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

 描述(申请人提供)：隐球菌性脑膜炎是影响艾滋病毒/艾滋病患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100万新病例发生，每年有70多万人死亡；在撒哈拉以南非洲，每年死于隐球菌性脑膜炎的艾滋病毒患者比死于结核病的人多。隐球菌病的黄金标准疗法是两性霉素B联合5-氟胞嘧啶。虽然这种疗法在世界上资源丰富的地区很容易实施，但在疾病负担最重的许多资源有限的地区却无法使用。在这些地区，氟康唑是一种效果较差的抑菌剂，因为它容易获得、安全和口服给药。不幸的是，接受流感治疗的患者死亡率要高得多；这被归因于流感的抑菌活性。因此，迫切需要治疗隐球菌病的新疗法，这是我们项目的长期目标。由于具有杀菌活性的分子在临床上优于抗真菌药物，我们开发了一种高通量筛选方法，直接鉴定具有杀菌活性的分子对新生葡萄球菌的作用。我们用该方法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筛选了350,000个化合物文库；2)从默克实验室收集了700个抗念珠菌分子。前者的目的是鉴定一种具有抗新生隐球菌细胞壁相关过程的活性的分子：由此筛选出一种新型的苯并咪唑硫脲支架，该支架具有较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，为后续优化其抗隐球菌活性奠定基础。从默克文库中，我们已经鉴定了22个对新生隐孢子菌具有杀菌活性的分子，其浓度为≤2微克/毫升。我们将在AIM 2中完成默克文库命中的二次筛选，并确定总共四个高优先级支架的作用机制。初步评估表明，一系列角鲨烯合成酶抑制剂是成功的药物之一。我们将验证这一假设，并从剩余的命中中确定三个高优先级脚手架的目标。从这个探索性项目中产生的机械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.