进展

关注并星标CPHI制药在线咳嗽是一种复杂的神经生理反射。咳嗽感受器以受体和离子通道的形式位于气道感觉神经末梢，在受到刺激后产生信号，经迷走神经传入脑干咳嗽中枢，再经传出神经活化相应肌群产生咳嗽。因而，将咳嗽感受器作为咳嗽治疗靶点成为镇咳新药研发的热点。 1、外周神经元靶点外源性刺激诱导外周感觉神经元的功能或表型变化，并上调宿主的咳嗽反应。对靶向外周神经药物的研究，可以有效的避免中枢神经系统带来的副作用。到目前为止，嘌呤能P2X3受体和瞬时受体家族是很具希望的外周治疗靶点。 1.1 瞬时受体电位通道（transient receptor potential chanels，TRP） TRP家族是一类分布广泛的非选择性阳离子通道，可作为细胞传感器对细胞环境中的各种刺激作出反应，如温度、化学物质、拉伸、渗透压、酸碱度和氧化等。与咳嗽相关的TRP通道包括 TRPV1，TRPA1，TRPV4 和TRPM8。 ①TRPV1/TRPA1。TRPV1和 TRPA1均为钙离子渗透通道，TRPV1主要分布在无髓鞘的C纤维和有髓鞘的Aδ 纤维上，可以由辣椒素、低PH、白藜芦醇毒素和内源性介质等直接刺激激活，而TRPA1主要分布于无髓鞘的C纤维上，可由天然产物肉桂醛、氧化应激产物和环境刺激物臭氧等直接激活。TRPV1和TRPA1通道在结构和功能上紧密相连，TRPV1通道的Ca2+流入可激活TRPA1通道；TRPA1拮抗剂AP-18可部分抑制吸入肉桂醛导致的豚鼠咳嗽，但当与 TRPV1拮抗剂联合使用时，则可全部消除肉桂醛诱导的咳嗽。联合阻断TRPV1和TRPA1通道或环氧合酶（COX）和12-脂氧合酶（12-lipoxygenase，12-LOX）受体能明显抑制吸入缓激肽诱导的咳嗽和气道阻塞，表明同时阻断TRPV1和TRPA1通道对咳嗽和气道阻塞具有协同抑制作用。 ②TRPV4。TRPV4 是一种渗透传感器，主要表达于感觉神经中的三叉神经节和背根神经节，在迷走神经伤害性感受器上表达较少，可以被内源性物质或外部化学物质激直接激活，也可通过细胞内信号通路被间接激活。研究表明，TRPV4 的激活与迷走神经终末端附近的附属细胞释放 ATP的相关性比与神经细胞的相关性更大，选择性的P2X3拮抗剂AF-353被证明可以抑制 TRPV4在动物和人类中引发的神经去极化和咳嗽，表明 TRPV4 可以介导ATP的释放。Pannexin 1是一个大电导离子孔，允许细胞内的ATP外流，在 Pannexin 1基因敲除小鼠的迷走神经组织中，对TRPV4 的反应被取消，表明TRPV4 介导ATP 的释放可能需要 Pannexin 1的参与，靶向Pannexin 1-TRPV4-ATP轴可能为慢性咳嗽提供一种新的治疗手段。 ③TRPM8。TRPM8通道属于配体门控冷感觉离子通道，具有电压传感和离子选择功能，广泛分布于背根神经节、三叉神经节及气道迷走神经节，通过控制细胞内Ca2+浓度调节咳嗽反射。在8℃～28℃条件下，TRPM8通道能被天然或人工合成的冷物质模拟剂如薄荷醇、桉油精、留兰香等物质激活。TRPM8通道激动剂薄荷醇已被广泛用于镇咳治疗，但薄荷醇还可影响除TRPM8通道以外的其他离子通道。有研究认为，鼻腔吸入薄荷醇的止咳效果是由于鼻三叉传入神经上表达TRPA1- /TRPV1- /TRPM8+的神经元亚群的激活，而非迷走支气管肺感觉神经的激活，不过该研究并不能排除通过其他机制所产生的作用。 1.2 P2X3受体 P2X3受体是由 ATP激活位于迷走神经感觉纤维上的阳离子通道，主要分布于感觉神经细胞中，包括三叉神经、背根神经节和结状神经节。在炎症呼吸道中，由于细胞的损伤、缺氧或者应激，ATP大量释放到细胞外空间，并作为信号分子促进疾病和炎症的发展。ATP也能通过替代免疫细胞上表达的嘌呤能受体来调节炎症。由于ATP与 P2X3受体结合，产生诱发咳嗽的动作电位，P2X3受体抑制剂如Gefapixant，Eliapixant和Sivopixant成为研究热点。Gefapixant Ⅲ期临床试验疗效结果显示，患者的咳嗽频率、严重程度、生活质量都得到了明显的改善，味觉不良反应事件较轻且停药后可逆，更重要的是保护性咳嗽不受治疗影响。因此，服用 Gefapixant对难治性或不明原因的慢性咳嗽有效，是一种可接受、具有安全性的治疗方式，近期Gefapixant已经被批准在日本上市，与此同时也在寻求欧盟和美国的上市批准。Eliapixant作为P2X3受体拮抗剂，被认为是治疗慢性咳嗽的有效途径，能够显著减少咳嗽频率和严重程度，且其选择性高，味觉不良反应发生率低，表现出良好的耐受性。研究人员推测 Eliapixant具有普适性，但还需进一步研究。Sivopixant也是一个具有高度选择性的P2X3 受体拮抗剂，在24h内每小时咳嗽频数显著减少，提高了患者生活质量，很少出现味觉相关不良反应，没有出现因味觉障碍而停止治疗的患者。 2、中枢神经元靶点重复的外周刺激会导致中枢神经系统的变化，但无论是何种方式激活迷走神经传入，都是向脑干核团，主要是孤束核（nucleus tractus solitarii，NTS）提供输入。参与呼吸道感觉处理的中枢神经可塑性改变是咳嗽的重要驱动因素。因此，中枢神经系统相比外周神经系统靶点而言具有潜在优势，可能具有调节脑干和更高级中枢通路的潜在优势。 2.1 电压门控钠通道（voltage-gated sodium channel,NaV） NaV是动作电位的启动和传导所必需的，NAV 1.8作为NAVS通道的一个亚型，在迷走感觉神经元的兴奋性和动作电位放电能力中起着重要的作用，在炎症和特定炎症介质存在的情况下可以激活 NAV 1.8。研究发现，使用神智清醒的豚鼠咳嗽模型，于静脉注射途径急性暴露于前列腺素E2（PGE2）导致的咳嗽反射敏化，同时使用EP3拮抗剂L-798，106能显著降低咳嗽反应，NAV 1.8 拮抗剂A-803467同样的呈剂量依赖性地抑制咳嗽反应。这项研究提示 PGE2 通过EP3受体依赖的 NAV 1.8 通道激活咳嗽反射。因此，靶向中枢EP3受体和 NAV 1.8 亚型通道可能是治疗咳嗽过敏综合征的一种新途径。 2.2 神经激肽-1受体（neurokinin-1，NK-1）速激肽是一类兴奋性神经肽，包括人血清P物质（SP）、神经激肽NKA和NKB等。速激肽主要由迷走神经纤维产生并释放到气道外周，广泛分布于中枢和外周神经系统。豚鼠长时间暴露在二手烟烟雾中会增强突触传递，这一现象能被NK-1拮抗剂所逆转，柠檬酸诱导的咳嗽反应也能被NK-1拮抗剂逆转。动物实验研究结果表明，在中枢神经系统，SP和NK-1受体在神经传递中发挥重要作用。此外，SP可以激活迷走神经纤维，该作用也能被NK-1拮抗剂阻断，表明这一机制在外周神经系统中也能发挥作用。特发性肺纤维化（IPF）和急性咳嗽患者吸入SP后咳嗽反应增加，咳嗽患者体内SP水平升高，提示SP可能是咳嗽反应的重要介质，而 NK-1受体是重要的治疗靶点。 2.3尼古丁胆碱能受体（neuronalnicotinic acetylcholine receptor，nAChR）α7 中枢神经系统中分布最多的是α4β2和 α7 两种亚型，α7 nAChR是nAChRs家族中的一种特殊的亚型，当激动剂结合到α7 nAChR的配体结合域时，可迅速引起中央离子通道的开放，且α7 nAChR对 Ca2+有非常高的通透性，可通过 Ca2+内流或非Ca2+依赖性途径引起细胞内信号转导引发咳嗽。研究证明烟碱受体亚型在豚鼠中的镇咳作用，其中亚型α4β2选择性激动剂Tc-6683对豚鼠的诱发性咳嗽反应没有影响，而亚型α7选择性激动剂PHA543613能剂量依赖性地抑制诱发性咳嗽。 2.4 γ-氨基丁酸受体（GABA） GABA 在中枢神经系统中被广泛分布和利用。加巴喷丁是GABA 的结构类似物。研究报道，加巴喷丁能够显著改善咳嗽症状，降低咳嗽频率和咳嗽严重程度，提高患者生活质量。不过调查发现仍有约40%的患者对加巴喷丁治疗无效，因此，需要加强对加吧喷丁治疗有效患者的特征筛选，以期提高该药使用的成功率。参考资料 [1]刘佳,罗云,潘云风等.慢性咳嗽潜在治疗靶点研究进展[J].中国药理学通报,2023,39(08):1426-1429. [2]姚玥,吴灏,张家硕等.咳嗽治疗靶点及其新药研究进展[J].中国新药杂志,2023,32(15):1538-1545. 作者简介：小米虫，药品质量研究工作者，长期致力于药品质量研究及药品分析方法验证工作，现就职于国内某大型药物研发公司，从事药品检验分析及分析方法验证。智药研习社近期课程报名来源：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~

2023-06-20

·GlobeNewswire-Health Care

Veralox Names Jonathan Mow as Chief Executive Officer as the Company Advances Development of First-in-Class Therapies for Immune-Mediated Diseases in Conjunction with Financing

-- New Financing of $24 Million Fully Funds the Phase 2 Clinical Program for Lead Candidate VLX-1005 -- Company Prepares to Evaluate VLX-1005 in Heparin-Induced Thrombocytopenia (HIT) FREDERICK, Md., June 20, 2023 (GLOBE NEWSWIRE) -- Veralox Therapeutics, a clinical-stage biotechnology company developing a new class of therapies targeting the 12-lipoxygenase (12-LOX) pathway to address some of medicine’s most persistent and serious immune-mediated diseases, today announced the appointment of Jonathan Mow as the company’s new chief executive officer. Mr. Mow’s appointment comes as Veralox secured $24 million in funding to advance VLX-1005 through a Phase 2a proof-of-concept study evaluating its impact on heparin-induced thromobcytopenia (HIT), a life-threatening rare disease caused by an aberrant immune response to heparin exposure. The investment round included new investors Pappas Capital and NYBC Ventures and existing investors Hatteras Venture Partners, Sanofi Ventures, JDRF T1D Fund and Genesys Capital, amongst others. In conjunction with the financing, the company welcomes Peter Young of Pappas Capital as a director and Meg Wood of NYBC Ventures as an observer. “VLX-1005 has great promise to revolutionize the treatment of HIT and other immune-mediated diseases,” Mr. Young said. “I am thrilled to join the board at such an exciting time, and to be working with a leader of Jonathan’s caliber to move into later stages of clinical development.” “This is an exciting time for Veralox as we head into our proof-of-concept Phase 2 study with VLX-1005 for HIT, a serious complication subsequent to heparin exposure that is accompanied by significant morbidity and mortality,” Mr. Mow said. “Our novel approach with 12-LOX inhibitors has great potential in this and other diseases and I would like to thank our investors for their financial support of our important mission and giving me the opportunity to lead this world-class effort and team.” Mr. Mow brings more than 25 years of accomplishments in biotechnology management to Veralox, most recently serving as CEO of PhaseBio Pharmaceuticals. At PhaseBio, he led the company’s scientific and business transformations, guiding the company from early-stage research to Phase 3 development, and through a successful initial public offering in 2018. Earlier in his career, Mr. Mow served as vice president, business development for Amylin Pharmaceuticals until its sale to Bristol-Myers Squibb in 2012; was co-founder and vice president, commercial and business development of Corus Pharma, Inc. until its acquisition by Gilead Sciences in 2006; and headed business development for PathoGenesis Corporation until its acquisition by Chiron Corporation in 2000. Mr. Mow has also held positions in marketing, marketing research and sales at Bristol-Myers Squibb, Wyeth/Lederle International and Syntex Laboratories. He holds a B.S. in mechanical engineering from University of California at Berkeley and M.B.A. from Carnegie Mellon University’s Tepper School of Business. About Veralox TherapeuticsVeralox Therapeutics Inc. is the clinical leader in developing first-in-class therapeutics targeting 12-lipoxygenase, pioneering a new class of therapies that treat the underlying pathologies of serious immune-inflammatory diseases with unmet medical needs. The company’s lead candidate, VLX-1005, is in development for the treatment of patients with heparin-induced thrombocytopenia (HIT). VLX-1005 has orphan drug designation in the United States and has been awarded Fast Track Designation by the U.S. Food and Drug Administration. Second generation therapeutic products are under development for type 1 diabetes and other immune-mediated and inflammatory diseases. For more information, visit our website: https://veralox.com/. Media Contact: Lisa GuitermanScient PR202-330-3431Lisa.guiterman@gmail.com

临床2期高管变更孤儿药快速通道

2022-10-17

·生物制品圈

新型mTOR抑制剂的设计与机制研究

小分子药物的使用通常会导致全身的药理作用，这会同时提高药效和毒性，并影响其治疗指数。虽然脱靶效应可以通过改善药物特异性的化学修饰来缓解，但靶向-组织外毒性是一项独特的挑战，需要精确控制组织分区。靶向-组织外毒性会影响常用药物，如他汀类药物(骨骼肌中HMG CoA还原酶抑制导致肌病)和第一代抗组胺药(大脑中H1受体被阻断导致嗜睡)，及其他有效治疗药物的安全使用。mTOR的化学抑制剂就是一个很好的例子。尽管在许多中枢神经系统(central nervous system, CNS)疾病中已经研发了变构和正构mTOR抑制剂，包括结节性硬化症、胶质母细胞瘤和酒精使用障碍，但mTOR的全身抑制与各种剂量限制不良反应相关（如免疫抑制、代谢障碍和儿童生长抑制）。如果这些mTOR抑制剂的药理作用能够局限于中枢神经系统，那么它们的治疗窗口将大大拓宽。 mTOR激酶的靶向治疗可以通过变构和正构两种方式实现。目前mTOR抑制剂主要分为三类：抗生素变构mTOR抑制剂(第一代)、ATP竞争性mTOR抑制剂(第二代)和其他新型mTOR抑制剂(第三代)。第一代mTOR抑制剂一般是指抗生素类变构mTOR抑制剂，主要是雷帕霉素(Rapamycin)及其衍生物(Rapalogs)。雷帕霉素并不直接抑制mTOR 活性，它与FK506结合蛋白12(FK506-binding protein 12, FKBP12)结合形成FKBP12-雷帕霉素复合物，该复合物再结合mTOR的FRB结构域，其中，雷帕霉素嵌入由 FKBP12 和 mTOR 的 FRB 结构域形成的空腔中，改变mTOR的构象，从而以变构的方式抑制mTORC1活性。因此FKBP12只是雷帕霉素介导的 mTORC1 抑制的辅助因子。第二代mTOR抑制剂主要是ATP竞争性抑制剂，与mTOR激酶结构域中的ATP结合位点结合。第三代mTOR抑制剂RapaLink-1是将雷帕霉素和mTOR激酶抑制剂MLN018整合到同一个分子上而获得的，其通过结合FKBP12的FRB结构域作用于mTOR，同时连接的小分子抑制剂MLN018作用于mTOR激酶结构域。尽管其分子量较大(1784 Da)，与早期的mTOR抑制剂相比，RapaLink-1在促进胶质母细胞瘤消退方面表现出了增强的体内疗效。由于RapaLink-1含有一个TOR激酶抑制剂(TOR kinase inhibitor, TORKi)部分，它与ATP口袋结合。为了知道RapaLink-1与FKBP12的相互作用是否对其抑制mTOR必不可少，研究人员用纯化的mTOR蛋白进行了体外激酶测定，发现无论FKBP12存在与否，RapaLink-1均表现出与MLN0128 (RapaLink-1的TORKi部分)相同的半最大抑制浓度(half-maximal inhibitory concentration , IC50)值。这表明在无细胞环境下，RapaLink-1可以不依赖FKBP12而作用mTOR的活性位点。然而，当研究者在细胞中测试RapaLink-1时，他们观察到FKBP12对mTOR的抑制有很强的依赖性。在表达dCas9-KRAB的K562细胞中，用两种不同的sgRNA敲除FKBP12基因极大地阻碍了其细胞活性。当他们使用FK506 (FKBP12的高亲和力天然配体)阻断FKBP12的配体结合位点时，这种作用更加明显。用western blot检测mTOR信号，观察到类似的RapaLink-1对FKBP12的依赖性。10 nM的RapaLink-1将磷酸化的 S6 (S240/244)和磷酸化的4EBP蛋白 (T37/46)降低到几乎无法检测到的水平，而10 nM的RapaLink-1和10 μ M FK506的组合对这两种标记均无影响。这些结果强调，尽管FKBP12不是RapaLink-1在体外结合和抑制mTOR活性位点所必需的，但它是细胞活性所必需的，其可能是RapaLink-1在细胞内积累的接收器。基于这一发现，研究者提出了一种化学策略，将已知的激酶抑制剂转化为FKBP12依赖的形式，可以实现对其他激酶靶点的脑特异性抑制：通过脑透性mTOR抑制剂(RapaLink-1)+FKBP12来在脑内发挥功能，而联合使用FKBP12的脑非渗透抑制剂(RapaBlock)来阻断脑外FKBP12对RapaLink-1的依赖作用。当RapaLink-1和RapaBlock这两种药物联合使用时，可促进在胶质母细胞瘤消退且检测不到全身毒性。为了确定具有适当通透性的候选FKBP12抑制剂(即细胞通透性但不渗透血脑屏障(blood–brain barrier, BBB))，他们考虑了已知的天然和合成的高亲和力FKBP12配体(例如FK506，rapamycin和SLF/Shield-1)，但所有这些化合物都很容易穿过血脑屏障。因此，他们合成了一组SLF和FK506的衍生物，其中一些完全阻断了RapaLink-1对mTOR的抑制。他们对四种具有不同侧链化学类型的化合物进行了额外的体内筛选，分别分析了用RapaLink-1和候选化合物(分别为1和40 mg/kg)组合处理的小鼠的大脑和骨骼肌组织中的mTOR信号。FK506的吡啶氮氧化物衍生物(06-041)可以保护周围组织不受RapaLink-1的影响，但允许大脑中mTOR的有效抑制，他们将其命名为“RapaBlock”。此外，修饰FK506的C21烯丙基还有一个额外的好处——它取消了与FK506的天然靶标钙调神经醛酸的结合，其抑制会导致活化T细胞核因子(Nuclear factor of activated T cells, NFAT)信号的抑制，从而导致免疫抑制。RapaBlock和FK506同FKBP12的结合具有相当的亲和力(抑制常数(Ki)分别为3.1 nM和1.7 nM)，但与FK506不同的是，RapaBlock对钙调磷酸酶没有表现出任何抑制活性。在细胞中，RapaBlock(最多10µM)本身不会影响mTOR信号，但会以剂量依赖性的方式减弱RapaLink-1的药理作用。RapaBlock在阻断雷帕霉素方面似乎更有效，其以100:1的化学计量率将磷酸化S6信号恢复到与未处理细胞相同的水平。这些数据表明，RapaBlock通过与细胞内FKBP12的竞争性结合，使RapaLink-1和雷帕霉素无法抑制mTOR。为了检验RapaLink-1和RapaBlock的联合作用是否能在体内对mTOR产生脑特异性抑制，他们用RapaLink-1(1 mg/kg)或RapaLink-1(1 mg/kg)和RapaBlock(40 mg/kg)的联合作用治疗健康BALB/cnu/nu小鼠，在4小时或24小时后用胰岛素(250 mU)刺激mTOR活性，并通过免疫印迹分析解剖组织。RapaLink-1和RapaBlock组合显示出显著的组织特异性效应：与只使用RapaLink-1的小鼠相比，大脑中的mTOR活性受到了相当程度的抑制，但骨骼肌中的mTOR活性没有受到抑制，未出现mTOR抑制剂常见的靶向副作用，如体重减轻、糖代谢受损和肝毒性。接下来，研究人员在裸鼠中建立表达萤火虫荧光素酶的U87MG细胞原位颅内异种移植瘤模型，并用RapaLink-1 (1 mg / kg)、RapaBlock (40 mg / kg)或两者的联合注射治疗这些小鼠，每5天注射一次。所有治疗均耐受良好，体重无明显变化。RapaLink-1单独使用或与RapaBlock联合使用均能显著抑制肿瘤生长并提高生存率，而单独使用RapaBlock对两者均无显著影响。在建立了这样一种二元治疗方法来实现对mTOR的脑特异性抑制后，研究人员想知道是否同样的策略可以适用于其他需要脑限制性药理学的药物。一个关键的挑战是，要使这种方法普遍化，“活性”组件(如RapaLink-1)必须依赖于FKBP12的可用性。他们首先用GNE7915进行尝试，GNE7915是一种富含亮氨酸重复激酶2 (leucine-rich repeat kinase 2, LRRK2)的有效和特异性抑制剂，在治疗帕金森病的临床前研究中。最近的研究表明，小分子抑制剂对LRRK2的系统性抑制可诱导II型肺细胞的细胞质空泡化，这表明这些化合物存在潜在的安全隐患。他们将FK506和GNE7915的药效团与哌嗪基团进行化学连接，合成了FK-GNE7915。在体外LRRK2激酶试验中，FK-GNE7915是GNE7915的次等抑制剂(IC50分别为107 nM和3.0 nM)，尽管在实验中加入10 μ M FKBP12后其活性增强(IC50为21 nM)。然而，在量化磷酸化的LRRK2 (S935)水平作为LRRK2抑制标记的细胞试验中，FK-GNE7915比母化合物GNE7915更有效超过十倍(IC50分别为6.7 nM和81 nM)。这两个结果的并置表明FK506部分在FK-GNE7915增强的细胞效力中发挥了作用。他们推断，高亲和力FK506-FKBP12相互作用可以促进FK-GNE7915在细胞内的积累，类似于RapaLink-1的情况。这一特性允许研究人员通过控制FKBP12的可用性来编程抑制LRRK2。综上，通过利用雷帕霉素类似物对FKBP12的独特功能依赖性，研究人员开发了一种方法，通过同时使用强效mTOR抑制剂(RapaLink-1)和FKBP12的细胞渗透性、BBB不渗透性配体(RapaBlock)，实现脑特异性mTOR抑制。这种双重药理学策略实现了组织限制性mTOR抑制，降低了外周组织的毒性，并在异种胶质母细胞瘤模型中保持了RapaLink-1的治疗效果。基于这些发现，作者预期，同样的药物组合可能在其他由mTOR活性失调引起的中枢神经系统疾病(如酒精使用障碍)中具有更广泛的价值。这种方法与这些先例的不同之处在于，它不涉及两种对同一靶点或通路具有抵消作用的药物;相反，RapaBlock通过直接衰减激酶抑制剂的活性来控制组织特异性。因此，目前的体系具有适用于多种靶标的优势，只需要一个不变的RapaBlock分子和一个FKBP12依赖的抑制剂，可以根据FK506和先导化合物的结构轻松设计。虽然在本研究中关注的是蛋白质激酶，但似乎有理由期望该方法也适用于其他类的治疗靶点，如GTPase和组蛋白修饰酶。参考文献Ziyang Zhang, Qiwen Fan, Xujun Luo, Kevin Lou, William A Weiss, Kevan M Shokat. Brain-restricted mTOR inhibition with binary pharmacology. Nature. 2022 Sep;609(7928):822-828. doi: 10.1038/s41586-022-05213-y

免疫疗法小分子药物