Characterization of activity and mechanism of novel fungicidal anti-cryptococcal molecules

 描述（由申请人提供）：隐球菌脑膜炎是影响艾滋病毒/艾滋病感染者的最重要的机会性感染之一。最近的流行病学研究表明，每年约有100万新病例，每年有70多万人死亡;在撒哈拉以南非洲，每年死于隐球菌脑膜炎的艾滋病毒患者多于结核病。隐球菌病的金标准疗法是阿替霉素B联合5-氟胞嘧啶。虽然这一方案在世界上资源丰富的地区很容易实施，但在许多疾病负担最高的资源有限地区却无法使用。在这些地区，使用氟康唑，一种不太有效的抑真菌剂，因为它容易获得，安全，口服。不幸的是，接受FLU治疗的患者死亡率要高得多;这是由于FLU的抑真菌活性。因此，隐球菌病的新疗法是迫切需要的，这是我们项目的长期目标。由于具有杀真菌活性的分子在临床上比抑真菌药物更上级，我们开发了一种高通量筛选测定法，该测定法直接鉴定具有抗念珠菌杀真菌活性的分子。新人类我们已经用该测定筛选了两个不同的文库：1）通过NIH资助的分子探针和文库筛选网络项目的350，000个化合物文库和2）来自默克实验室收集的抗念珠菌分子的700个分子的集中组。前一个筛选被设计用于鉴定具有针对C. neoformans：一种新型的苯并咪唑硫脲支架，具有低的人体毒性和良好的抗隐球菌活性。我们将在目标1中确定其作用机制，为随后优化其抗隐球菌活性奠定基础。从默克图书馆，我们已经确定了22个分子与杀真菌活性对C。浓度≤ 2 µg/mL的新生儿。我们将完成目标2中默克文库命中集的二次筛选，并确定总共四个高优先级支架的作用机制。初步评估表明，一组角鲨烯合酶抑制剂是命中。我们将测试这一假设，并从剩余的命中中确定三个高优先级支架的目标。从这个探索性项目中出现的机制信息将被用作后续应用的基础，以优化支架的抗隐球菌活性。

 DESCRIPTION (provided by applicant): Cryptococcal meningitis is one of the most important opportunistic infections affecting people living with HIV/AIDS. Recent epidemiologic studies indicate that approximately 1 million new cases occur each year and over 700,000 people die each year; in sub-Saharan Africa, more HIV patients die of cryptococcal meningitis each year than tuberculosis. The gold standard therapy for cryptococcosis is amphotericin B combined with 5-flucytosine. Although this regimen is easily administered in resource-rich regions of the world, it is not available in many resource-limited regions with the highest burden of disease. In these regions, fluconazole, a less effective, fungistatic agent, is used because it is readily available, safe, and administered orally. Unfortunately, patients treated with FLU have much higher death rates; this has been attributed to the fungistatic activity of FLU. Thus, new therapies for cryptococcosis are urgently needed and this is the long-term goal of our project. Because molecules with fungicidal activity are clinically superior to fungistatic drugs, we have developed a high throughput screening assay that directly identifies molecules with fungicidal activity toward C. neoformans. We have screened two distinct libraries with the assay: 1) a 350,000 compound library through an NIH-funded Molecular Probes and Library Screening Network project and 2) a focused set of 700 molecules from Merck Laboratories collection of anti-candidal molecules. The former screen was designed to identify a molecule with activity against cell wall related processes in C. neoformans: a novel benzimidazole thiourea scaffold with low human toxicity and good anti-cryptococcal activity emerged from this screen. We will identify its mechanism of action in Aim 1 to set the stage for subsequent optimization of its anti-cryptococcal activity. From the Merck Library, we have identified 22 molecules with fungicidal activity against C. neoformans at concentrations ≤ 2 µg/mL. We will complete the secondary screening of the set of Merck library hits in Aim 2 and identify the mechanism of action for a tota of four high priority scaffolds. Initial evaluation indicates that a set of squalene synthase inhibitors are among the hits. We will test that hypothesis and identify the target of three high priority scaffolds from the remaining hits. The mechanistic information that emerges from this exploratory project will be used as a basis for a subsequent application to optimize the anti-cryptococcal activity of the scaffolds.