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

 描述（由申请人提供）：大约一半的艾滋病毒感染患者可以获得联合抗逆转录病毒治疗（cART），但仍患有某种形式的认知障碍，严重影响生活质量。这一发现传达了对连续性神经保护治疗的需要，以充分保护大脑免受感染。来自具有认知障碍的HIV患者的脑已显示出神经酰胺的蓄积;神经酰胺的一个主要来源是通过中性鞘磷脂酶2（nSmase 2）催化的鞘磷脂的水解。尽管短暂的nSMase 2上调是正常脑功能的一部分，但实验证据开始表明，慢性nSMase 2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nSmase 2正在成为HAND和其他神经退行性疾病发病机制中的重要参与者，但目前nSmase 2抑制剂的药物不足以探索该酶的作用。目前的nSMase 2抑制剂表现出非药物样性质，包括高分子量、差的溶解度和差的药代动力学。本提案的目的是进行高通量筛选（HTS），以鉴定和表征nSMase 2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nSMase 2激活在HIV相关神经认知障碍（HAND）中的作用。该项目的目标1是对NIH国家转化科学推进中心（NCATS）化学库中的约430，000种化合物进行qHTS，并进行后续靶向减分析和正交放射性分析，以鉴定nSMase 2抑制剂。我们将使用1536孔基于荧光的测定，其使用人nSmase 2，其是HTS就绪的，Z' = 0.8。将从多种化学型中选择约100种最有效的药物样抑制剂用于目标2的评价。在目标2中，我们将在多个领域对选定的抑制剂进行表征，包括它们成为中枢神经系统药物的潜力、它们的代谢稳定性、它们在体外的神经保护能力以及它们的脱靶倾向。在完成目标2后，我们预计将鉴定出10至20种属于不同化学型的nSMase 2抑制剂，这些抑制剂有可能成为将推进目标3的化学探针。在目标3中，我们将进行初步的SAR优化，重点是消除目标2中抑制剂表征过程中遇到的最易解决的问题。我们对化学探针的标准是：IC 50 = 1 µM，CNS-多参数优化评分= 4，与小鼠和人肝微粒体孵育1小时后化合物稳定性= 60%，剂量依赖性神经元保护和10 µM时已知具有安全性风险的44个靶点的抑制率= 20%。通过实施本提案中的目标确定的化学探针将可供研究界用于研究nSMase 2在HAND动物模型中的作用，以及可能的其他神经退行性疾病，其中nSMase 2被怀疑发挥作用。

 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.