Targeting DUSP-5 to Treat Vascular Anomalies

描述（由申请人提供）：血管异常（VAs），胚胎血管发育中的先天性错误分为两类：血管瘤和血管畸形（vm）。目前的VAs治疗方法疗效有限，且有明显的并发症。因此，为了改善对这些疾病患者的治疗，寻找新药或重新利用fda批准的药物来靶向VAs是至关重要的。我们的长期目标是了解导致VAs发病机制的潜在机制，以便针对这种情况产生更好的治疗方法。为了实现这一目标，目标是确定靶向双特异性磷酸酶-5 （Dusp-5）的小分子（SMs）， 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。我们已经在DUSP-5 （S147P）的147 AA处发现了一个丝氨酸到脯氨酸的突变，该突变导致磷酸酶活性降低，无法使p-ERK去磷酸化。这导致持续的p-ERK水平，这通常与VAs等细胞的增殖增加有关。我们的中心假设是，“人类S147P蛋白重现了斑马鱼S148P蛋白的功能，即突变扰乱了与p-ERK的相互作用，使得DUSP5磷酸酶结构域（PD）不能被正确定位以使p-ERK去磷酸化。”小分子如SM1842、fda批准的化合物（苏拉明）和SM1842类似物可以逆转这种效应，从而在分子水平（体外）和细胞功能方面允许WT和S147P功能之间的切换。”这一假设是基于我们小组的初步数据提出的，该数据通过对DUSP5 -5与ERK相互作用的计算建模研究，预测了DUSP5 PD结构域与p-ERK的不正确定位，这表明了导致S147P<s活性低下的分子机制。此外，通过10,500个SM化合物与Dusp-5酶c端PD的计算对接方法，鉴定出SM1842和其他在ECs中作为Dusp-5有效拮抗剂的SM。SM1842是已知命中命中最有效的，在p-ERK试验中作为Dusp-5拮抗剂，影响内皮细胞中vegf刺激的p-ERK和Dusp-5水平，并在体外生化试验中恢复S148P功能。苏拉明- fda批准的化合物（与SM1842结构相似）也影响内皮细胞内源性Dusp-5和p-ERK水平。我们将通过三个具体目标来验证这一假设：1)确定SM1842影响Dusp-5和S147P功能的结构机制；2)确定SM1842的最佳化学模拟物；3)表征FDA批准的类似SM1842的化合物（苏拉明）在体内和体外的活性。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揭示SM1842及其类似物在体内和体外影响Dusp-5<s活性的机制基础。这种方法是创新的，因为像SM1842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标的研究模式。拟议的研究意义重大，因为该项目的好处将为VA患者提供治疗选择方面的直接临床影响，重要的是将基础科学发现立即转化为有形的临床效益。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because vascular anomalies (VAs) represent an important clinical problem that has few therapeutic options. The successful development of a small molecule or FDA- approved compound that targets mutated Dusp-5 over WT Dusp-5 protein will provide the much needed therapy alternatives for patients with VAs. Thus, the proposed research is directly relevant to NIH<s mission of reducing the burden of debilitating health conditions from diseases affected by deregulated vasculature.