Phenprocoumon

1HIV蛋白酶在我们讨论HIV蛋白酶抑制剂之前，我们先来看看艾滋病毒的生命周期，以帮助我们了解HIV蛋白酶在病毒复制中的作用。HIV蛋白酶是一种参与HIV生命全周期的病毒特异性酶。而蛋白酶抑制剂则通过阻断病毒成熟而发挥作用。艾滋病病毒本身是一种病毒粒子，它通过性交、输血或垂直途径(从母亲到婴儿)进入血液。为了使病毒粒子穿透宿主细胞，其包膜突刺蛋白gp120与T细胞膜上的CD4+发生接触。这里gp120代表HIV包膜蛋白上的分子量为120K道尔顿的糖蛋白。这种接触的过程叫做向性。一旦gp120和CD4发生接触，病毒粒子在另一种包膜糖蛋白gp41的帮助下，将其RNA遗传信息“注射”到宿主细胞中。但是RNA并不是单独活动的。它的“旅伴”包括三种酶：逆转录酶、蛋白酶和整合酶。抑制逆转录酶和蛋白酶已被证明是最成功的有效治疗方法，大多数已批准的艾滋病药物都以这两种酶为靶点。HIV病毒一旦进入细胞中，就会脱掉它的蛋白质外壳，将RNA链的遗传信息和逆转录酶一起释放到细胞质中。在细胞质内，病毒可以利用底物的优势，通过逆转录酶将RNA基因组逆转录为双链DNA副本。HIV蛋白酶是制造新病毒所必需的一种酶，负责将多蛋白基因产物加工成功能成熟的蛋白质。从本质上来说，一些大的病毒蛋白质必须被蛋白酶分解成具有调节功能的小蛋白质。它在病毒复制中起着关键作用，无论是在功能还是结构上都是病毒蛋白质的最佳特征。它是一种天门冬氨酸蛋白酶，其催化机制类似于人类肾素。其他人类天冬氨酸蛋白酶包括胃蛋白酶、胃泌素和组织蛋白酶。HIV蛋白酶是由单体亚基对称二聚形成的，每个亚基都有一个催化的天冬氨酸残基。因此，许多制药公司最初筛选他们的老肾素抑制剂(用于调节血压)来寻找HIV蛋白酶抑制剂就不足为奇了。蛋白酶抑制剂看的设计明显促进了X射线晶体结构的可用性，允许直接观察结合到酶的抑制剂。蛋白酶作用于病毒生命周期的最后一步。蛋白酶抑制剂在核苷逆转录酶抑制剂发现之后不久被发现。1996年，随着第一代HIV蛋白酶抑制剂沙奎那韦、利托那韦和茚地那韦的问世，HIV蛋白酶抑制剂的时代就此开始。2第一代HIV蛋白酶抑制剂1沙奎那韦罗氏的沙奎那韦(saquinavir, Invirase)是美国市场上第一个HIV蛋白酶抑制剂。早在1986年，罗氏就开展了一项雄心勃勃的国际合作，以解决HIV蛋白酶的问题。由Ian Duncan和Sally Redshaw领导的在英国Welwyn的一个化学小组，在选择比色分析法作为体外试验后，使用“过渡态模拟”策略设计抑制剂，这种策略之前在生产强效肾素抑制剂方面非常成功。由于反应的过渡态具有最高的反应热力学能量，因此，过渡态模拟物能够抑制酶的功能。沙奎那韦依赖于羟基部分作为四面体过渡态的等构体，这一设计元素源于对肾素抑制剂的广泛探索，肾素是另一种哺乳动物天冬氨酸蛋白酶。化学家们很快取得了一个重要的里程碑式结果：通过定义最小的模拟肽，达到可以实现接受的抑制水平。他们发现，三肽在效力和生物利用度方面都是理想的。因此，三肽成为许多蛋白酶抑制剂共同遵循的主题。罗氏团队在微调三肽方面做出了巨大的努力，通过逐个修饰每个氨基酸残基，系统地探索了他们的先导化合物。他们的辛勤工作在1991年得到了回报，沙奎那韦在1995年12月获得FDA批准，成为市场上第一种用于治疗艾滋病的HIV蛋白酶抑制剂。虽然沙奎那韦是一种拟肽药，很容易就能被代谢掉，但后来发现它与另一种蛋白酶抑制剂利托那韦作为药物增强剂共同使用更有益。由于利托那韦抑制了肝脏中降解沙奎那韦的细胞色素P450 3A4(CYP3A4)酶，因此两种药物联合使用可以显著提高沙奎那韦的血浆水平。CYP3A4是肝脏中含量最丰富的CYP同工酶。此外，沙奎那韦的出现标志着将蛋白酶抑制剂引入临床的鸡尾酒药物组合疗法的开始。[1]2利托那韦雅培的利托那韦(ritonavir, Norvir)是市场上第二种蛋白酶抑制剂。利托那韦被发现的环境非常独特。雅培团队由X射线晶体学家John Erickson和药物化学家Dale Kempf领导。据传默克有30名化学家参与他们的HIV蛋白酶抑制剂项目，而Kempf只有3名。雅培没有像大多数制药公司那样筛选他们的肾素抑制剂，而是利用了Erickson对HIV蛋白酶的X射线晶体学的研究，这将被证明对他们的药物设计有帮助。结合基于结构的药物设计(SBDD)和传统的药物化学，他们制备了一系列基于对称性的抑制剂，以匹配HIV蛋白酶的C2-对称性质。因为他们的抑制剂结合到了酶的两侧, Kempf将它们称为分子花生酱(molecular peanut butter)。通过这种方法，他们找到了含有吡啶的四肽A-77003。虽然A-77003在结合和细胞试验中是有效的，但它有极高的人类胆道清除率。通过降低分子量和用更可溶性的氨基酸取代现有的氨基酸，他们实现了生物利用度的提高。 最终发现，吡啶末端被肝细胞色素P450氧化为N-氧化物。简单地用代谢更强的噻唑取代吡啶，并进行一些微调。利托那韦的生物利用度为78%，而吡啶模拟物A-77003的生物利用度为26%。1996年3月，雅培的利托那韦获得了FDA的批准。[2]雅培发现利托那韦无疑令人印象深刻。但发现它之后的两件事，一样有趣。其中一项涉及它的晶体形态，另一项涉及吉利德发现的利托那韦类似物cobicistat，用作生物利用度增强剂。1996年，雅培的利托那韦获得了FDA批准，并以商品名Norvir进行销售之后，使用原始结晶I生产了该药物的活性药物成分(API)。由于利托那韦在固体状态下没有生物利用度，所以它被制为口服溶液或半固体胶囊，都是在乙醇-水溶液中。 1998年初，利托韦那上市两年后，原料药的生产开始出现问题：许多批次开始变成一种新的、不同的、极难溶解的晶体形式II，尽管晶体形式I最开始也很难溶。新的晶体形式II有一个广泛的分子间氢键网络，它在热力学上比原来的I更稳定。一旦有了II的晶体种子，无论它是多么细小和微观，大自然总是会把API转换成热力学上更稳定的晶型II。一组接触过晶体II的科学家参观了雅培在意大利的制造工厂，调查了利托那韦的批量生产过程。后来，在工厂的生产过程中，大量的晶型II开始出现在原料药的生产过程中。这种情况确实让雅培陷入了营销危机。雅培的制药化学家们费尽心思才想出了一种只生产原始剂型I的方法：为了防止剂型II的形成，半固态胶囊或口服剂型在使用前需要冷藏等措施。[3]利托那韦成功后的另一个故事发生在加州Foster City的吉利德。有趣的是，当许多其他蛋白酶抑制剂被CYP3A4代谢时，利托那韦基于将药物运送到细胞外这一机制，有效地抑制了CYP3A4和P-糖蛋白(Pgp)。因此，它可以与CYP3A4蛋白酶抑制剂或其他药物共同使用，增加血浆水平。因此，双蛋白酶抑制剂在疗效和降低耐药性方面已证明是强有效的治疗方案。低剂量利托那韦现在主要用作HIV抑制剂的药代动力学(PK)增强剂，也被称为药物增强剂。药物增强剂是一种本身通常对治疗靶点不具活性，但可以抑制使活性药物代谢的酶。因此，药物增强剂可以改善抗病毒药物的PK谱，以更低的剂量和更少的次数达到足够的谷浓度(药物在下一次给药前所达到的最低浓度)。吉利德与日本Tobacco公司合作开发了HIV整合酶抑制剂挨替拉韦(elvitegravir，Vitekta, 2014)。遗憾的是，尽管挨替拉韦是一种有效的整合酶链转移抑制剂(INSTI)，但它在肝脏和肠道中被CYP3A4广泛代谢，不可能进行一天一次的治疗。尽管使用亚治疗剂量的利托那韦有可能引起对其他蛋白酶抑制剂的耐药性，但当与利托那韦一起给药时，出现了较好的药物暴露增加。因此吉利德决定生产一种只能通过抑制CYP3A4介导的代谢来提高生物利用度，但完全不抑制HIV蛋白酶的药物；同时，高水溶性和良好的理化性质也有利于进行药物制备。虽然利托那韦的水溶性较差，但在药物化学负责人Manoj Desai的领导下，由Lianhong Xu领导的团队以利托那韦为起点，研究出了一种有效的、口服生物利用度高的选择性CYP3A4抑制剂cobicistat。科学家们敲掉了利托那韦上的一个羟基，因为它模拟了酰胺水解的过渡状态：通过与HIV蛋白酶活性位点催化域的两个氨基酸形成氢键，而没有关键羟基的分子几乎无法与HIV蛋白酶结合。Cobicistat于2012年获得FDA批准，吉利德将其以Tybost的商品名出售，已与多种抗逆转录病毒药物联合使用。[4]在药物发现中，当两种药物被相同的肝脏CYP酶代谢时，就会发生药物-药物相互作用(DDI)。它们通常有充分的理由被认为是一种“负债”。例如，拜耳的他汀类药物cerivastatin (Baycol)在与降低胆固醇的纤维类药物一起服用时，会导致严重的肝损伤。因为这两种药物都是由CYP3A4代谢，肝脏却没有足够的CYP3A4酶来同时代谢这两种药物，从而就会产生毒性。但有了利托那韦和cobicistat这样的药物增强剂，我们可以把药物的“负债”变成“资产”。当药物增强剂使肝脏的CYP酶忙碌时，抗病毒药物可以专注于杀死病毒，而不用提防自己。Cobicistat可以使每日一次的含蛋白酶抑制剂的单片治疗成为可能。3茚地那韦默克的茚地那韦(indinavir, Crixivan)是市场上第三个蛋白酶抑制剂。默克于1986年开始了对HIV蛋白酶抑制剂的研究，当时分子生物学部门的高级主任Irving Sigal是该项目的带头人。他们最初也筛选了肾素抑制剂对HIV蛋白酶的抑制作用，然后利用已知的HIV蛋白酶晶体结构进行合理的药物设计，该晶体结构由默克首先发现，NIH的科学家优化。1990年，化学家Wayne Thompson发现了L-689,502，它能有效抑制HIV蛋白酶，但缺乏肾素活性。然而，它只有注射才有效，没有生物可利用性。随后，受到文献中可行的口服药物沙奎那韦启发，Joseph Vacca成功地将沙奎那韦的一个片段植入了L-689,502。 1989年，Vacca团队的新员工Bruce Dorsey和他的同事Rhonda Levin成功合成了茚地那韦。虽然在单药治疗试验中，大约40%的患者在服用该药6个月后，RNA低于400拷贝/毫升，但在一些患者中，HIV病毒对茚地那韦产生了耐药性。幸运的是，他们发现，茚地那韦和AZT或拉米夫定(Epivir)结合能够非常有效地抑制病毒水平。默克对联合疗法的研究率先证明了鸡尾酒疗法的疗效，并成为业界标准。茚地那韦在1996年1月向FDA提交申请后，于1996年3月在加速审查过程中获得批准。[5]其他重要的早期蛋白酶抑制剂包括Agouron(现辉瑞)的奈非那韦(nelfinavir, Viracept, 1997年3月获批)和Vertex的amprenavir(Agenerase, 1999年4月批准)。这些统称为第一代蛋白酶抑制剂。Vertex公司在蛋白酶抑制剂领域起步较晚。借助晶体学和计算机辅助药物设计，Vertex和他们在Kissei公司的日本合作者发现了amprenavir，这是一种非常有效的皮摩尔HIV蛋白酶抑制剂。1993年，Vertex与Burroughs Wellcome合作，研究Kissei公司的amprenavir的PK特性。被AZT的成功冲昏头脑的Burroughs Wellcome在蛋白酶抑制剂领域起步较晚，需要借助Kissei/Vertex的药物作为跳板。Amprenavir足够小，足以穿透血脑屏障，杀死其中的病毒。显然，他们对这一结果很满意，Burroughs Wellcome关闭了自己的HIV蛋白酶项目，并同意支付Vertex 2亿美元药物临床试验费用。 Amprenavir于1999年获得FDA批准，Vertex和Burroughs Wellcome(1995年更名为Glaxo Wellcome, 2000年更名为Glaxo SmithKline)联合销售该药物，商品名为Agenerase。该药物尽管具有100%的生物利用度，但后来因其水溶性较差(0.04 g/mL)而停止使用。它需要较高的赋形剂与药物的比例，来保证胃肠道的溶解度和最终的吸收。Vertex继续制备其磷酸酯前药福沙那韦(fosamprenavir)，其水溶液溶解度增加了8倍(0.31 g/mL)。前药福沙那韦于2005年获得批准，Vertex和GSK将其以Lexva的商品名进行出售。蛋白酶抑制剂可能是抗逆转录病毒类中最有效的，一部分原因是它们通常具有较高的耐药性基因屏障。早期使用的第一代蛋白酶抑制剂沙奎那韦、利托那韦、茚地那韦、奈非那韦并没有利用对CYP3A4抑制的促进作用。尽管如此，这些抑制剂的药代动力学特性并不理想。尽管具有很高的遗传障碍，但这些第一代蛋白酶抑制剂最终确实出现了耐药性。后来，利托那韦提供的药代动力学增强使得给药频率降低，许多药物可以每天给药一次，而且由于每日血浆谷浓度大大提高，耐药性的遗传屏障更高。 3第二代HIV蛋白酶抑制剂虽然最初的利托那韦促进方案是与第一代蛋白酶抑制剂一起进行的，但更有效和更安全的第二代抑制剂的开发预示着鸡尾酒方案新时代的到来。这些第二代抑制剂包括福沙那韦(amprenavir的前药)、洛匹那韦(lopinavir)、阿扎那韦(atazanavir)、替普那韦(tipranavir)和达芦那韦(darunavir)。许多第一代蛋白酶抑制剂都是拟肽药。它们是基于一种改良的仿肽模型设计的，其中肽基底物的可分割键被不可分割的过渡态拟态取代。我们总是需要大量的药物化学研究来有条不紊地修饰肽的每一个细节，为了获得一种有效的、具有生物有效性的药物。由于早期蛋白酶抑制剂对代谢的易损性，几乎所有这些药物都必须与利托那韦一起作为PK增强剂。Upjohn则采取了不同的方法。 密歇根州Kalamazoo的Upjohn不想采用拟肽法。相反，他们在20世纪90年代早期对5000种不同的现有历史化合物进行了中等通量筛选。与今天的高通量筛选(HTS)常规地筛数百万种化合物相比，5000种化合物似乎小得荒谬。尽管如此，他们还是鉴定出血液稀释剂华法林是HIV蛋白酶的弱抑制剂。巧合的是，他们在密歇根州Ann Arbor的Parke–Davis的邻居从相关的筛选工作中发现了类似的基质。尽管华法林的效力远不如仿胃蛋白酶抑制剂，但它因其广泛的生物利用度，仍具有吸引力。本质上，Upjohn更倾向于药物代谢动力学而不是药效。随后，他们对与华法林类似的化合物进行了重点筛选，确定了华法林类似物Phenprocoumon，是一种已知的口服活性抗凝剂。Phenprocoumon的效力是华法林的30倍，并且在细胞实验中已经开始显示抗病毒活性。Phenprocoumon-蛋白酶复合物的晶体结构对未来蛋白酶抑制剂的药物设计有很大的帮助。利用计算机辅助，基于结构的药物发现平台(SBDD)， Upjohn得到了他们的第一代临床候选药物。但后来它被更有效的类似物取而代之。他们的第二代临床候选药物再次被放弃，因为它虽然具有非常高的蛋白质结合度，但与当代最活跃的消化性模拟药物相比，其效力仍然有限。第三代就成功了。他们最终研制出了安全性和有效性都更好的第三代临床候选药物替普那韦，商品名为Aptivus，于2005年获得FDA批准。遗憾的是，与其他蛋白酶抑制剂相比，替普那韦副作用严重。[6]最新的HIV蛋白酶抑制剂是Janssen公司的达芦那韦(darunavir, Prezista)，于2006年获得FDA批准。Arun Ghosh发现达芦那韦的经历是一段漫长而曲折的旅程。1988年，Ghosh在哈佛大学完成了E.J. Corey的博士后培训后，在默克位于宾夕法尼亚州West Point的基地工作了6年，参与了HIV蛋白酶抑制剂的发现。但在1994年，他决定在伊利诺伊州立大学的芝加哥分校开始他的学术生涯，2005年前往普渡大学。为了更好的发现HIV蛋白酶抑制剂，Ghosh采用了“骨架结合”的设计概念，以最大限度地与HIV蛋白酶的活性位点相互作用，特别是促进与蛋白质主干原子的广泛氢键。他的团队最初在罗氏的沙奎那韦上安装了一个四氢呋喃(THF)环。后来，通过结合默克茚地那韦的左旋特性，Ghosh得到了一种非常好的含有四氢呋喃的蛋白酶抑制剂。同时，Vertex/Kissei也加入了类似的3-(S)-THF基序作为其磺胺化合物的P2配体。有趣的是，实际上Searle在含磺胺的蛋白酶抑制剂上有优先权，尽管最后因蛋白质结合和代谢问题失败，但Vertex不得不在1995年向Searle支付2500万美元来解决专利问题。在药物发现领域，人们总是相互学习。虽然Vertex“借用”了Ghosh的THF片段，但Ghosh也毫不害羞地“借用”了Vertex的磺胺部分。对Ghosh来说，为了巩固自己的知识产权地位，并通过更多的氢键增强效力，他发明了一个双THF基团来取代THF片段。最终得到了达芦那韦，而通用名称中的“arun”无疑是对发明者的致敬。达芦那韦的研发是由Janssen的子公司Tibotec进行的。该药于2006年获得批准，Janssen公司以Prezista的商品名销售该药。[7]PK增强的第二代蛋白酶抑制剂覆盖了最初的蛋白酶耐药，使得对第一代药物产生耐药的患者可以连续使用蛋白酶抑制剂。对于更有效的第二代抑制剂尤其如此。最后，几乎所有的HIV蛋白酶抑制剂，包括最新和最好的达鲁那韦，都需要与药代动力学增强剂(如利托那韦或cobicistat)一起服用，以便在期望的剂量和频率下达到有效的血浆水平。令人欣慰的是，蛋白酶抑制剂的使用将艾滋病患者的死亡率降低了70%。— 总结 —综上所述，目前市场上的10种HIV蛋白酶抑制剂是：沙奎那韦(saquinavir, Invirase, Roche)，1995茚地那韦(indinavir, Crixivan, Merck)，1996利托那韦(ritonavir, Norvir, Abbott), 1996nelfinavir (Viracept, Agouron/Eli Lilly)，1997amprenavir (Agenerase, Vertex/GSK，停产)，1999-2008年洛匹那韦(lopinavir, Kaletra与利托那韦，雅培)，2000阿扎那韦(atazanavir, Reyataz, Ciba Geigy)，2003福沙那韦(fosamprenavir, amprenavir的前药，lexva, GSK/Vertex)，2005替普那韦(tipranavir，Aptivus, Upjohn/Pfizer/Boehringer Ingelheim)，2005达芦那韦(darunavir, Prezista, Janssen)，2006英文原文（上下滑动查看更多）HIV Protease inhibitors for Treating AIDS—Protease Inhibitors, Part-3This series on protease inhibitors covers five classes:ACE Inhibitors for Treating HypertensionDPP-4 inhibitors for Treating DiabetesHIV Protease inhibitors for Treating AIDSHCV NS3/4A Serine Protease Inhibitors for Treating HCV3CL Protease inhibitors for treating COVID-19Today, we review HIV protease inhibitors for treating AIDS.1.HIV proteaseBefore we talk about HIV protease inhibitors, it is an opportune time for us to look at the virus’s life-cycle. This will give us a perspective of HIV protease’s functions in the virus’s replication.HIV protease is a virus-specific enzyme involved in processing and maturation HIV’s life cycle. Protease inhibitors, in turn, function by blocking further viral processing and maturation.An HIV by itself is a virion, which enters the blood stream through sexual intercourse, blood transfusion, or the vertical route (from mother to infant). In order for the virion to penetrate into the host cell, its envelope spike protein gp120 makes contacts with CD4+ on T cell membrane. Here, gp stands for glycoprotein on HIV’s envelope protein and 120 is the glycoprotein’s molecular weight of 120K Dalton. The process of making contact is called tropism. Once contact is made between gp120 and CD4, the virion “injects” its RNA genetic information into the host cell with the help of another envelope glycoprotein, gp41. But the RNA does not travel alone. Its travel companions include three enzymes serving as its accomplices: reverse transcriptase, protease, and integrase. Inhibition of reverse transcriptase and protease has proven to be the most successful approach to effective therapies and most of the approved AIDS drugs target these two enzymes. Once HIV has gained the entry into the cell, the virus sheds its protein coat, releasing its genetic information of RNA strand along with the reverse transcriptase into the cytosol. Inside the cytosol, the virus can take advantage of the availability of substrates to reversely transcribe the RNA genome into a double-stranded DNA copy using reverse transcriptase. HIV protease, the enzyme that HIV needs to make new virus, is responsible for processing a couple of polyprotein gene products into mature and functional proteins. In essence, some large viral proteins must be broken down to smaller proteins with regulatory functions by the protease. It plays a critical role in virus replication and is the best characterized of the virus’s proteins, both functionally and structurally. It is an aspartyl protease similar to renin in humans in its catalytic mechanism. Other human aspartic proteases include pepsin, gastricsin, and cathepsins. The HIV protease is formed by the symmetrical dimerization of monomeric subunits, each of which contributes a catalytic aspartate residue. Therefore, it is not surprising that many drug firms initially screened their old renin inhibitors (for regulation of blood pressure) to look for hit for HIV protease inhibitors. Protease inhibitor design was markedly facilitated by the availability of X-ray crystallographic structures that allowed direct observation of inhibitors bound to the enzyme. Although protease acts at the last step of the virus life cycle, protease inhibitors were discovered shortly after nucleoside reverse transcriptase inhibitors. The era of HIV protease inhibitor drugs began in 1996 with the introduction of saquinavir, indinavir, and ritonavir as the first-generation HIV protease inhibitors. 2.First-generation HIV protease inhibitorsRoche’s saquinavir (Invirase) was the first HIV protease inhibitor on the United States market. Back in 1986, Roche undertook an ambitious international collaboration to tackle the HIV protease. The chemistry team in Welwyn, England was led by Ian Duncan and Sally Redshaw. After choosing a colorimetric assay as their in vitro assay, chemists designed inhibitors using the “transition-state mimic” strategy, which was previously highly successful in producing potent renin inhibitors. Since the transition state of a reaction has the highest energy for the reaction thermodynamics, therefore, a transition-state mimic is capable of inhibiting the functions of the enzyme. Saquinavir relied upon the hydroxyethylene moiety as an isostere of the tetrahedral transition state, a design element originating rom the extensive exploration of inhibitors of renin, another mammalian aspartyl protease. The chemists soon achieved an important milestone by defining the smallest peptide mimetic with which they could achieve acceptable level of inhibition. They found that a tripeptide was ideal in terms of both potency and bioavailability. As a consequence, tripeptides became a common theme in many protease inhibitors to follow. The Roche team made a heroic effort in fine-tuning the tripeptide, exploring their lead compound systematically by modifying each amino acid residue one by one. Their hard work paid off in 1991 when the team arrived saquinavir, which became the first HIV protease inhibitor on the market for the treatment of AIDS when it was approved by the FDA in December 1995. Although saquinavir, a peptomimetic, was metabolized easily, it was later found more beneficial to co-administrate with another protease inhibitor ritonavir by Abbott as a pharmacoenhancer. The combination could boost the plasma level of saquinavir significantly because ritonavir inhibited the cytochrome P450 3A4 (CYP3A4) enzyme in the liver that degraded saquinavir. CYP3A4 is the most abundant CYP isozyme in the liver. Moreover, the availability of saquinavir marked the beginning of combination therapy as cocktail drugs by introducing protease inhibitors to the clinic.[1] Abbott’s ritonavir (Norvir) was the second protease inhibitor on the market. The circumstances under which ritonavir was discovered was quite unique. The Abbott team was led by X-ray crystallographer John Erickson and medicinal chemist Dale Kempf. While it was rumored that Merck had 30 chemists on their HIV protease inhibitor project, Kempf had three. Instead of screening their renin inhibitors like most drug firms did, Abbott took advantage of Erickson’s X-ray crystallography work on the HIV protease, which would prove to be instrumental to their drug design. Integrating structure-based drug design (SBDD) and traditional medicinal chemistry, they prepared a series of symmetry-based inhibitors to match the C2-symmetric nature of the HIV protease. Kempf dubbed their inhibitors molecular peanut butter because they bound to both sides of the enzyme. Using that approach, they arrived at pyridine-containing A-77003, a tetrapeptide. Although A-77003 was potent in binding and cellular assays, it was not bioavailable with extremely high human biliary clearance. By reducing the molecular weight and replacing the existing amino acids with more soluble ones, they achieved an increase in bioavailability. They finally hit the jackpot when they identified that the pyridine termini were oxidized into N-oxide by hepatic cytochrome P450. Simply replacing the pyridines with metabolically more robust thiazoles and a little fine-tuning gave rise to ritonavir with bioavailability of 78% in comparison to 26% for the pyridyl analog A-77003. In March 1996, Abbott’s ritonavir (Norvir) won approval by the FDA. [2] Abbott’s discovery of ritonavir was certainly impressive. But two ensuing events after its discovery were equally, if not more, intriguing. One involved its crystalline forms and the other led to cobicistat, a ritonavir analog discovered by Gilead as a bioavailability booster.   After gaining the FDA’s blessing to sell ritonavir with the trade name Norvir in 1996, Abbott manufactured the drug’s active pharmaceutical ingredient (API) with the original crystalline form I. Since ritonavir was not bioavailable in its solid state, it was formulated as either an oral solution or semi-solid capsule, both in an ethanol–water-based solution. In early 1998, after two years on the market, things began to go wrong in manufacturing the API: many lots started to become a new, different, and extremely insoluble crystalline form II even though form I was not that soluble itself to start with. There was an extensive intermolecular hydrogen bond network for the new crystalline form II, which was thermodynamically more stable than the original crystalline form I. Once there was a crystal seed of form II, no matter how minute and microscopic the seed was, Mother Nature would always convert the API to the thermodynamically more stable form II. A team of scientists who had been exposed to the crystalline form II visited Abbott’s manufacturing facility in Italy to investigate ritonivir’s bulk manufacturing process. Afterward, significant amount of crystalline form II started showing up in bulk drugs during the manufacturing process done at the plant. The situation really put Abbott at a marketing crisis. It took a Herculean effort of Abbott’s process chemists to figure out a way to make only the original form I. In order to prevent formation of form II, the semi-solid capsule or oral formulation required refrigeration prior to use, among other measures to be taken.  [3] Another story following ritonavir’s success took place at Gilead Sciences in Foster City, California. Intriguingly, while many other protease inhibitors are metabolized by CYP3A4, ritonavir is a potent mechanism-based inhibitor of both CYP3A4 and P-glycoprotein (Pgp), which shuttles the drug outside the cell. Therefore, it could be used to increase plasma levels of co-dosed protease inhibitors or any other drugs that are metabolized by CYP3A4. As a result, dual protease inhibitor therapy has proven to be a powerful regimen in terms of efficacy and minimizing drug resistance. Low doses of ritonavir are now used primarily as a pharmacokinetic (PK) booster of HIV inhibitors, also known as a PK enhancer or pharmacoenhancer. A pharmacoenhancer, which itself is often not active against the therapeutic target, but can inhibit the enzyme that metabolizes the active drug. Therefore, a pharmacoenhancer can improve the PK profiles of an antiviral drug to achieve adequate trough concentrations at lower dosage and with less frequent dosing.30 The trough concentration is the lowest concentration reached by a drug before the next dose is administered. Gilead worked with Japan Tobacco on the HIV integrase inhibitor elvitegravir (Vitekta, 2014). Regrettably, despite being a potent integrase strand transfer inhibitor (INSTI), elvitegravir was extensively metabolized, primarily by CYP3A4 in both liver and intestine, making a once-daily regimen impossible. They obtained good boost of drug exposure when given together with ritonavir, although using sub-therapeutic dose ritonavir had the potential to cause drug resistance against other protease inhibitors. Gilead decided to make a drug that would only boost bioavailability by inhibiting CYP3A4-mediated metabolism but was completely devoid of HIV protease inhibition. Meanwhile, a high aqueous solubility and good physiochemical properties were desirable to facilitate drug formulation. Ritonavir had a poor aqueous solubility. Under the leadership of Manoj Desai, head of Medicinal Chemistry, a team led by Lianhong Xu arrived at a potent, orally bioavailable and selective CYP3A4 inhibitor cobicistat using ritonavir as their starting point. The secret to their success was removal of a hydroxyl group on ritonavir because it mimicked the transition state of amide hydrolysis through hydrogen bond with the two amino acids at the catalytic domain in the active site of the HIV protease. The molecule without the key hydroxyl group had little binding to the HIV protease. Cobicistat was approved by the FDA in 2012 and Gilead sold it under the trade name Tybost, which has been used in combination with a variety of antiretroviral drugs. [4] In drug discovery, drug-drug interaction (DDI) occurs when two drugs are metabolized by the same isoform of the liver CYP enzyme. They are often regarded as a liability for good reasons. For example, Bayer’s statin cerivastatin (Baycol) caused severe liver damage when it was given with fibrate drugs to lower cholesterol. Since both of them are metabolized by CYP3A4, the liver does not have enough CYP3A4 enzyme to metabolize both of the drugs at the same time, thus causing toxicities. But with pharmacoenhancers such as ritonavir and cobicistat, we can turn a drug’s liability into an asset. While the pharmacoenhancers keep the liver CYP enzymes busy, the antiviral drugs can focus on killing viruses without watching their own back. Cobicistat can potentially make a once daily, protease inhibitor-containing single tablet regimen possible.  Merck’s indinavir (Crixivan) was the third protease inhibitor on the market. Merck began their research on HIV protease inhibitors in 1986 with Irving Sigal, a senior director in the Department of Molecular Biology, as the project champion. They initially also screened their renin inhibitors for HIV protease inhibition and then carried out rational drug design by taking advantage of the known crystal structure of HIV protease, first discovered by Merck and refined by the NIH scientists. In 1990, chemist Wayne Thompson arrived at L-689,502, which was active in inhibiting the HIV protease but was devoid of renin activity. Unfortunately, it was not bioavailable and only effective by injection. By that time, Roche’s saquinavir surfaced in literature as a viable oral drug. Inspired by saquinavir’s success, Joseph Vacca successfully incorporated a fragment of saquinavir into L-689,502. Bruce Dorsey, a new hire in 1989 in Vacca’s group, and his associate, Rhonda Levin, succeeded in synthesizing indinavir. Although in the monotherapy trials, around 40% of the patients were below 400 copies of RNA after six months on the drug, HIV developed resistance to indinavir in some patients. Fortunately, it was found that the combination of indinavir and AZT or lamivudine (Epivir) was quite effective in substantially suppressing the virus levels. Merck’s studies of combination therapy were the first to prove the efficacy of the cocktail approach and became the standard for the industry. After filing with the FDA in January 1996, indinavir received approval in March 1996 in an accelerated review process.[5] Other important early protease inhibitors included nelfinavir (Viracept, approved in March 1997) by Agouron (now Pfizer) and amprenavir (Agenerase, approved in April 1999) by Vertex. These are collectively known as the first-generation protease inhibitors. Vertex was a bit late to the game of protease inhibitors. Heavily leveraging crystallography and computer-aided drug design, Vertex and their Japanese collaborators at Kissei Pharmaceuticals discovered amprenavir, a very potent, picomolar inhibitor of HIV protease. With the drug from Kissei, Vertex then collaborated with Burroughs Wellcome to investigate the PK properties of amprenavir in 1993. Drunken with the financial success of AZT, Burroughs Wellcome was late to the field of protease inhibitors and needed Kissei/Vertex’s drug as a jumping board. Amprenavir was small enough to penetrate to brain where the virus often hid. Apparently liked what they saw, Burroughs Wellcome shut down their own small protease program and agreed to pay for the full cost of the $200 million for the clinical trials of the drug. Amprenavir was approved by the FDA in 1999 and Vertex and Burroughs Wellcome (which became later Glaxo Wellcome in 1995 and eventually Glaxo SmithKline in 2000) jointly sold the drug with a brand name Agenerase. The drug was later discontinued in use because it had a poor aqueous solubility (0.04 g/mL) despite having a relative 100% bioavailability. It required a high ratio of excipient to drug to afford gastrointestinal tract solubility and eventual absorption. Vertex proceeded to prepare its phosphate ester prodrug, fosamprenavir, which had an eight-fold increase of aqueous solubility (0.31 g/mL). The prodrug fosamprenavir was approved in 2005 and Vertex and GSK sold it under the brand name Lexiva. Protease inhibitors are perhaps the most efficacious of the antiretroviral classes, in part because of their generally high genetic barrier to resistance. The early use of the first-generation protease inhibitors saquinavir, ritonavir, indinavir, and nelfinavir did not take advantage of boosting by CYP3A4 inhibition. Nevertheless, the suboptimal pharmacokinetic properties of these inhibitors meant that although the genetic barriers were high, resistance did ultimately emerge to these first-generation protease inhibitors. Later on, pharmacokinetic boost provided by ritonavir allowed for less frequent dosing, once a day for many drugs, and a higher genetic barrier to resistance, a consequence of the much-improved daily trough plasma concentrations.3.Second-generation HIV protease inhibitorsAlthough the initial rotinavir boosting regimens were implemented with the first-generation protease inhibitors, the development of more potent and safer second-generation inhibitors that heralded a new era of cocktail regimens. These second-generation inhibitors include fosamprenavir (the prodrug of amprenavir), lopinavir, atazanavir, tipranavir, and darunavir. Many of the first-generation protease inhibitors were peptomimetics. They were designed based on the basis of a modified peptomimetic motif wherein the scissile bond of a peptidic substrates replaced by a non-cleavable transition-state mimetic. It invariably took a tremendous amount of medicinal chemistry to methodically modify the peptide in every minute detail to achieve a drug that was potent and, more importantly, bioavailable. Due to the vulnerability of the early protease inhibitors toward metabolism, nearly all of them had to be given with rotinavir as a PK booster. Upjohn took a different approach.   Upjohn in Kalamazoo, Michigan did not want to take the peptomimetic approach. Instead, they carried out a medium throughput screen on a dissimilarity set of 5,000 of their existing historical compounds in the early 1990s. A library of 5,000 compounds seems absurdly small for a screen in light of today’s high throughput screen (HTS) routinely going through millions of compounds. Nonetheless, they identified warfarin, a blood thinner, as a weak inhibitor of HIV protease. Coincidently, their neighbors at Parke–Davis in Ann Arbor, Michigan identified a similar substrate from related screening efforts. Even though warfarin was far less potent than peptomimitc protease inhibitors, it was still attractive because warfarin was vastly bioavailable. In essence, Upjohn favored pharmacokinetics over potency for their screening hits. They subsequently carried out a focused screen of compounds similar to warfarin and identified phenprocoumon, a warfarin analog and a known orally active anticoagulant. Phenprocoumon was thirty-fold more potent than warfarin and already began to show antiviral activities in a cellular assay. A crystal structure of the phenprocoumon–protease complex was very helpful to future drug designs of better protease inhibitors. Taking advantage of computer-aided, structure-based drug design (SBDD), Upjohn arrived at their first-generation clinical candidate. But it was discontinued in favor of more potent analogs. Their second-generation clinical candidate was once again abandoned because it had a very high degree of protein binding and its potency was still modest, particularly in comparison to the most active contemporary peptomimetics. Third time was the charm. They eventually came up with their third-generation clinical candidate tipranavir with superior safety and efficacy. The drug, brand named Aptivus, was approved by the FDA in 2005. Regrettably, tipranavir seems to have more severe side effects compared to other protease inhibitors. [6]  The latest entrant of HIV protease inhibitor was Janssen’s darunavir (Prezista), approved by the FDA in 2006. The discovery of darunavir by Arun Ghosh took a long and windy journey. After finishing his postdoctoral training with E. J. Corey at Harvard in 1988, Ghosh worked at Merck’s West Point, Pennsylvania site for six years, involving in the discovery of HIV protease inhibitors. But he decided to start his independent academic career at the University of Illinois at Chicago in 1994 before he moved to Purdue University in 2005. To discover better HIV protease inhibitors, Ghosh employed a “backbone binding” design concept to maximize the interactions with the active site of HIV protease, particularly, to promote extensive hydrogen bonding with protein backbone atoms. His group initially installed a tetrahydrofuran (THF) ring onto Roche’s saquinavir. Later on, by combining the left-handed feature of Merck’s indinavir, Ghosh arrived at a reasonably good THF-containing protease inhibitor. Meanwhile, Vertex/Kissei also incorporated the similar 3-(S)-THF motif as the P2 ligand of their sulfonamide compound. Interestingly, Searle actually had a priority on the sulfonamide-containing protease inhibitors and Vertex had to pay Searle $25 million in 1995 to resolve the patent issue even though Searle’s own program flopped due to protein binding and extensive metabolism issues.In the field of drug discovery, one always learns from each other. While Vertex “borrowed” Ghosh’s THF fragment, Ghosh was not shy either to “borrow” Vertex’s sulfonamide moiety. For Ghosh, to solidify his intellectual property position and boost potency via more hydrogen bonding, he invented a bis-THF group in place of the THF fragment. The result was darunavir, whereas “arun” in the generic name was undoubtedly a nod to the inventor. Development of darunavir was carried out by Tibotec, a subsidiary of Janssen. The drug was approved in 2006 and Janssen sells it with a brand name of Prezista. [7] The coverage of initial protease resistance by PK-boosted second-generation protease inhibitors allowed for the sequential use of protease inhibitors in patients who developed resistance to first generation agents. This is especially true for the more potent second-generation inhibitors. At the end of the day, almost all HIV protease inhibitors, including the latest and the best darunavir, need to be given with a pharmacokinetic enhancer such as ritonavir or cobicistat in order to achieve effective plasma drug levels at the desired dose and frequency. Gratifyingly, the availability of protease inhibitors has dropped the fatality rate for AIDS patients by 70%. 4.SummaryTo summarize, ten HIV protease inhibitors currently on the market are: saquinavir (Invirase, Roche), 1995indinavir (Crixivan, Merck), 1996ritonavir (Norvir, Abbott), 1996nelfinavir (Viracept, Agouron/Eli Lilly), 1997amprenavir (Agenerase, Vertex/GSK, discontinued), 1999–2008lopinavir (Kaletra with ritonavir, Abbott), 2000atazanavir (Reyataz, Ciba Geigy), 2003fosamprenavir (a prodrug of amprenavir, Lexiva, GSK/Vertex), 2005tipranavir (Aptivus, Upjohn/Pfizer/Boehringer Ingelheim), 2005darunavir (Prezista, Janssen), 2006参考文献（向下滑动查看更多）1.Duncan, Ian B.; Redshaw, Sally Discovery and Early Development of Saquinavir Infectious Diseases Therapy 2002, 25(Protease Inhibitors in AIDS Therapy), 27–47. 2.Kempf, Dale J. Discovery and Early Development of Ritonavir and ABT-378 Infectious Diseases Therapy 2002, 25(Protease Inhibitors in AIDS Therapy), 49–64. 3.Chemburkar, S. R.; Bauer, J.; Deming, K.; Spiwek, H.; Patel, K.; Morris, J.; Henry, R.; Spanton, S.; Dziki, W.; Porter, W.; Quick, J.; Bauer, P.; Donaubauer, J.; Narayanan, B. A.; Soldani, M.; Riley, D.; McFarland, K. Dealing with the Impact of Ritonavir Polymorph on the Last Stage of Bulk Drug Process Development, In Organic Process Research & Development 2000, 4, 413-417. 4.Xu, L.; Liu, H.; Murray, B. P.; Callebaut, C.; Lee, M. S.; Hong, A.; Strickley, R. G.; Tsai, L. K.; Stray, K. M.; Wang, Y.; Rhodes, G. R.; Desai, M. C. Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer. ACS Med. Chem. Lett. 2010, 1, 209–213. 5.Dorsey, B. D.; Vacca, J. P. Discovery and Early Development of Indinavir. Infectious Diseases Therapy 2002, 25(Protease Inhibitors in AIDS Therapy), 65–83.6.Romines, K. R. Discovery and development of tipranavir, In Antiviral Drugs, ed., Kazmierski, W. M., Wiley: Hoboken, NJ (2011), pp 47–57. 7.(a) Ghosh, A. K.; Sridhar, P. R.; Kumaragurubaran, N.; Koh, Y.; Weber, I. T.; Mitsuya, H. Bis-tetrahydrofuran: a privileged ligand for darunavir and a new generation of HIV protease inhibitor that combat drug resistance, ChemMedChem 2006, 1, 939–950. (b) Ghosh, A. K. Recent Progress in the Development of HIV-1 Protease Inhibitors for the Treatment of HIV/AIDS. J. Med. Chem. 2016, 59, 5172–5208. 识别微信二维码，添加生物制品圈小编，符合条件者即可加入微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。