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。 隐孢子虫病是儿童腹泻的第二大常见原因， 肠道内的真核寄生虫，隐孢子虫或C.人，和 特别是对营养不良的儿童造成伤害和死亡。与其他顶复门（如疟原虫）相比， 或弓形虫），不需要昆虫载体或动物宿主，因为寄生虫可以直接转移 通过粪口途径传播给人类尽管发病率高， 营养不良的儿童，隐孢子虫病没有有效的治疗方法。 我们之前已经筛选了GSK Tres Cantos专有的约200万种化合物库， P.恶性疟原虫N-肉豆蔻酰转移酶（NMT）。NMT是一种转移肉豆蔻酸酯的酶， 氨基酸，与蛋白质的N-末端甘氨酸残基协同作用，这有助于靶向底物 蛋白质到膜区域。NMT已被验证为真菌和寄生虫疾病的药物靶标， 包括疟疾和利什曼病我们假设NMT高通量筛选（HTS）有效地 对疟原虫也将积极对隐孢子虫和测试前八名命中对 微小隐孢子虫NMT（CpNMT）。在这前八名中，有三个对纯化的 酶和一个在体外显示出抗寄生虫的活性。后续的合成化学和结构- 的药物设计程序进一步发展这些命中到一个领先的系列（称为系列-2）的高效 （~ 10 nM IC 50）抑制剂，对人酶的选择性为500-1000 X。两条最有希望的线索 然后在小鼠感染模型中进行测试，发现两种分子 完全清除了感染这些数据用于化学验证NMT作为药物治疗的可药用靶标。 隐孢子虫病的治疗在本提案中，我们打算进一步开发先导化合物， 改善临床前药物开发制剂中药物样特征。

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.