Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen; Nakano, Hiroyasu

doi:10.1371/journal.pone.0184843

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Journal Article · Thu Sep 21 00:00:00 EDT 2017 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0184843· OSTI ID:1405009

^[1]; Brault, Amy ^[1]; Vincent, Fabien ^[1]; Weng, Shawn ^[2]; Wang, Hong ^[1]; Dumlao, Darren ^[1]; Aulabaugh, Ann ^[1]; Aivazian, Dikran ^[3]; Castro, Dana ^[3]; Chen, Ming ^[1]; Culp, Jeffrey ^[1]; Dower, Ken ^[4]; Gardner, Joseph ^[1]; Hawrylik, Steven ^[1]; Golenbock, Douglas ^[5]; Hepworth, David ^[4]; Horn, Mark ^[3]; Jones, Lyn ^[4]; Jones, Peter ^[4]; Latz, Eicke ^[6] more »

Pfizer, Groton, CT (United States)
Pfizer Centers for Therapeutic Innovation (CTI), Boston, MA (United States)
Pfizer Centers for Therapeutic Innovation (CTI), San Diego, CA (United States)
Pfizer, Cambridge, MA (United States)
Univ. of Massachusetts Medical School, Worcester, MA (United States)
Univ. of Massachusetts Medical School, Worcester, MA (United States); Univ. Hospitals Bonn (Germany)
Toho Daigaku (Japan)

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (K_D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE

OSTI ID:: 1405009

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 9 Vol. 12; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

References (41)

Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization Nikolovska-Coleska, Zaneta; Wang, Renxiao; Fang, Xueliang Analytical Biochemistry, Vol. 332, Issue 2 https://doi.org/10.1016/j.ab.2004.05.055	journal	September 2004
Trex1 Prevents Cell-Intrinsic Initiation of Autoimmunity Stetson, Daniel B.; Ko, Joan S.; Heidmann, Thierry Cell, Vol. 134, Issue 4 https://doi.org/10.1016/j.cell.2008.06.032	journal	August 2008
Structure-Guided Reprogramming of Human cGAS Dinucleotide Linkage Specificity Kranzusch, Philip J.; Lee, Amy S. Y.; Wilson, Stephen C. Cell, Vol. 158, Issue 5 https://doi.org/10.1016/j.cell.2014.07.028	journal	August 2014
The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop Zhang, Xu; Wu, Jiaxi; Du, Fenghe Cell Reports, Vol. 6, Issue 3 https://doi.org/10.1016/j.celrep.2014.01.003	journal	February 2014
An RNA-Based Fluorescent Biosensor for High-Throughput Analysis of the cGAS-cGAMP-STING Pathway Bose, Debojit; Su, Yichi; Marcus, Assaf Cell Chemical Biology, Vol. 23, Issue 12 https://doi.org/10.1016/j.chembiol.2016.10.014	journal	December 2016
The interferon response to intracellular DNA: Why so many receptors? Unterholzner, Leonie Immunobiology, Vol. 218, Issue 11 https://doi.org/10.1016/j.imbio.2013.07.007	journal	November 2013
The AIM2-like Receptors Are Dispensable for the Interferon Response to Intracellular DNA Gray, Elizabeth E.; Winship, Damion; Snyder, Jessica M. Immunity, Vol. 45, Issue 2 https://doi.org/10.1016/j.immuni.2016.06.015	journal	August 2016
The cGAS-STING Pathway for DNA Sensing Xiao, T. Sam; Fitzgerald, Katherine A. Molecular Cell, Vol. 51, Issue 2 https://doi.org/10.1016/j.molcel.2013.07.004	journal	July 2013
Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2′,3′ cGAMP Signaling Kranzusch, Philip J.; Wilson, Stephen C.; Lee, Amy S. Y. Molecular Cell, Vol. 59, Issue 6 https://doi.org/10.1016/j.molcel.2015.07.022	journal	September 2015
PRRs are watching you: Localization of innate sensing and signaling regulators Chow, Jonathan; Franz, Kate M.; Kagan, Jonathan C. Virology, Vol. 479-480 https://doi.org/10.1016/j.virol.2015.02.051	journal	May 2015
Development of a Rule-Based Method for the Assessment of Protein Druggability Perola, Emanuele; Herman, Lee; Weiss, Jonathan Journal of Chemical Information and Modeling, Vol. 52, Issue 4 https://doi.org/10.1021/ci200613b	journal	April 2012
Identifying and Characterizing Binding Sites and Assessing Druggability Halgren, Thomas A. Journal of Chemical Information and Modeling, Vol. 49, Issue 2 https://doi.org/10.1021/ci800324m	journal	January 2009
Long-Range ¹ H– ¹⁵ N J Couplings Providing a Method for Direct Studies of the Structure and Azide–Tetrazole Equilibrium in a Series of Azido-1,2,4-triazines and Azidopyrimidines Shestakova, Tatyana S.; Shenkarev, Zakhar O.; Deev, Sergey L. The Journal of Organic Chemistry, Vol. 78, Issue 14 https://doi.org/10.1021/jo4008207	journal	June 2013
Building unique bonds to fight misplaced DNA Fagundes, Caio T.; O'Neill, Luke A. J. Cell Research, Vol. 23, Issue 9 https://doi.org/10.1038/cr.2013.81	journal	June 2013
Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort Namjou, B.; Kothari, P. H.; Kelly, J. A. Genes & Immunity, Vol. 12, Issue 4 https://doi.org/10.1038/gene.2010.73	journal	January 2011
Structural mechanism of cytosolic DNA sensing by cGAS Civril, Filiz; Deimling, Tobias; de Oliveira Mann, Carina C. Nature, Vol. 498, Issue 7454 https://doi.org/10.1038/nature12305	journal	May 2013
Structure-based maximal affinity model predicts small-molecule druggability Cheng, Alan C.; Coleman, Ryan G.; Smyth, Kathleen T. Nature Biotechnology, Vol. 25, Issue 1 https://doi.org/10.1038/nbt1273	journal	January 2007
Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 are associated with systemic lupus erythematosus Lee-Kirsch, Min Ae; Gong, Maolian; Chowdhury, Dipanjan Nature Genetics, Vol. 39, Issue 9 https://doi.org/10.1038/ng2091	journal	July 2007
STING and the innate immune response to nucleic acids in the cytosol Burdette, Dara L.; Vance, Russell E. Nature Immunology, Vol. 14, Issue 1 https://doi.org/10.1038/ni.2491	journal	December 2012
Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing Chen, Qi; Sun, Lijun; Chen, Zhijian J. Nature Immunology, Vol. 17, Issue 10 https://doi.org/10.1038/ni.3558	journal	September 2016
Discriminating self from non-self in nucleic acid sensing Schlee, Martin; Hartmann, Gunther Nature Reviews Immunology, Vol. 16, Issue 9 https://doi.org/10.1038/nri.2016.78	journal	July 2016
Translating nucleic acid-sensing pathways into therapies Junt, Tobias; Barchet, Winfried Nature Reviews Immunology, Vol. 15, Issue 9 https://doi.org/10.1038/nri3875	journal	August 2015
STING-Associated Vasculopathy with Onset in Infancy — A New Interferonopathy Crow, Yanick J.; Casanova, Jean-Laurent New England Journal of Medicine, Vol. 371, Issue 6 https://doi.org/10.1056/nejme1407246	journal	August 2014
cGMP-AMP synthase paves the way to autoimmunity Rodero, Mathieu P.; Crow, Yanick J. Proceedings of the National Academy of Sciences, Vol. 112, Issue 42 https://doi.org/10.1073/pnas.1517578112	journal	October 2015
Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING Wu, Xiaomei; Wu, Fei-Hua; Wang, Xiaoqiang Nucleic Acids Research, Vol. 42, Issue 13 https://doi.org/10.1093/nar/gku569	journal	June 2014
Cyclic GMP-AMP Is an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA Wu, J.; Sun, L.; Chen, X. Science, Vol. 339, Issue 6121 https://doi.org/10.1126/science.1229963	journal	December 2012
Gene-Targeted Mice Lacking the Trex1 (DNase III) 3'->5' DNA Exonuclease Develop Inflammatory Myocarditis Morita, M.; Stamp, G.; Robins, P. Molecular and Cellular Biology, Vol. 24, Issue 15 https://doi.org/10.1128/mcb.24.15.6719-6727.2004	journal	July 2004
The Type I Interferonopathies Lee-Kirsch, Min Ae Annual Review of Medicine, Vol. 68, Issue 1 https://doi.org/10.1146/annurev-med-050715-104506	journal	January 2017
Cytosolic sensing of aberrant DNA: arming STING on the endoplasmic reticulum Wang, Qiang; Liu, Xing; Zhou, Qin Expert Opinion on Therapeutic Targets, Vol. 19, Issue 10 https://doi.org/10.1517/14728222.2015.1067303	journal	July 2015
cGAS-cGAMP-STING: The three musketeers of cytosolic DNA sensing and signaling: Cytosolic DNA Sensing and Signaling Tao, Jianli; Zhou, Xiang; Jiang, Zhengfan IUBMB Life, Vol. 68, Issue 11 https://doi.org/10.1002/iub.1566	journal	October 2016
Binding screen for cystic fibrosis transmembrane conductance regulator correctors finds new chemical matter and yields insights into cystic fibrosis therapeutic strategy: CFTR Correctors in CF Therapeutics Hall, Justin D.; Wang, Hong; Byrnes, Laura J. Protein Science, Vol. 25, Issue 2 https://doi.org/10.1002/pro.2821	journal	January 2016
Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase Gao, Pu; Ascano, Manuel; Wu, Yang Cell, Vol. 153, Issue 5 https://doi.org/10.1016/j.cell.2013.04.046	journal	May 2013
Cytosolic DNA Sensing: The Field Narrows Vance, Russell E. Immunity, Vol. 45, Issue 2 https://doi.org/10.1016/j.immuni.2016.08.006	journal	August 2016
Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus Crow, Yanick J.; Hayward, Bruce E.; Parmar, Rekha Nature Genetics, Vol. 38, Issue 8 https://doi.org/10.1038/ng1845	journal	July 2006
Aicardi–Goutières syndrome and the type I interferonopathies Crow, Yanick J.; Manel, Nicolas Nature Reviews Immunology, Vol. 15, Issue 7 https://doi.org/10.1038/nri3850	journal	June 2015
Activated STING in a Vascular and Pulmonary Syndrome Liu, Yin; Jesus, Adriana A.; Marrero, Bernadette New England Journal of Medicine, Vol. 371, Issue 6 https://doi.org/10.1056/NEJMoa1312625	journal	August 2014
Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases Gao, Daxing; Li, Tuo; Li, Xiao-Dong Proceedings of the National Academy of Sciences, Vol. 112, Issue 42 https://doi.org/10.1073/pnas.1516465112	journal	September 2015
Type I interferonopathies: a novel set of inborn errors of immunity: Type I interferonopathies Crow, Yanick J. Annals of the New York Academy of Sciences, Vol. 1238, Issue 1 https://doi.org/10.1111/j.1749-6632.2011.06220.x	journal	November 2011
Cyclic GMP-AMP Synthase Is a Cytosolic DNA Sensor That Activates the Type I Interferon Pathway Sun, L.; Wu, J.; Du, F. Science, Vol. 339, Issue 6121 https://doi.org/10.1126/science.1232458	journal	December 2012
Gene-Targeted Mice Lacking the Trex1 (DNase III) 3'->5' DNA Exonuclease Develop Inflammatory Myocarditis Morita, M.; Stamp, G.; Robins, P. Molecular and Cellular Biology, Vol. 24, Issue 15 https://doi.org/10.1128/MCB.24.15.6719-6727.2004	journal	July 2004
Cutting Edge: cGAS Is Required for Lethal Autoimmune Disease in the Trex1-Deficient Mouse Model of Aicardi–Goutières Syndrome Gray, Elizabeth E.; Treuting, Piper M.; Woodward, Joshua J. The Journal of Immunology, Vol. 195, Issue 5 https://doi.org/10.4049/jimmunol.1500969	journal	July 2015

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A simple model for determining affinity from irreversible thermal shifts Hall, Justin Protein Science, Vol. 28, Issue 10 https://doi.org/10.1002/pro.3701	journal	August 2019
Small molecules targeting the innate immune cGAS‒STING‒TBK1 signaling pathway Ding, Chunyong; Song, Zilan; Shen, Ancheng Acta Pharmaceutica Sinica B, Vol. 10, Issue 12 https://doi.org/10.1016/j.apsb.2020.03.001	journal	December 2020
STING palmitoylation as a therapeutic target Hansen, Anne Louise; Mukai, Kojiro; Schopfer, Francisco J. Cellular & Molecular Immunology, Vol. 16, Issue 3 https://doi.org/10.1038/s41423-019-0205-5	journal	February 2019
Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression Lama, Lodoe; Adura, Carolina; Xie, Wei Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-08620-4	journal	May 2019
Compound C Reducing Interferon Expression by Inhibiting cGAMP Accumulation Lai, Junzhong; Luo, Xuan; Tian, Shuoran Frontiers in Pharmacology, Vol. 11 https://doi.org/10.3389/fphar.2020.00088	journal	February 2020
cGAS/STING Pathway in Cancer: Jekyll and Hyde Story of Cancer Immune Response Bose, Debojit International Journal of Molecular Sciences, Vol. 18, Issue 11 https://doi.org/10.3390/ijms18112456	journal	November 2017
Interrupting cyclic dinucleotide-cGAS–STING axis with small molecules Sintim, Herman O.; Mikek, Clinton G.; Wang, Modi MedChemComm, Vol. 10, Issue 12 https://doi.org/10.1039/c8md00555a	journal	January 2019
Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation Xie, Wei; Lama, Lodoe; Adura, Carolina Proceedings of the National Academy of Sciences https://doi.org/10.1073/pnas.1905013116	journal	May 2019
The cGAS–cGAMP–STING pathway connects DNA damage to inflammation, senescence, and cancer Li, Tuo; Chen, Zhijian J. The Journal of Experimental Medicine, Vol. 215, Issue 5 https://doi.org/10.1084/jem.20180139	journal	April 2018
Human DNA-PK activates a STING-independent DNA sensing pathway Burleigh, Katelyn; Maltbaek, Joanna H.; Cambier, Stephanie https://doi.org/10.1101/587501	posted_content	March 2019
cGAS in action: Expanding roles in immunity and inflammation Ablasser, Andrea; Chen, Zhijian J. Science, Vol. 363, Issue 6431 https://doi.org/10.1126/science.aat8657	journal	March 2019
Human DNA-PK activates a STING-independent DNA sensing pathway Burleigh, Katelyn; Maltbaek, Joanna H.; Cambier, Stephanie Science Immunology, Vol. 5, Issue 43 https://doi.org/10.1126/sciimmunol.aba4219	journal	January 2020
MALDI-TOF Mass Spectrometry-Based High-Throughput Screening for Inhibitors of the Cytosolic DNA Sensor cGAS Simon, Roman P.; Winter, Martin; Kleiner, Carola SLAS DISCOVERY: Advancing the Science of Drug Discovery, Vol. 25, Issue 4 https://doi.org/10.1177/2472555219880185	journal	October 2019
Suramin potently inhibits cGAMP synthase, cGAS, in THP1 cells to modulate IFN-β levels Wang, Modi; Sooreshjani, Moloud A.; Mikek, Clinton Future Medicinal Chemistry, Vol. 10, Issue 11 https://doi.org/10.4155/fmc-2017-0322	journal	June 2018

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59 BASIC BIOLOGICAL SCIENCES
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Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

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