Discovery of New Anti-amoeba Therapeutics

新抗阿米巴疗法的发现

• 批准号: 9480203
• 负责人: William David Nes
• 金额: $ 31.1万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-22 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9480203
• 关键词: ADME Study; Acanthamoeba; Acanthamoeba Keratitis; Active Sites; Amebiasis; Amebicides; Amino Acid Sequence; Amoeba genus; Amphotericin B; Anabolism; Animals; Antifungal Agents; Azoles; Binding; Binding Sites; Biochemical; Biological Availability; Catalysis; Characteristics; Cholesterol; Collaborations; Combined Modality Therapy; Complex; Cornea; Crystallization; Cultured Cells; Cyst; Disease; Drug Targeting; Drug usage; Effectiveness; Electronics; Enzymes; Ergosterol; Genome; Goals; Growth; Growth Inhibitors; Health; Health Sciences; Healthcare; Human; In Vitro; Incubated; Infection; Ketoconazole; Lanosterol; Lead; Medical; Medical center; Metabolic Control; Metabolism; Methyltransferase; Mus; Naegleria; Naegleria fowleri; Orthologous Gene; Outcome; Parasites; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phase; Property; Protein Isoforms; Protozoa; Reaction; Recombinant Proteins; Research; Research Personnel; Rodent Model; Roentgen Rays; Series; Side; Specificity; Steroids; Sterols; Structure; Testing; Texas; Therapeutic; Toxic effect; Universities; Use Effectiveness; Vision; analog; base; cholesterol biosynthesis; design; drug candidate; enzyme biosynthesis; fungus; improved; in vitro testing; in vivo; inhibitor/antagonist; medical schools; methyl group; molecular recognition; mouse model; novel; pathogen; preference; primary amebic meningoencephalitis; public health relevance

 DESCRIPTION (provided by applicant): Amebiasis and related vision-threatening infections caused by amoebae is a major contributor to diarrheal diseases, primary amoebic meningoencephalitis and cornea problems. Sequencing of amoeba genomes has evoked a broad search for new, pathogen-specific drug targets, of which the sterol C24-methyltransferase (24-SMT) is a clear candidate since it is synthesized in protozoa but not in animals. Notably, 24-SMT is responsible for the introduction of the methyl group at C24 into the ergosterol side chain. The cholesterol side chain is missing this structural feature which is crucial to ergosterol function. In the R21 phase, we will incubate a series of mechanism-based inhibitors of 24-SMT that differ in the sterol frame and electronics of the side chain with cultured cells of Acanthamoeba and Naegleria and determine which compounds are the most potent inhibitors of ergosterol biosynthesis, trophozoites growth and cyst formation. Additionally, to make improvements to existing drug therapy for treating amoeba infections we will further evaluate in vitro representative anti-fungal azoles that target sterol 14-demethylase (14-SDM; CYP51). Therefore, we will characterize the substrate preference and product outcome of cloned enzymes and use these enzymes to determine inhibitor specificity, binding and covalent inactivation properties. The R33 phase will be undertaken with the proof-of-concept demonstrated. We will test in vitro whether our lead molecules that inhibit 24-SMT in combination with traditional chemotherapeutics have synergistic activities. Additionally, in collaboration with investigators at Meharry Medical College and UTSouthwestern Medical Center, we will evaluate our lead candidate drugs in a mouse model of Acanthamoeba keratitis or primary amebic meningoencephalitis due to Naegleri fowleri. With the aid of a collaborator at Texas Tech University Health Sciences Center, crystal structures of 24-SMT and 14- SDM complexed with relevant inhibitors will be generated to identify binding sites with certainty and should reveal interactions involved with catalysis. ADME/toxicity properties will be evaluated by the bioavailability and metabolism of 3H-inhibitor fed to healthy mice. The overall goal of these studies is to establish mechanism-based inhibitors as a novel class of anti- amoeba agents and to develop synergistic partners of steroidal inhibitors and antifungal agents (medical azole or amphotericin B) that target ergosterol biosynthesis and processing. Specific therapeutic combinations of these compounds could achieve optimal amebacidal effectiveness that thereby, provide for better healthcare.

 描述(申请人提供)：阿米巴引起的阿米巴病和相关的危及视力的感染是腹泻疾病、原发性阿米巴脑膜脑炎和角膜问题的主要诱因。对阿米巴基因组的测序引起了对新的病原体特异性药物靶点的广泛搜索，其中甾醇C24-甲基转移酶(24-SMT)是一个明显的候选基因，因为它是在原生动物中合成的，而不是在动物中合成的。值得注意的是，24-SMT负责将C24上的甲基引入麦角甾醇侧链。胆固醇侧链缺少这种对麦角固醇功能至关重要的结构特征。在R21阶段，我们将与棘阿米巴和Naegleria的培养细胞孵育一系列基于机制的24-SMT抑制剂，这些抑制剂的甾醇框架和侧链电子结构不同，并确定哪些化合物对麦角甾醇生物合成、滋养体生长和包囊形成最有效。此外，为了改进现有的治疗阿米巴感染的药物疗法，我们将进一步在体外评估针对固醇14-去甲基酶(14-SDM；CYP51)的具有代表性的抗真菌唑。因此，我们将表征克隆酶的底物选择性和产物结果，并使用这些酶来确定抑制剂的特异性、结合和共价失活性质。R33阶段将进行概念验证演示。我们将在体外测试我们的铅分子是否与传统化疗药物联合抑制24-SMT具有协同活性。此外，我们将与梅哈里医学院和德克萨斯大学西南医学中心的研究人员合作，在棘阿米巴角膜炎或因福氏奈格尔氏杆菌引起的原发性阿米巴脑膜脑炎的小鼠模型上评估我们的主要候选药物。在德克萨斯理工大学健康科学中心的一名合作者的帮助下，将产生与相关抑制剂络合的24-SMT和14-SDM的晶体结构，以确定结合部位，并应揭示与催化有关的相互作用。ADME/毒性特性将通过给健康小鼠喂养的~3H-抑制剂的生物利用度和代谢来评估。这些研究的总体目标是建立基于机制的抑制剂作为一类新型的抗阿米巴药物，并开发以麦角甾醇生物合成和加工为靶点的类固醇抑制剂和抗真菌药物(医用唑或两性霉素B)的协同伙伴。这些化合物的特定治疗组合可以实现最佳的阿米巴杀菌效果，从而提供更好的医疗保健。