High throughput screening (HTS) to discover chemical probes for neutral sphingomyelinase2

 描述(由申请人提供)：在接受联合抗逆转录病毒疗法(CART)的艾滋病毒感染患者中，约有一半仍患有某种形式的认知障碍，严重影响生活质量。这一发现表明，需要辅助性神经保护疗法来充分保护大脑免受感染。有认知障碍的HIV患者的大脑被证明有神经酰胺的积累；神经酰胺的一个主要来源是中性鞘磷脂酶2(NSmase2)催化的神经鞘蛋白的水解。尽管短暂的nSMase2上调是正常大脑功能的一部分，但实验证据开始表明，慢性nSMase2上调会导致包括神经炎症和氧化应激在内的负面影响。虽然nSmase2在手部和其他神经退行性疾病的发病机制中扮演着重要的角色，但目前的nSMase2抑制剂尚不足以探索该酶的作用。目前的nSMase2抑制剂表现出非药物性质，包括高相对分子质量、较差的溶解性和较差的药代动力学。这项建议的目的是进行高通量筛选(HTS)来鉴定和表征nSMase2的小分子抑制剂。这些抑制剂将被用作化学探针，以了解慢性nSMase2激活在艾滋病毒相关神经认知障碍(HAND)中所起的作用。该项目的目标1是对NIH国家先进翻译科学中心(NCATS)的化学库中的约43万种化合物进行QHTS，并进行后续靶标减去分析和正交放射性分析，以确定nSMase2抑制剂。我们将使用基于1536孔荧光的分析，使用人类nSmase2，它是HTS就绪的，Z‘=0.8。在目标2中，我们将从多个化学类型中选择大约100个最有效和最类似药物的抑制剂进行评估。在目标2中，我们将在几个方面对所选抑制剂进行表征，包括它们作为中枢神经系统药物的潜力、它们的代谢稳定性、它们在体外具有神经保护的能力以及它们的脱靶倾向。在AM 2完成后，我们预计将鉴定属于不同化学类型的10到20个nSMase2抑制剂，它们有可能成为化学探针，并将推进到目标3。在目标3中，我们将进行初步的SAR优化，重点是消除在AIM 2中的抑制剂鉴定过程中遇到的最可寻址的易感性。我们的化学探针的标准将是：IC50=1微米，CNS-多参数优化分数=4，与小鼠和人肝微粒体孵育1小时后化合物稳定性=60%。剂量依赖的神经元保护和=20%的对44个已知存在安全风险的靶点的抑制在10微米。通过实施本提案中的目标确定的化学探针将可用于研究nSMase2在手部动物模型中的作用，以及可能在nSMase2可能发挥作用的其他神经退行性疾病中的作用。

 DESCRIPTION (provided by applicant): Approximately half of patients infected with HIV that have access to combinational antiretroviral therapy (cART) still suffer from some form of cognitive impairment that profoundly affects quality of life. This finding conveys the need for adjunctive neuroprotective therapy to fully protect the brain from infection. Brains from HIV patients with cognitive impairment have been shown to exhibit accumulation of ceramides; one major source of ceramide is through the hydrolysis of sphingomyelin catalyzed by neutral sphingomyelinase 2 (nSmase2). Even though transient nSMase2 upregulation is part of normal brain functioning, experimental evidence is beginning to indicate that chronic nSMase2 upregulation results in negative effects including neuroinflammation and oxidative stress. While nSmase2 is emerging as an important player in the pathogenesis of HAND and other neurodegenerative diseases, the current armamentarium of nSMase2 inhibitors is inadequate to explore the role of the enzyme. Current nSMase2 inhibitors exhibit non-drug like properties including high molecular weight, poor solubility and poor pharmacokinetics. The objective of this proposal is to conduct a high throughput screen (HTS) to identify and characterize small molecule inhibitors of nSMase2. These inhibitors would be used as chemical probes to understand the role that chronic nSMase2 activation plays in HIV-associated neurocognitive disorder (HAND). Aim 1 of the project is to conduct qHTS of about 430,000 compounds from the chemical library at the NIH National Center for Advancing Translational Sciences (NCATS) with follow-up target-minus assay and orthogonal radioactivity assays to identify nSMase2 inhibitors. We will use a 1536-well fluorescence- based assay using human nSmase2 that is HTS-ready with a Z' = 0.8. Approximately 100 of the most potent and drug-like inhibitors from multiple chemotypes will be selected for evaluation in aim 2. In aim 2 we will characterize the selected inhibitors in several areas including their potential to be CNS drugs, their metabolic stability, their ability to be neuroprotective in vitro and their off target liabilities. Upon completion of am 2 we anticipate identifying 10 to 20 nSMase2 inhibitors belonging to different chemotypes with the potential to become chemical probes that will advance to aim 3. In aim 3 we will conduct preliminary SAR optimization that will focus on eliminating the most addressable liabilities encountered during inhibitor characterization in aim 2. Our criteria for a chemical probe will be: IC50 = 1 µM, CNS-Multi-parameter Optimization score = 4, compound stability = 60% after 1 h incubation with mouse and human liver microsomes, dose dependent neuronal protection and = 20% inhibition of 44 targets known to be a safety risk at 10 µM. Chemical probes identified through the implementation of the aims in this proposal will be available to the research community to investigate the role of nSMase2 in animal models of HAND and possibly other neurodegenerative diseases where nSMase2 is suspected to play a role.