Structural Analysis and Inhibitor Optimization of Cryptosporidium n-myristoyltransferase for Drug Discovery

摘要 每年有超过525,000名五岁以下的儿童死于传染病引起的腹泻。 隐孢子虫病是导致儿童腹泻的第二大原因，是由 真核寄生虫微小隐孢子虫或人隐孢子虫对肠道的定植 尤其是损害和杀害营养不良的儿童。与其他复合顶体(如疟原虫)形成对比 或弓形虫)，不需要昆虫媒介或动物宿主，因为寄生虫可以直接传播 通过粪便-口腔途径从人传给人。尽管发病率很高，而且对 对于营养不良的儿童，目前还没有有效的治疗隐孢子虫病的方法。 我们之前对GSK Tres Cantos专有的库进行了筛选，库中有大约200万种化合物 恶性疟原虫N-肉豆蔻酰基转移酶(NMT)。NMT是一种转移肉豆蔻酸的酶，肉豆蔻酸是一种14碳脂肪 酸，与蛋白质的N-末端甘氨酸残基共翻译，有助于靶向底物 蛋白质到膜区。NMT已被确认为治疗真菌和寄生虫病的药物靶点， 包括疟疾和利什曼病。我们假设NMT高通量筛选(HTS)有效 对抗疟原虫也会积极对抗隐孢子虫，并测试了前八个打击针对 微小隐孢子虫NMT(CpNMT)。在前八名中，有三名对纯化的 一种酶和一种酶在体外显示出抗寄生虫活性。合成化学和结构的续篇- 基于药物设计的计划进一步将这些药物发展成高效的先导系列(称为系列-2) (~10 nm IC50)抑制剂，比人类酶具有500-1000倍的选择性。两条最有希望的线索 然后在感染的小鼠模型中进行测试，发现这两个分子 完全清除了感染。这些数据用于从化学上验证NMT作为药物靶点的作用 治疗隐孢子虫病。在这项提议中，我们打算进一步开发先导化合物，以便 改善类药物特性，为临床前药物开发做准备。

ABSTRACT Each year over 525,000 children under age five are killed by diarrhea caused by infectious disease. Cryptosporidiosis, the second most frequent cause of childhood diarrhea, is an infection caused by colonization of the intestines by the eukaryotic parasites, Cryptosporidium parvum or C. hominis, and particularly damages and kills malnourished children. In contrast to other Apicomplexans (such as Plasmodium or Toxoplasma), there is no required insect vector or animal host, since parasites can be transferred directly from human to human through the fecal-oral route. Despite the high incidence and significant impact on malnourished children, there are no effective treatments for cryptosporidiosis. We had previously screened the GSK Tres Cantos proprietary library of ~2 million compounds against P. falciparum N-myristoyltransferase (NMT). NMT is an enzyme which transfers myristate, a 14-carbon fatty acid, to the N-terminal glycine residue of proteins co-translationally, which contributes to targeting the substrate protein to membrane regions. NMT has been validated as a drug target in fungal and parasitic diseases, including malaria and leishmaniasis. We hypothesized that NMT high-throughput screening (HTS) hits effective against Plasmodium would also be active against Cryptosporidium and tested the top eight hits against Cryptosporidium parvum NMT (CpNMT). Of those top eight hits, three were effective against the purified enzyme and one showed activity against the parasite in vitro. A follow-on synthetic chemistry and structure- based drug design program further developed these hits into a lead series (called Series-2) of highly effective (~10nM IC50) inhibitors with 500-1000 X selectivity over the human enzyme. The two most promising leads from the Series-2 scaffold were then tested in a mouse model of infection and found that both molecules completely cleared infection. These data serve to chemically validate NMT as a druggable target for the treatment of Cryptosporidiosis. In this proposal we intend to further develop lead compounds in order to improve drug-like characteristics in preparation for preclinical drug development.