Targeting DUSP-5 to Treat Vascular Anomalies

描述（由申请人提供）：血管异常（VA），胚胎血管发育中的先天性缺陷分为两个不同的组：血管瘤和血管畸形（VM）。目前VA的治疗方法疗效有限，并有严重的并发症。因此，为了改善对患有这些疾病的患者的治疗，找到新的药物或重新使用FDA批准的药物来靶向VA至关重要。我们的长期目标是了解导致VA发病机制的潜在机制，以便能够产生针对这种情况的更好的治疗方法。为了实现这一目标，我们的目标是鉴定靶向双特异性磷酸酶-5（Dusp-5）的小分子（SM），Dusp-5是丝裂原活化蛋白激酶（MAPK）家族的一员，在血管瘤和VM患者中发生突变。我们已经鉴定了DUSP-5中147 AA处的丝氨酸至脯氨酸突变（S147 P），其导致不能使p-ERK去磷酸化的低活性磷酸酶。这导致持续的p-ERK水平，这通常与细胞增殖增加有关，例如VA中的细胞。我们的中心假设是，“人类S147 P蛋白重现了斑马鱼S148 P蛋白的功能，从而突变干扰了与p-ERK的相互作用，使得DUSP 5磷酸酶结构域（PD）不能正确定位以使p-ERK去磷酸化。小分子如SM 1842、FDA批准的化合物（苏拉明）和SM 1842类似物可以逆转这种作用，从而允许WT和S147 P功能在分子水平（体外）和细胞功能方面的转换。“这一假设是基于我们小组的初步数据制定的，这些数据预测了DUSP 5 PD结构域与p-ERK的不正确定位，使用Dusp-5与ERK相互作用的计算建模研究，这表明了导致S147 P活性低下的分子机制。此外，用10，500种SM化合物与Dusp-5酶的C-末端PD的计算对接方法鉴定了SM 1842和在EC中充当有效Dusp-5拮抗剂的其他SM。SM 1842是鉴定的命中中最有效的，在p-ERK测定中作为Dusp-5拮抗剂，影响内皮细胞中VEGF刺激的p-ERK和Dusp-5水平，并在体外生化测定中恢复S148 P功能。苏拉明-FDA批准的化合物（与SM 1842类似的结构）也影响内皮细胞中的内源性Dusp-5和p-ERK水平。将通过追求三个特定目标来检验所提出的假设：1）确定SM 1842影响Dusp-5和S147 P功能的结构机制; 2）鉴定SM 1842的最佳化学类似物;和3）表征与SM 1842相似的FDA批准的化合物（苏拉明）的体内和体外活性。在这些目标中，我们将采用各种生物物理学，细胞生物学，分子和发育生物学方法来阐明SM 1842及其类似物影响Dusp-5体内和体外活性的机制基础。这种方法是创新的，因为像SM 1842这样的分子选择性地影响突变蛋白而不是WT蛋白，功能受到大型制药公司的高度追捧，这种应用有可能改变基于靶点的研究范式。拟议的研究意义重大，因为该项目的益处将在治疗选择方面为VA患者提供即时的临床影响，重要的是将基础科学发现瞬间转化为切实的临床益处。

DESCRIPTION (provided by applicant): Vascular anomalies (VAs), inborn errors in embryonic vascular development are classified into two distinct groups: hemangiomas and vascular malformations (VMs). Current therapies for VAs are limited in efficacy and have significant complications. Therefore, to improve therapy for patients afflicted with these conditions, it is critical to find new drugs or repurpose FDA-approved drugs to target VAs. Our long-term goal is to understand the underlying mechanisms that lead to pathogenesis of VAs so that better therapeutics targeting this condition can be generated. In order to pursue that goal, the objective is to identify small molecules (SMs) that will target dual-specific phosphatase-5 (Dusp-5), a member of the mitogen-activated protein kinase (MAPK) family, which is mutated in patients with hemangiomas and VMs. We have identified a serine to proline mutation at 147 AA in DUSP-5 (S147P), which results in a hypoactive phosphatase that is unable to dephosphorylate p-ERK. This results in sustained p-ERK levels, which is often associated with increased proliferation of cells such as those in VAs. Our central hypothesis is that, "human S147P protein recapitulates zebrafish S148P protein function, whereby mutation perturbs the interaction with p-ERK such that DUSP5 phosphatase domain (PD) cannot be properly positioned to de-phosphorylate p-ERK. Small molecules such as SM1842, FDA-approved compounds (Suramin), and SM1842 analogs can reverse this effect, thereby permitting a switch between WT and S147P function both at the molecular level (in vitro), and in terms of cellular function." This hypothesis is formulated based on preliminary data from our group that predicts the incorrect positioning of the DUSP5 PD domain in relation to p-ERK using computational modeling studies on Dusp-5 interaction with ERK, which suggests the molecular mechanism that leads to the S147P's hypoactivity. Further, computational docking approach with 10,500 SM compounds to the C-terminal PD of Dusp-5 enzyme identified SM1842, and other SMs that act as potent Dusp-5 antagonist in ECs. SM1842 is the most potent of the identified hits, acts as Dusp-5 antagonist in p-ERK assay, affects VEGF-stimulated p-ERK and Dusp-5 levels in endothelial cells, and restores S148P function in biochemical assays in vitro. Suramin - FDA-approved compounds (similar structure to SM1842) also affect endogenous Dusp-5 and p-ERK levels in endothelial cells. The proposed hypothesis will be tested by pursuing three specific aims: 1) Determine the structural mechanism for SM1842 in affecting Dusp-5 and S147P function; 2) Identify the optimal chemical analog for SM1842; and 3) Characterize the activity of FDA- approved compounds (suramin) similar to SM1842 in vivo and in vitro. In each of these aims, we will employ a variety of biophysical, cell biology, molecular and developmental biology approaches to unravel mechanistic basis for SM1842 and its analog to affect Dusp-5's activity in vivo and in vitro. The approach is innovative because molecules like SM1842 that selectively affect mutant protein over WT protein, function are highly sought after by big Pharma, and this application has the potential to shift paradigm in target-based research. The proposed research is significant because benefits of this project will provide immediate clinical impact for VA patients in terms of therapy options, and importantly will translate basic science discovery into tangible clinical benefits instantaneously.