Novel Methods for the Inhibition of Phosphatase Function

磷酸酪氨酸磷酸酶1B （PTP1B）调节葡萄糖摄取，已被证实是治疗II型糖尿病和肥胖的药物靶点。尽管受到广泛关注，但PTP1B小分子抑制剂的发展受到两个主要问题的阻碍；由于大多数已知抑制剂的极性性质而缺乏生物利用度，并且由于PTP家族中高度保守的活性位点，抑制剂对其他磷酸酶的选择性较差。因此，需要新的方法来抑制PTP1B和其他磷酸酶的功能。在本建议中，我们概述了实现这一目标的两种不同战略。首先，我们建议开发一种新的小分子筛选策略，用于直接发现磷酸酶的变构配体。变弹性小分子很可能克服磷酸酶活性位点抑制剂的局限性，因为它可能结合到磷酸酶的一个区域，这个区域不像活性位点那样高度保守或极性。我们将利用已有的钒酸盐离子结合磷酸酶活性位点的倾向，形成磷酸盐转移过程中关键过渡态的稳定类似物。我们建议通过实施小分子微阵列（SMM）技术来测试这一概念，以筛选PTP1B的钒酸盐稳定过渡态构象的小分子变构配体。抑制磷酸酶功能的第二种策略是通过将泛素-蛋白酶体途径重定向到目标磷酸酶来靶向磷酸酶的降解。我们设想通过将含有已知PTP1B配体的嵌合小分子与低分子量药物样分子连接，从而实现PTP1B的这一目标，该分子可以结合von Hippel-Lindau肿瘤抑制因子（VHL）， VHL是VBC-Cul2 E3泛素连接酶复合物的识别亚基。这种靶向嵌合体的蛋白水解（PROTAC）会诱导PTP1B的多泛素化，导致其被蛋白体降解。这两种方法都力求通过使用小分子来控制磷酸酶的功能。虽然我们最初将在研究充分的磷酸酶PTP1B （II型糖尿病的有效药物靶点）上开发这些方法，但我们随后计划将这些研究扩展到与癌症和其他疾病有关的其他磷酸酶。

Phosphotyrosine Phosphatase 1B (PTP1B) regulates glucose uptake and has been validated as a drug target for the treatment of type II diabetes as well as obesity. Despite widespread interest, the development of small molecule PTP1B inhibitors has been hindered by two main issues; the lack of bioavailability due to the polar nature of most known inhibitors and poor selectivity of the inhibitor over other phosphatases due to the highly conserved active sites across the PTP family. Because of this a need exists for new methodologies to inhibit the functions of PTP1B and other phosphatases. In this proposal we outline two different strategies to accomplish this goal. First, we propose to develop a novel small molecule screening strategy for the direct discovery of allosteric ligands of phosphatases. An allosteric small molecule is likely to overcome the stated limitations of phosphatase active site inhibitors since it would likely bind to a region of the phosphatase that is not as highly conserved or polar as the active site. We will capitalize on the well precedented propensity of vanadate ions to bind the active site of phosphatases and form stable analogs of key transition states in phosphate transfer. We propose to test this concept by implementing Small Molecule Microarray (SMM) technology to screen for small molecule allosteric ligands of a vanadate stabilized transition state conformation of PTP1B. A second strategy for inhibiting phosphatase function is to target the phosphatase for degradation by redirecting the ubiquitin-proteasome pathway towards a target phosphatase. We envision achieving this for PTP1B by implementing a chimeric small molecule containing a known PTP1B ligand tethered to a low molecular weight drug-like molecule that can bind the von Hippel-Lindau tumor suppressor (VHL), the recognition subunit of the VBC-Cul2 E3 ubiquitin ligase complex. Such a PROteolysis TArgeting Chimera (PROTAC) should induce the poly-ubiquitination of PTP1B, leading to its degradation by the proteosome. Both of these methodologies strive to control the function of phosphatases through the use of small molecules. While we will initially develop these methodologies on the well studied phosphatase PTP1B, a validated drug target for type II diabetes, we subsequently plan to expand these studies to other phosphatases which have been implicated in cancer and other diseases.