作者: Luo, Dahai ; Harris, Reuben S ; Anindita, Paulina Duhita ; Lattmann, Simon ; Spicer, Timothy P ; Kang, CongBao ; Scampavia, Louis ; Ngo, Khac Huy ; Liew, Chong Wai ; Otsuka, Yuka

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors.

2020-12-08·Proceedings of the National Academy of Sciences of the United States of America1区 · 综合性期刊

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors

1区 · 综合性期刊

Article

作者: Simeonov, Anton ; Abrams, Rachel P. M. ; Steiner, Joseph P. ; Jadhav, Ajit ; Teramoto, Tadahisa ; Nath, Avindra ; Li, Wenxue ; Yasgar, Adam ; Balasubramanian, Anuradha ; Zakharov, Alexey V. ; Hall, Matthew D. ; Padmanabhan, Radhakrishnan ; Malik, Nasir ; Bachani, Muzna ; Lee, Myoung-Hwa ; Dorjsuren, Dorjbal ; Eastman, Richard T. ; Brimacombe, Kyle

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

2019-01-11·Oncotarget

Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase

Article

作者: Ghosh, Jagadananda ; Sarveswaran, Sivalokanathan ; Morisetty, Shravan ; Varma, Nadimpalli R.S.

The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44 ⁽⁺⁾, CD133 ⁽⁺⁾ cell subpopulations were isolated from human prostate tumors which exhibit stem-like properties showing therapeutic-resistance, capacity of self-renewal, and exact recapitulation of the original tumor in vivo. Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.

项与喹夫拉朋相关的新闻（医药）

2023-09-10

·CPHI制药在线

5-脂氧合酶及其抑制剂研究进展

关注并星标CPHI制药在线脂氧合酶是一类非血红素含铁双加氧酶，存在于动、植物的组织和细胞中，可催化花生四烯（AA）、二十二碳六烯酸和各种多不饱和脂肪酸(polyunsaturated fatty acid，PUFA)的氧化反应。脂氧合酶主要分为1、3、5、8、12、15-脂氧合酶6种亚型。其中5-脂氧合酶(5-lipoxygenase，5-LOX)是脂氧合酶同工酶中最常见的、研究较为深入的非血红素铁蛋白氧化代谢酶，是许多高活性氧化脂质生物合成中的关键酶。5-LOX作用于AA氧化通过两步完成：第一步使5-LOX氧化成5-羟过氧化二十碳四烯酸（5-HPETE）再转化为5-羟基二十碳四烯酸（5-HETE）。第二步催化5-HPETE生成白三烯A4(leukotriene A4，LTA 4 )。LTA 4既可转化为致炎物白三烯B4(leukotriene B4，LTB4)，也可生成白三烯C4(leukotriene C4，LTC4)进而生成白三烯D4(leukotriene D4，LTD4)、白三烯E4(leukotrieneE4，LTE4)等导致过敏反应的反应物质。白三烯物质（LT）是哮喘、慢性阻塞性肺疾病、炎症性肠病、关节炎、动脉粥样硬化、皮炎和癌症等多种慢性炎症性疾病相关的促炎性脂质介质，通过进一步的双加氧作用和对外源化学物的协同氧化作用，在炎症、哮喘、脑缺血、部分恶性肿瘤及阿尔茨海默症等疾病的发生和发展中具有极其重要的作用。5-LOX抑制剂通过阻断LT的产生，抑制AA转变为LT，从而减少炎症的产生。因此，开发有效的5-LOX抑制剂对治疗上述疾病至关重要。根据抑制剂的作用机制可将5-LOX抑制剂分为直接抑制剂和间接抑制剂。直接抑制剂直接抑制剂主要针对5-LOX分子结构特点进行研发，比如干扰5-LOX C-端催化结构域中Fe2+与Fe3+正常的转换，或者是与铁离子络合来抑制5-LOX的活性。直接抑制剂包括氧化还原抑制剂、非氧化还原抑制剂、铁离子配体抑制剂。间接抑制剂主要是围绕FLAP进行设计，在5-LOX途径中需要FLAP的参与，抑制了FLAP 就能阻断 5-LOX 途径。 1、氧化还原抑制剂氧化还原抑制剂一般是亲脂性的小分子芳香族化合物，例如酚类化合物和醌类化合物。1,4-苯醌AA-861是第一批氧化还原型5-LOX抑制剂，经多项研究证实其在体内可产生抗炎作用，并已经通过了预防季节性过敏性鼻炎的临床试验。苯醌衍生物在人类单核细胞、中性粒细胞和整个人类血液中可作为5-LOX的有效活性抑制剂，对两种LT相关炎症标准化动物模型的实验研究表明，新型1,4-苯醌RF-Id能够通过抑制LT的产生，在体内抑制导致抗炎反应的5-LOX。此外，氧化还原抑制剂还包括去甲二氢愈创木酸 (NDGA)、香豆素、黄酮类化合物等几类物质，它们均在体外实验证实具有高效的5-LOX 抑制作用。氧化还原抑制剂能把Fe3+还原为Fe2+，从而干扰了铁离子的正常催化循环，阻止5-LOX 的激活，导致5-LOX途径不能正常进行。此外，黄酮类的某些化合物，例如槲皮素、芦丁由于B环上具有邻位的酚羟基，他们还能与Fe3+络合，从而加大了对 5-LOX 的抑制作用。另外，研究人员用荧光法测定了氧化还原抑制剂的氧化还原电势，并揭示了氧化还原抑制剂的作用机制，当氧化还原抑制剂存在时，它降低了Fe2+与Fe3+之间的转换，抑制了酶催化反应，从而需要更多的脂质过氧化物去氧化Fe2+转变成 Fe3+，因此，在酶催化反应过程中，真正参与催化反应的脂质过氧化物的量是很少的，酶催化反应即被抑制。 2、非氧化还原抑制剂非氧化还原抑制剂属于竞争型抑制剂，它能与底物AA竞争结合到5-LOX 的活性中心，导致5-LOX途径受阻，相对于其他抑制剂具有更高的选择性，但是磷酸化作用和Ca2+会干扰这些抑制剂。ZD2138 和ZM230487属于非氧化还原抑制剂中的两个代表。它们属于甲氧基四氢吡喃衍生物，临床研究表明，ZD2138 能够有效抑制关节炎和哮喘病。实验研究表明，一些天然产物和ZD2138 的杂交体 L-697198，以及通过对L-697198结构进行优化而得到的L-708780和L-739010，表现出良好的生物利用度。但后续动物实验发现，该种抑制剂有多种毒性，限制了它的临床使用。此外，L-739,010、CJ-13,610和PF-419183也是非氧化还原型5-LOX抑制剂，在细胞测定中显示IC50的低纳摩尔范围。L-739,010可以减少LT介导的炎症反应，且其抗痛觉亢进活性与炎症大脑中LT水平降低有关，在急性卡拉胶模型和慢性炎症模型中表现出优异的药效和药代动力学，但难以形成共价蛋白结合代谢物。CJ-13,610在大鼠内侧半月板横切面模型上对骨关节炎疼痛有抑制作用，口服CJ-13,610可逆转该动物模型的两种疼痛模式，包括触觉异位痛和负重差异。通过大鼠气囊模型证明了PF-4191834对LTB4产生的抑制作用，PF-4191834也完成了哮喘的Ⅱ期临床试验，但其用于膝骨关节炎的Ⅱ期临床试验因出现晕厥、急性肝炎和胃溃疡出血等严重不良事件而被终止。 3、铁离子配体抑制剂铁离子配体抑制剂大体包括两种，异羟基肟酸类和羟基脲类，羟肟酸衍生物BWA4C和羟基脲衍生物Zileuton是有效的口服5-LOX抑制剂。BWA4C是溃疡性结肠炎患结肠组织中LTB4合成的有效选择性抑制剂，以BWA4C为代表的乙酰异羟肟酸5-脂氧合酶抑制剂有助于评价LTB4在溃疡性结肠炎中的临床意义，并为该病的治疗提供新的途径。Zileuton使急性和慢性气道功能得到了改善，也减少了β受体激动剂或糖皮质激素的需求，并在美国被批准用于治疗哮喘，但是对于变应性鼻炎、类风湿关节炎和炎症性肠病的治疗潜力较低，并且它有许多缺点，如肝毒性和不良药代动力学特征等。以Zileuton结构为基础设计出了一种新型抑制剂VIA-2291，可以在支气管痉挛动物模型中表现出比Zileuton高5倍的效力，口服半衰期16 h，对哮喘患者的运动性支气管收缩有一定的疗效。VIA-2291已成功完成动脉粥样硬化和心血管疾病的Ⅱ期临床试验，成为临床开发中领先的5-LOX抑制剂之一。RBX-7796是一种具有十二烷基链的尿素衍生物，可抑制大鼠血液中LTB4的体外合成，并对各种炎症和支气管收缩动物模型有活性。间接抑制剂间接抑制剂不直接抑制5-LOX的活性。FLAP是5-LOX 激活蛋白，它能结合底物 AA 并且把它转移给5-LOX。在细胞中，5-LOX 与 FLAP 是共同表达的。当给予足够的底物 AA，如果缺少FLAP，细胞也不能合成白三烯等物质。MK886 是研发出来的第一代间接抑制剂，同时也是第一个用于临床的间接抑制剂。它能与AA竞争FLAP 上的结合位点，导致5-LOX 无法与FLAP 相互结合，5-LOX 途径因此受阻。此外，抑制剂AM803正在进行临床实验，并已经完成了对哮喘病人的二期阶段试验，它的衍生物AM103也成功通过了一期临床实验。不过研发的某些抑制剂也存在问题，如NGDA和MK0591 等也存在两方面的缺陷，一是不能特异性针对5-LOX，对人体正常的代谢也有一定的干扰，从而限制了临床应用；二是稳定性不够，直接影响抑制剂的抑制效力。参考资料 [1]李丹凤,范涛,鹿欣雨.5-脂氧合酶抑制剂的研究进展[J].生命的化学,2023,43(05):666-673. [2]黄凯,蒋世云,陈柳军.5-脂氧合酶及其抑制剂[J].中国生物化学与分子生物学报,2014,30(12):1190-1196. 作者简介：小泥沙，食品科技工作者，食品科学硕士，现就职于国内某大型药物研发公司，从事营养食品的开发与研究。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~

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研发状态

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适应症	最高研发状态	国家/地区	公司	日期
哮喘	临床2期	美国	Merck GmbH	-
哮喘	临床2期	加拿大	Merck GmbH	-
炎症性肠病	临床2期	-	Merck Sharp & Dohme Corp.	-
前列腺癌	临床前	美国	Henry Ford Health System	2014-10-01