Selective inhibitors of ubiquitin E3 ligase to treat high cholesterol

描述(申请人提供)：高胆固醇血症是心血管疾病的主要因素。在美国，超过3500万人总胆固醇偏高，因此罹患心脏病的风险是正常水平的两倍。低密度脂蛋白受体(LDLR)对胆固醇调节至关重要，因为LDLR突变会增加血清胆固醇水平和心血管疾病的风险。提高低密度脂蛋白受体的水平或活性是目前市场上几种降胆固醇药物使用的一种有效的药理机制。例如，他汀类药物抑制HMG辅酶A还原酶，从而干扰胆固醇的合成，从而上调肝脏LDLR的活性，增加低密度脂蛋白从血液中的清除。他汀类药物被广泛使用，但在许多患者中与不良反应有关；因此，目前正在寻找具有不同LDLR机制的新药，这些新药具有减少副作用的可能性和/或使用减少他汀类药物剂量的联合治疗的可能性。泛素环指E3连接酶被称为IDOL(LDLR的可诱导降解物；也被称为MYLIP和MIR)，泛素化LDLR的细胞质结构域，导致其降解。因此，Idol是降胆固醇药物发现的一个有希望的新靶点，因为Idol抑制剂有望提高LDLR水平。此外，Idol抑制提供了一条机械上独特的途径来增加LDLR的含量/活性。拟议的项目旨在通过提高低密度脂蛋白受体水平和增强低密度脂蛋白清除来发现IDOL的小分子抑制剂，以降低血清胆固醇。Progenra开发了一种新的方法，可以检测几乎任何E3连接酶的活性；该方法已被用于寻找选择性E3连接酶抑制剂，其中几种正在进行临床前开发。因此，将对该分析进行调整和验证，以高通量发现Idol的抑制剂，并将进行中试筛选，以寻找具有潜在实用价值的降胆固醇治疗药物。这项工作将分为三个具体目标。在为期一年的第一阶段中，该试验将验证自身泛素化(IDOL)抑制剂和已知人类IDOL底物泛素化抑制剂的有效性。然后，将使用从筛选中选择的抑制剂进行实验，以获得概念的细胞验证。在第二阶段，将进行更广泛的筛选和化学优化、ADME和体内概念验证和有效性研究，商业目标是开发和销售具有单剂或组合降胆固醇活性的药物。 公共卫生相关性：血液胆固醇升高是心血管疾病的一个主要因素；在美国，这种情况影响了3500万人，他们患心脏病的风险相应增加。目前降低胆固醇的首选药物是他汀类药物，这种药物通过增加“坏”胆固醇与其受体的结合来发挥作用，从而促进胆固醇从血液中清除。尽管他汀类药物被广泛开出，但在许多患者中，它与不良反应有关；因此，目前正在寻找新的药物，这些药物要么通过某种机制发挥作用，导致副作用比他汀类药物产生的副作用更少或更轻，要么可以作为联合治疗方案的一部分。Progenra已经为降胆固醇药物开发确定了一个有前景的新靶点，一种被称为Idol的酶，它将泛素与坏胆固醇受体(LDLR)偶联，标志着蛋白酶体中的受体降解。这一靶点的抑制剂有望通过阻止该受体的降解来增加LDLR水平。Progenra将调整和验证一种用于高通量筛选Idol抑制剂的试验，筛选其化合物集合，并证明筛选中的抑制剂如预测的那样影响细胞中的LDLR。在第二阶段，选定的抑制剂将进行临床前开发，商业目标是开发和销售具有单剂或组合降胆固醇活性的药物。

DESCRIPTION (provided by applicant): Hypercholesterolemia is a major factor in cardiovascular disease. In the USA, more than 35 million individuals have high total cholesterol and thus twice the normal risk of heart disease. The LDL receptor (LDLR) is critical to cholesterol regulation, as mutation of LDLR increases serum cholesterol levels and risk of cardiovascular disease. Increasing the level or activity of LDLR is an effective pharmacological mechanism employed by several cholesterol-lowering drugs now on the market. Statins, for example, inhibit HMG CoA reductase thus interfering with cholesterol synthesis, which upregulates hepatic LDLR activity and increases clearance of LDL from the bloodstream. Statins are widely prescribed, but are associated with adverse reactions in many patients; thus, new drugs of differing LDLR mechanism with reduced potential to cause side effects and/or potential for combination treatment employing reduced statin doses are currently being sought. An ubiquitin RING-finger E3 ligase known as Idol ( inducible degrader of the LDLR; also known as MYLIP and Mir), ubiquitylates LDLR on its cytoplasmic domain, resulting in its degradation. Idol is thus a promising new target for cholesterol-lowering drug discovery, as Idol inhibitors are expected to increase LDLR levels. Moreover, Idol inhibition provides a mechanistically distinct pathway to increase LDLR content/activity. The proposed project aims to discover small molecule inhibitors of Idol for lowering serum cholesterol by increasing LDLR levels and enhancing LDL clearance. Progenra has developed a novel assay that can detect the activity of virtually any E3 ligase; the assay has been used to find selective E3 ligase inhibitors, several of which are in pre-clinical development. Accordingly, the assay will be adapted and validated for high-throughput discovery of inhibitors of Idol, and a pilot screen will be conducted for inhibitors that are of potential utility as cholesterol- lowering therapeutic agents. The work will be divided into three Specific Aims. In the 1 year Phase I, the assay will be validated for inhibitors of both auto-ubiquitylation (Idol) and for inhibitors of ubiquitylation of the known substrate of human Idol. Cellular proof of concept will then be obtained experimentally using selected inhibitors from the screens. In Phase II, a more extensive screen and chemical optimization, ADME, and in vivo proof of concept and efficacy studies will be conducted, with the commercial goal of developing and marketing a pharmaceutical agent with single agent or combinatorial cholesterol lowering activity. PUBLIC HEALTH RELEVANCE: Elevated blood cholesterol is a major factor in cardiovascular disease; in the USA this condition affects more than 35 million individuals, who accordingly face increased risk of developing heart disease. The current drug of choice for lowering cholesterol is the family of statins, which work by increasing the binding of "bad" cholesterol to its receptors and thereby promote clearance of cholesterol from the bloodstream. Although widely prescribed, statins are associated with adverse reactions in many patients; thus, new drugs that would either work by a mechanism that leads to fewer or less severe side effects than those produced by the statins or be usable as part of a combination treatment regimen are currently being sought. Progenra has identified a promising new target for cholesterol-lowering drug discovery, an enzyme known as Idol, which conjugates ubiquitin to bad cholesterol receptors (LDLR), marking the receptor for degradation in the proteasome. Inhibitors of this target are expected to increase LDLR levels by preventing degradation of the receptor. Progenra will adapt and validate an assay for high throughput screening for inhibitors of Idol, screen its compound collection, and demonstrate that inhibitors from the screen affect LDLR in cells as predicted. In Phase II, selected inhibitors will undergo pre-clinical development, with the commercial goal of developing and marketing a pharmaceutical agent with single agent or combinatorial cholesterol lowering activity.