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方法，可单独使用或在 与其他抗真菌药物联合使用改善患者的治疗结果 来自隐球菌性脑膜炎(CM)。 全世界由隐球菌引起的感染造成的疾病负担超过100万例 每年一次。这种疾病通常是由吸入这种真菌引起的，这种真菌从肺部传播到 免疫受损个体的中枢神经系统，在那里被归类为隐球菌 脑膜炎(CM)。如果不治疗，CM总是致命的。一线治疗仅限于两性霉素B/5- 氟胞嘧啶，这在发展中国家基本上是买不到的。 我们最近在寻找治疗CM的新疗法方面的努力主要集中在针对真菌 蛋白质Gwt1酰基转移酶，是组装 糖基磷脂酰肌醇(GPI)锚定的蛋白质，后来附着在真菌细胞壁上。 Amplyx PharmPharmticals已经从卫材获得了一系列有效的Gwt1抑制剂的许可，包括 APX001，已经完成一期临床试验的第一类分子。APX001A，有源的 APX001的部分成分对隐球菌有一定的抑制作用，但对小鼠动物有一定的抑制作用 模型最好与氟康唑联合观察。我们最近进行了综合和评估 额外的Gwt1抑制剂，并已经确定了几个分子有显著改善 微生物活性(&gt；30倍)对新生隐球菌属和加蒂亚隐球菌属。在这份提案中，林奈 Bioscience Inc.将开发一种基于显微镜的新生芽孢杆菌检测方法，并用它来证实 新分子靶向Gwt1，评估与其他抗真菌药物的协同作用，并在体外和体内进行探索 活体表型效应。杜克大学医学院的约翰·完美博士将对化合物进行评估 观察其对CM小鼠模型的疗效。这项提议的最终结果将是选出一个 支持IND研究的候选人，证明在这两个CM中都比APX001有显著改进 存活和真菌负荷的小鼠模型。 我们将首先开发一种用于确定作用机制的快速显微分析方法 隐球菌中Gwt1抑制剂的(MOA)，并将其应用于APX001A类似物的研究。到时候我们会的 描述APX001A分子的30个类似物，它们的效力显著高于(4到32倍) APX001AVS隐球菌属。我们计划使用我们新的基于显微镜的分析来确保这些更多 强大的分子正瞄准目标。我们还将表征这些分子的潜在细胞毒性。 和新陈代谢稳定性。将选出排名前10位的化合物，并对其进行进一步研究，以表征其 微生物活性，包括测定它们对一组C. 新生杆菌和加蒂亚氏杆菌分离株。我们将成功放大合成8-10个精选类似物 形成并评估用于动物疗效研究的适当制剂。我们会 表征8-10种化合物的PK、MTD和安全性，识别口服含量超过40%的分子 具有MTD、潜在的HERG抑制和潜在的细胞色素P450的生物利用度 等同于或优于APX001(APX001A)的异构体相互作用。在最后一步，我们将 展示5-10个化合物在新生隐球菌感染动物模型中的疗效。 我们将测试分子在肺和脑模型中减轻真菌负担的能力，并改善 在CM模型中存活。我们还将确定这些化合物在使用时是否会减少真菌负担 与氟康唑(或用于治疗CM的其他抗真菌药物)联合使用。我们会 使用我们基于显微镜的方法来评估Gwt1抑制剂在体内的有效性和作用模式 脑和肺感染模型。完成后，我们将宣布候选人已准备好启用IND 学习。

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.