Discovery of New Anti-amoeba Therapeutics

新抗阿米巴疗法的发现

• 批准号: 8960084
• 负责人: William David Nes
• 金额: $ 20.89万
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8960084
• 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; 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; Life; Medical; Medical center; Metabolic Control; Metabolism; Methyltransferase; Mus; Naegleria; Naegleria fowleri; Orthologous Gene; Outcome; Parasites; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Phase; Process; Property; Protein Isoforms; Protozoa; Reaction; Recombinant Proteins; Relative (related person); Research; Research Personnel; Rodent Model; Roentgen Rays; Series; Side; Specificity; Sterols; Structure; Testing; Texas; Therapeutic; Toxic effect; Universities; Use Effectiveness; Vision; analog; base; cholesterol biosynthesis; design; drug candidate; enzyme biosynthesis; feeding; fungus; improved; in vitro testing; in vivo; inhibitor/antagonist; interest; 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（由申请人提供）：阿米巴病和相关的由阿米巴引起的威胁视力的感染是导致角膜疾病、原发性阿米巴脑膜脑炎和角膜问题的主要原因。变形虫基因组测序已经引起了广泛的搜索新的，病原体特异性的药物靶点，其中甾醇C24-甲基转移酶（24-SMT）是一个明确的候选人，因为它是在原生动物中合成，而不是在动物中。值得注意的是，24-SMT负责将C24处的甲基引入麦角固醇侧链中。胆固醇侧链缺少对麦角固醇功能至关重要的这种结构特征。在R21阶段，我们将孵育一系列基于机制的24-SMT抑制剂，其在侧链的甾醇框架和电子方面与阿米巴和耐格里属的培养细胞不同，并确定哪些化合物是麦角甾醇生物合成、滋养体生长和包囊形成的最有效抑制剂。此外，为了改善现有的治疗阿米巴感染的药物疗法，我们将进一步评估靶向固醇14-脱甲基酶（14-SDM; CYP 51）的体外代表性抗真菌唑类药物。因此，我们将表征克隆酶的底物偏好和产物结果，并使用这些酶来确定抑制剂特异性、结合和共价失活特性。R33阶段将在概念验证的基础上进行。我们将在体外测试我们的抑制24-SMT的先导分子与传统化疗药物组合是否具有协同活性。此外，与Meharry医学院和UT西南医学中心的研究人员合作，我们将在由Naegleri fowleri引起的阿米巴角膜炎或原发性阿米巴脑膜脑炎的小鼠模型中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合作者的帮助下，将生成与相关抑制剂复合的24-SMT和14- SDM的晶体结构，以确定结合位点，并应揭示与催化有关的相互作用。将通过饲喂健康小鼠的3 H-抑制剂的生物利用度和代谢评价ADME/毒性特性。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的抗阿米巴药物，并开发靶向麦角固醇生物合成和加工的甾体抑制剂和抗真菌剂（医用唑或阿替霉素B）的协同合作伙伴。这些化合物的特定治疗组合可以实现最佳的杀阿米巴效果，从而提供更好的医疗保健。