Development of a new class of antifungals effective against Cryptococcus

目标：创造一种耐受性良好的口服治疗药物，可单独使用或与其他药物联合使用。 与其他抗真菌剂联合使用，以改善患有 隐球菌性脑膜炎（CM） 隐球菌引起的感染的全球疾病负担超过100万例 每年。这种疾病通常是由吸入真菌引起的，真菌从肺部传播到 免疫功能低下个体的中枢神经系统，在那里它被归类为隐球菌 脑膜炎（CM）。如果不治疗，CM总是致命的。一线治疗仅限于阿霉素B/5- 氟胞嘧啶，这在发展中国家基本上无法获得。 我们最近的努力，以确定新的治疗方法来治疗CM集中在针对真菌 蛋白Gwt 1酰基转移酶，一种组装 糖基磷脂酰肌醇（GPI）锚定蛋白，其随后附着于真菌细胞壁。 Ampoxix Pharmaceuticals已获得来自Kidai的一系列强效Gwt 1抑制剂的许可，包括 APX 001，已完成1期临床试验的首个同类分子。APX 001 A，主动 APX 001的一部分，对隐球菌有一定的活性，然而，其在小鼠动物中的效力 模型与氟康唑联合使用时观察效果最好。我们最近综合并评估了 另外的Gwt 1抑制剂，并已确定了几种分子与显着改善 微生物活性（> 30倍）对C.新型隐球菌和加提。在这份提案中，林奈 生物科学公司将开发一种基于显微镜的C.用它来证实 新的分子靶向Gwt 1，评估与其他抗真菌药物的协同作用，并在体外和体内探索 体内表型效应。杜克大学医学院的John Perfect博士将评估化合物 在CM小鼠模型中的疗效。这一建议的最终结果将是选择一个 IND使能研究的候选药物，证明与APX 001相比，在两种CM中均具有显著改善 存活和真菌负荷小鼠模型。 我们将开始通过发展一种快速的显微镜检测来确定作用机制 (MOA)Gwt 1抑制剂在隐球菌中的表达，并将其应用于APX 001 A类似物的研究。然后我们将 表征了APX 001 A分子的30种类似物，其比APX 001 A分子显著更有效（4至>32倍）， APX 001 A vs隐球菌。我们计划使用我们新的基于显微镜的检测方法，以确保这些更多 有效的分子就在目标上我们还将表征这些分子的潜在细胞毒性 和代谢稳定性。将选择前10种化合物并进一步研究，以表征其 微生物活性，包括确定它们对一组C. 新型隐球菌和gattii分离物。我们将成功地扩大合成8-10个选定的类似物 并评估用于动物功效研究的合适制剂。我们将 表征8-10种化合物的PK、MTD和安全性，鉴定超过40%口服给药的分子， 具有MTD、hERG抑制潜力和细胞色素P450潜力的生物利用度 同种型相互作用等同于或优于APX 001（APX 001 A）。在最后一步，我们将 证明了5-10种化合物在新型隐球菌感染动物模型中的功效。 我们将测试分子在肺和脑模型中减少真菌负担的能力， CM模型中的存活率。我们还将确定这些化合物在使用时是否能减少真菌负担 与氟康唑（或其他用于治疗CM的抗真菌药）联合使用。我们将 使用我们的基于显微镜的测定来评估Gwt 1抑制剂的有效性和体内作用模式， 脑和肺感染模型。完成后，我们将宣布候选人准备IND启用 问题研究

Goal: To create a well-tolerated, orally available therapeutic that can be employed alone or in combination with other antifungal agents to improve treatment outcomes for patients suffering from cryptococcal meningitis (CM). The worldwide disease burden of infections caused by Cryptococcus is over one million cases annually. The disease is normally caused by inhalation of the fungus, which spreads from the lungs to the central nervous system in immunocompromised individuals, where it is classified as cryptococcal meningitis (CM). CM is always fatal if left untreated. First-line therapy is limited to amphotericin B/5- flucytosine, which is largely unavailable in the developing world. Our recent efforts to identify novel therapeutics to treat CM have focused on targeting the fungal protein Gwt1 acyl transferase, an enzyme that is required for the assembly of glycosylphosphatidylinositol (GPI) anchored proteins that are later attached to the fungal cell wall. Amplyx Pharmaceuticals has in-licensed a portfolio of potent Gwt1 inhibitors from Eisai, including APX001, the first-on-class molecule which has completed Phase 1 clinical trials. APX001A, the active moiety of APX001, has some activity against Cryptococcus, however, its potency in mouse animal models is best observed in combination with fluconazole. We have recently synthesized and evaluated additional Gwt1 inhibitors and have identified several molecules with significantly improved microbiological activity (> 30-fold) against C. neoformans and C. gattii. In this proposal, Linnaeus Bioscience Inc. will develop a microscopy based assay for C. neoformans and use it to confirm that the new molecules target Gwt1, evaluate synergy with other antifungal agents, and explore in vitro and in vivo phenotypic effects. Dr. John Perfect, Duke University School of Medicine, will evaluate compounds for efficacy in mouse models of CM. The ultimate outcome of this proposal will be the selection of a candidate for IND-enabling studies that demonstrates a significant improvement vs APX001 in both CM survival and fungal burden mouse models. We will begin by developing a rapid microscopy assay for determining the mechanism of action (MOA) of Gwt1 inhibitors in Cryptococcus and apply it to studies of APX001A analogs. We will then characterize 30 analogs of APX001A molecules that are significantly more potent (4 to >32 fold) than APX001A vs Cryptococcus. We plan to use our new microscopy based assay to ensure these more potent molecules are on target. We will also characterize these molecules for their potential cytotoxicity and metabolic stability. The top 10 compounds will be selected and further studied to characterize their microbiological activities, including determining their spectrum of activity against a panel of C. neoformans and C. gattii isolates. We will successfully scale-up the synthesis of 8-10 selected analogs in prodrug form and assess appropriate formulations for use in animal efficacy studies. We will characterize the PK, MTD and safety for 8-10 compounds, identifying molecules with over 40% oral bioavailability that have an MTD, potential for hERG inhibition, and potential for cytochrome P450 isoform interaction that is equivalent to or better than APX001 (APX001A). In the final step, we will demonstrate the efficacy of 5-10 compounds in Cryptococcus neoformans animal models of infection. We will test molecules for their ability to reduce fungal burden in lung and brain models and to improve survival in CM models. We will also determine if these compounds reduce fungal burden when used in combination with fluconazole (or other antifungals that are used for the treatment of CM). We will use our microscopy based assay to assess Gwt1 inhibitor effectiveness and mode of action in vivo in brain and lung infection models. When completed we will declare a candidate ready for IND enabling studies.