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Title: Dual Allosteric Inhibition of SHP2 Phosphatase

Abstract

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 sitemore » 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.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]; ORCiD logo [2];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1] more »;  [1];  [1];  [1];  [2];  [1]; ORCiD logo [1] « less
  1. Novartis Inst. for Biomedical Research, Cambridge, MA (United States)
  2. Harvard Medical School, Boston, MA (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1435825
Grant/Contract Number:  
AC02–06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 13; Journal Issue: 3; Journal ID: ISSN 1554-8929
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; Peptides and proteins; Small molecules; Inhibitors; Inhibition; Screening assays

Citation Formats

Fodor, Michelle, Price, Edmund, Wang, Ping, Lu, Hengyu, Argintaru, Andreea, Chen, Zhouliang, Glick, Meir, Hao, Huai-Xiang, Kato, Mitsunori, Koenig, Robert, LaRochelle, Jonathan R., Liu, Gang, McNeill, Eric, Majumdar, Dyuti, Nishiguchi, Gisele A., Perez, Lawrence B., Paris, Gregory, Quinn, Christopher M., Ramsey, Timothy, Sendzik, Martin, Shultz, Michael David, Williams, Sarah L., Stams, Travis, Blacklow, Stephen C., Acker, Michael G., and LaMarche, Matthew J. Dual Allosteric Inhibition of SHP2 Phosphatase. United States: N. p., 2018. Web. doi:10.1021/acschembio.7b00980.
Fodor, Michelle, Price, Edmund, Wang, Ping, Lu, Hengyu, Argintaru, Andreea, Chen, Zhouliang, Glick, Meir, Hao, Huai-Xiang, Kato, Mitsunori, Koenig, Robert, LaRochelle, Jonathan R., Liu, Gang, McNeill, Eric, Majumdar, Dyuti, Nishiguchi, Gisele A., Perez, Lawrence B., Paris, Gregory, Quinn, Christopher M., Ramsey, Timothy, Sendzik, Martin, Shultz, Michael David, Williams, Sarah L., Stams, Travis, Blacklow, Stephen C., Acker, Michael G., & LaMarche, Matthew J. Dual Allosteric Inhibition of SHP2 Phosphatase. United States. https://doi.org/10.1021/acschembio.7b00980
Fodor, Michelle, Price, Edmund, Wang, Ping, Lu, Hengyu, Argintaru, Andreea, Chen, Zhouliang, Glick, Meir, Hao, Huai-Xiang, Kato, Mitsunori, Koenig, Robert, LaRochelle, Jonathan R., Liu, Gang, McNeill, Eric, Majumdar, Dyuti, Nishiguchi, Gisele A., Perez, Lawrence B., Paris, Gregory, Quinn, Christopher M., Ramsey, Timothy, Sendzik, Martin, Shultz, Michael David, Williams, Sarah L., Stams, Travis, Blacklow, Stephen C., Acker, Michael G., and LaMarche, Matthew J. 2018. "Dual Allosteric Inhibition of SHP2 Phosphatase". United States. https://doi.org/10.1021/acschembio.7b00980. https://www.osti.gov/servlets/purl/1435825.
@article{osti_1435825,
title = {Dual Allosteric Inhibition of SHP2 Phosphatase},
author = {Fodor, Michelle and Price, Edmund and Wang, Ping and Lu, Hengyu and Argintaru, Andreea and Chen, Zhouliang and Glick, Meir and Hao, Huai-Xiang and Kato, Mitsunori and Koenig, Robert and LaRochelle, Jonathan R. and Liu, Gang and McNeill, Eric and Majumdar, Dyuti and Nishiguchi, Gisele A. and Perez, Lawrence B. and Paris, Gregory and Quinn, Christopher M. and Ramsey, Timothy and Sendzik, Martin and Shultz, Michael David and Williams, Sarah L. and Stams, Travis and Blacklow, Stephen C. and Acker, Michael G. and LaMarche, Matthew J.},
abstractNote = {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.},
doi = {10.1021/acschembio.7b00980},
url = {https://www.osti.gov/biblio/1435825}, journal = {ACS Chemical Biology},
issn = {1554-8929},
number = 3,
volume = 13,
place = {United States},
year = {Fri Jan 05 00:00:00 EST 2018},
month = {Fri Jan 05 00:00:00 EST 2018}
}

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Cited by: 76 works
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Figures / Tables:

Figure 1 Figure 1: A. Structure, biochemical and cellular activity, and thermal stabilization of SHP099 (1). B. Model of engineered double mutant (SHP2T253M/Q257L) which disrupts the binding of 1. SHP2WT colored in wheat, SHP2T253/Q257 sidechains colored in magenta.

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Works referencing / citing this record:

Exploring the effect of E76K mutation on SHP2 cause gain‐of‐function activity by a molecular dynamics study
journal, August 2018


Synthesis, activity evaluation, and pro-apoptotic properties of novel 1,2,4-triazol-3-amine derivatives as potent anti-lung cancer agents
journal, January 2019


Design, Synthesis, and In Vitro Activity of Pyrazine Compounds
journal, December 2019


Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2
journal, October 2018


Exploring the effect of N308D mutation on protein tyrosine phosphatase‐2 cause gain‐of‐function activity by a molecular dynamics study
journal, October 2018


Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition
journal, October 2018


Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2
journal, October 2018


Structural reorganization of SHP2 by oncogenic mutations and implications for oncoprotein resistance to allosteric inhibition
journal, October 2018


Synthesis of small peptide compounds, molecular docking, and inhibitory activity evaluation against phosphatases PTP1B and SHP2
journal, December 2018


Beyond the Cell Surface: Targeting Intracellular Negative Regulators to Enhance T cell Anti-Tumor Activity
journal, November 2019


Design, Synthesis, and In Vitro Activity of Pyrazine Compounds
journal, December 2019


Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.