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Dual Allosteric Inhibition of SHP2 Phosphatase
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-01-05 00:00:00 , DOI: 10.1021/acschembio.7b00980 Michelle Fodor 1 , Edmund Price 1 , Ping Wang 1 , Hengyu Lu 1 , Andreea Argintaru 1 , Zhouliang Chen 1 , Meir Glick 1 , Huai-Xiang Hao 1 , Mitsunori Kato 1 , Robert Koenig 1 , Jonathan R. LaRochelle 2 , Gang Liu 1 , Eric McNeill 1 , Dyuti Majumdar 1 , Gisele A. Nishiguchi 1 , Lawrence B. Perez 1 , Gregory Paris 1 , Christopher M. Quinn 1 , Timothy Ramsey 1 , Martin Sendzik 1 , Michael David Shultz 1 , Sarah L. Williams 1 , Travis Stams 1 , Stephen C. Blacklow 2 , Michael G. Acker 1 , Matthew J. LaMarche 1
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-01-05 00:00:00 , DOI: 10.1021/acschembio.7b00980 Michelle Fodor 1 , Edmund Price 1 , Ping Wang 1 , Hengyu Lu 1 , Andreea Argintaru 1 , Zhouliang Chen 1 , Meir Glick 1 , Huai-Xiang Hao 1 , Mitsunori Kato 1 , Robert Koenig 1 , Jonathan R. LaRochelle 2 , Gang Liu 1 , Eric McNeill 1 , Dyuti Majumdar 1 , Gisele A. Nishiguchi 1 , Lawrence B. Perez 1 , Gregory Paris 1 , Christopher M. Quinn 1 , Timothy Ramsey 1 , Martin Sendzik 1 , Michael David Shultz 1 , Sarah L. Williams 1 , Travis Stams 1 , Stephen C. Blacklow 2 , Michael G. Acker 1 , Matthew J. LaMarche 1
Affiliation
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SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site—a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.
中文翻译：
SHP2磷酸酶的双重变构抑制作用
SHP2是由PTPN11基因编码的胞质蛋白酪氨酸磷酸酶，参与细胞增殖，分化和存活。最近，我们报道了一种抑制变构机制，该机制稳定了SHP2的自抑制构象。SHP099（1）被鉴定并表征为中等有效的，口服生物利用的，变构小分子抑制剂，该抑制剂与SHP2的三个结构域汇合形成的隧道状袋结合。在本报告中，我们描述了进一步的筛选策略，这些策略能够识别第二个不同的小分子变构位点。SHP244（2）被确定为SHP2的弱抑制剂，具有适度的酶热稳定性。X射线晶体学表明2在一个独特的，先前未开发的结合位点（在N末端SH2和PTP结构域的界面处形成的裂口）结合并稳定了SHP2的无活性，闭合构象。使用基于结构的设计2的衍生化导致SHP2热稳定性，生化抑制和随后的MAPK途径调节增加。在KYSE-520癌细胞中观察到下游MAPK通路标志物DUSP6 mRNA的下调。值得注意的是，两个变构位点同时占据了1和2以协同生化抑制实验和X射线晶体学为特征，这是可能的。将变构位点1抑制剂与变构位点2抑制剂组合导致细胞中增强的药理途径抑制作用。这项工作说明了双重变构靶向蛋白质抑制的罕见例子，展示了鉴定变构抑制剂的筛选方法和策略，并可以进一步研究SHP2在癌症和相关病理学中的作用。
更新日期：2018-01-05
中文翻译：
SHP2磷酸酶的双重变构抑制作用
SHP2是由PTPN11基因编码的胞质蛋白酪氨酸磷酸酶，参与细胞增殖，分化和存活。最近，我们报道了一种抑制变构机制，该机制稳定了SHP2的自抑制构象。SHP099（1）被鉴定并表征为中等有效的，口服生物利用的，变构小分子抑制剂，该抑制剂与SHP2的三个结构域汇合形成的隧道状袋结合。在本报告中，我们描述了进一步的筛选策略，这些策略能够识别第二个不同的小分子变构位点。SHP244（2）被确定为SHP2的弱抑制剂，具有适度的酶热稳定性。X射线晶体学表明2在一个独特的，先前未开发的结合位点（在N末端SH2和PTP结构域的界面处形成的裂口）结合并稳定了SHP2的无活性，闭合构象。使用基于结构的设计2的衍生化导致SHP2热稳定性，生化抑制和随后的MAPK途径调节增加。在KYSE-520癌细胞中观察到下游MAPK通路标志物DUSP6 mRNA的下调。值得注意的是，两个变构位点同时占据了1和2以协同生化抑制实验和X射线晶体学为特征，这是可能的。将变构位点1抑制剂与变构位点2抑制剂组合导致细胞中增强的药理途径抑制作用。这项工作说明了双重变构靶向蛋白质抑制的罕见例子，展示了鉴定变构抑制剂的筛选方法和策略，并可以进一步研究SHP2在癌症和相关病理学中的作用。
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