Development of Flavivirus Antivirals Targeting Dengue Virus and WNV Protease

描述（由申请人提供）：黄病毒家族的蚊媒成员包括登革热病毒（DENV1-4）和西尼罗河病毒（WNV）的四种血清型，由于最近流行的频率和严重程度，被归类为再出现的病原体。据估计，每年报告的DENV感染病例达1亿例，导致近2.5万人死亡。超过25亿人面临登革热病毒感染的风险。登革热的症状包括发烧、皮疹和关节痛。在更严重的情况下，病毒感染可发展成危及生命的登革出血热或登革休克综合征。登革休克综合征发生时，渗漏和/或出血足以引起休克，这往往导致死亡，特别是儿童，由于缺乏适当的支持性护理。西尼罗河病毒感染可导致脑膜炎、脑炎或瘫痪，导致住院患者约10%的死亡率。2012年，西尼罗河病毒感染人数突然激增，在48个州出现50 000例病例，其中228人死亡。尽管黄病毒感染在一部分患者中导致高发病率和死亡率，但目前尚无针对任何黄病毒感染的有效化疗治疗方法。黄病毒病原体对全球社会经济的综合影响迫切需要新的有效的抗病毒治疗方法。黄病毒RNA基因组编码3个结构蛋白（C、prM和E）和7个非结构蛋白（NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5）。NS3蛋白是一种具有蛋白酶、解旋酶和RNA三磷酸酶活性的多功能酶。NS3的n端区域有一个丝氨酸蛋白酶结构域，在NS2B辅因子（NS2B）存在下，是一个活性丝氨酸蛋白酶。这种蛋白酶（NS2B-NS3pro）在病毒前体多蛋白的切割中起着重要作用，这种功能的破坏对病毒复制是致命的。因此，通过抑制典型的病毒蛋白酶来阻断病毒复制可能导致有效的广谱抗病毒治疗的发展，这是我们研究计划的重点。我们提案的创新之处是发现了两种先导抑制剂，它们具有广谱（DENV1-4和WNV）抑制活性，并且含有儿茶酚部分，这也是17种FDA批准的药物中存在的亚结构。此外，建模数据表明，这些化合物与酶底物结合袋结合，从而作为竞争性抑制剂。因此，我们的线索的这些属性支持的可行性

DESCRIPTION (provided by applicant): Mosquito-borne members of the Flavivirus family include four serotypes of dengue virus (DENV1-4) and West Nile Virus (WNV) and are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. There are an estimated 100 million DENV infections reported each year resulting in nearly ~25,000 deaths. More than 2.5 billion people are at risk of infection by DENV. Symptoms of dengue fever include fever, rash, and arthralgia. In more severe cases, viral infections can develop into life-threatening dengue hemorrhagic fever or dengue shock syndrome. Dengue shock syndrome occurs when leakage and/or bleeding is sufficient to induce shock, which often leads to death, especially in children, due to the lack of adequate supportive care. WNV infections can result in meningitis, encephalitis, or paralysis leading to ~10% mortality among hospitalized patients. Year 2012 witnessed a sudden surge in the number of WNV infections, comprising >5,000 cases in 48 states with ~228 deaths. Despite the high morbidity and mortality resulting from flavivirus infections in a subset of patients, there is currently no effective chemotherapeutic treatment for infections for any of the flaviviruses. The combined global socioeconomic impact of flavivirus pathogens merits an urgent need for new and effective antiviral therapeutics. The Flavivirus RNA genome encodes for three structural proteins (C, prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). The NS3 protein is a multifunctional enzyme that contains protease, helicase and RNA triphosphatase activities. The N-terminal region of NS3 has a serine protease domain, which in the presence of the NS2B cofactor (NS2B, is an active serine protease. This protease (NS2B-NS3pro) plays an essential role in the cleavage of the viral precursor polyprotein and disruption of this function is lethal t viral replication. Therefore, blocking viral replication by inhibiting the quintessential viral protease could lead to the development of effective broad-spectrum antiviral therapeutics and is the focus of our research proposal. The innovation of our proposal is the discovery of two lead inhibitors, which have broad-spectrum (DENV1-4 and WNV) inhibitory activity and contain a catechol moiety, which is also a substructure present in 17 FDA approved drugs. In addition, the modeling data suggest the compounds bind to the enzyme substrate binding pocket and thus act as competitive inhibitors. Therefore, these properties of our leads support the feasibility for development into effective broad-spectrum antiviral therapeutics. The specific aims for Phase I of the proposal are: 1. Design and synthesize broad-acting inhibitors of DENV1-4 and WNV NS3pro; Milestone: Compounds with IC50d100nM against DENV1-4 and WNV protease will be advanced into cell-based assays. 2. Evaluate the therapeutic indices of optimized lead compounds using in vitro cell-based replicon, infectivity (EC50), and cytotoxicity (CC50) assays; Milestone: Identify 15-20 lead compounds with EC50d200nM and CC50e200uM representing the catechol and non-catechol series. 3. Conduct in vitro ADME-based lead optimization of compounds with broad-spectrum in vitro activity and acceptable therapeutic indices; Milestone: Identify 5-10 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers for evaluation in in vivo animal models for acute toxicity, efficacy and pharmacokinetics (Phase II application).