Inhibitors of HIV-induced downregulation of HLA class I

描述（由申请人提供）：HIV诱导的HLA I类下调的抑制剂多项研究表明，HLA I类（MHC-I）的下调在HIV感染中起重要作用。病毒蛋白Nef诱导MHC-I下调并干扰病毒抗原的呈递，使HIV感染的细胞对CD 8阳性CTL的杀伤不太敏感。感染SIV的猕猴缺乏这种活性， 消除MHC-I。Nef下调MHC-I的机制已被广泛研究，然而，尽管其在发病机制中起重要作用，但该活性仍是抗病毒开发的目标。进一步强调了这种功能在免疫逃避中的重要性，在许多疱疹病毒、痘病毒、乳头瘤病毒和腺病毒中也认识到了对抗原呈递的干扰。我们建议筛选一个小分子库，以确定HIV诱导的MHC-I下调的抑制剂。一个高含量的筛选已经完全优化，来自3,440个小分子的测试试点的初步结果已经验证了该检测方法，并确定了阻断这个有希望的目标的小分子。我们的研究结果证明了用小分子对MHC-I下调进行有效和特异性干扰的概念验证是可能的。我们提出以下目标：（1）筛选由高度多样化结构组成的46，280种药物样分子的文库，包括选择靶向激酶的分子和已知具有抗HIV活性的其他化合物（Lopac文库）;（2）验证和表征hts的效力、特异性和毒性;和（3）鉴定增强CTL杀死HIV感染细胞的能力的化合物。我们将使用具有多个内置对照的多模式HTS测定，以同时确认初级筛选中的命中，并消除具有不良毒性特征的假阳性，以及通过已知机制阻断HIV感染的其他命中。该I期应用的成功将通过鉴定一种或多种真正的先导系列药物样化合物来定义，其EC 50低于5 μ M，具有最小的细胞毒性（CC 50至少是EC 50的30倍），并且能够使HIV感染的细胞对CTL杀伤敏感。在II期提案中，将选择来自该筛选的化学先导化合物用于药物化学SAR优化，以获得效价、特异性和口服生物利用度。我们将进一步研究其作用机制，鉴定耐药菌株， 评估与已批准的抗逆转录病毒药物的拮抗作用的潜力，并评估与其他药物的交叉耐药性。此外，将在重现病毒特异性CTL应答的HIV感染的小动物模型中测试选定数量的化合物。具有改进的特征和特征作用机制的化合物将进入临床前开发研究，以支持未来的人体试验。施用抑制MHC-I下调的抗病毒药物可以延长HAART治疗开始前所需的时间，或者与其他药物组合可以通过改善患者对病毒的免疫应答来增强其抗病毒作用。这些研究将构成药物发现计划的基础，该计划可能导致新型抗病毒药物家族的一流药物，具有针对其他病毒的更广泛活性的潜力。PHS 398/2590（Rev.06/09）

DESCRIPTION (provided by applicant): Inhibitors of HIV-induced downregulation of HLA class I Multiple studies suggest that downmodulation of HLA class I (MHC-I) plays an important role in HIV infection. The viral protein Nef induces MHC-I downmodulation and interferes with the presentation of viral antigens, making HIV-infected cells less sensitive to killing by CD8-positive CTLs. Macaques infected with SIV deficient in this activity rapidly restore the ability to eliminate MHC-I. The mechanism by which Nef downmodulates MHC-I has been extensively investigated, however, despite its important role in pathogenesis this activity remains to be targeted for antiviral development. Further highlighting the importance of this function in immune evasion, interference with antigen presentation has also been recognized in many herpesviruses, poxviruses, papillomaviruses and adenoviruses. We propose screening a library of small molecules to identify inhibitors of the HIV-induced MHC-I downmodulation. A high content screen has been fully optimized and preliminary findings from a test pilot of 3,440 small-molecules have validated the assay and identified small-molecules blocking this promising target. Our results demonstrate proof-of-concept that potent and specific interference of MHC-I downmodulation with small molecules is possible. We propose the following aims: (1) to screen a library of 46,280 drug-like molecules composed of highly diverse structures, including molecules selected to target kinases, and other compounds known to be pharmacologically active (Lopac library); (2) to validate and characterize the potency, specificity and toxicity of hts, and; (3) to identify compounds that enhance the ability of CTLs to kill HIV- infected cells. We wil use a multi-modal HTS assay with multiple built-in controls to simultaneously confirm hits in primary screens and eliminate false positives with poor toxicity profiles, and other hits blocking HIV infection by known mechanisms. Success in this Phase I application will be defined by identifying one or more bona fide lead series of drug-like compounds with EC50s below 5¿M, minimal cytotoxicity (CC50 at least 30-fold greater than EC50), and the ability to sensitize HIV-infected cells to CTL killing. In a Phase II proposal, chemical leads from this screen will be selected for medicinal chemistry SAR optimization for potency, specificity and oral bioavailability. We wil further study the mechanism of action, characterize drug-resistant strains, ases the potential for antagonism with approved antiretrovirals, and evaluate cross-resistance with other drugs. Additionally, a selected number of compounds will be tested in a small-animal model of HIV infection that recapitulates the virus-specific CTL responses. Compounds with improved profiles and characterized mechanism of action will be advanced into preclinical development studies to support future human trials. Administration of antivirals inhibiting MHC-I downmodulation may lengthen the time required before initiation of HAART therapy, or alternatively, in combination with other drugs may enhance their antiviral effect by improving the patient's immune response against the virus. These studies will constitute the foundation of a drug discovery program that may lead to first-in-class drugs of a novel family of antivirals with the potential for broader activity against other viruses. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page