Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID

Chulhwan Kwak; Cheolhun Park; Minjeong Ko; Chun Young Im; Heegyum Moon; Young Hoon Park; So Young Kim; Seungyeon Lee; Myeong Gyun Kang; Ho Jeong Kwon; Eunmi Hong; Jeong Kon Seo; Hyun Woo Rhee

doi:10.1016/j.chembiol.2022.10.001

Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID

Chulhwan Kwak, Cheolhun Park, Minjeong Ko, Chun Young Im, Heegyum Moon, Young Hoon Park, So Young Kim, Seungyeon Lee, Myeong Gyun Kang, Ho Jeong Kwon, Eunmi Hong, Jeong Kon Seo, Hyun Woo Rhee

Research output: Contribution to journal › Article › peer-review

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Abstract

Direct identification of the proteins targeted by small molecules can provide clues for disease diagnosis, prevention, and drug development. Despite concentrated attempts, there are still technical limitations associated with the elucidation of direct interactors. Herein, we report a target-ID system called proximity-based compound-binding protein identification (PROCID), which combines our direct analysis workflow of proximity-labeled proteins (Spot-ID) with the HaloTag system to efficiently identify the dynamic proteomic landscape of drug-binding proteins. We successfully identified well-known dasatinib-binding proteins (ABL1, ABL2) and confirmed the unapproved dasatinib-binding kinases (e.g., BTK and CSK) in a live chronic myeloid leukemia cell line. PROCID also identified the DNA helicase protein SMARCA2 as a dasatinib-binding protein, and the ATPase domain was confirmed to be the binding site of dasatinib using a proximity ligation assay (PLA) and in cellulo biotinylation assay. PROCID thus provides a robust method to identify unknown drug-interacting proteins in live cells that expedites the mode of action of the drug.

Original language	English
Pages (from-to)	1739-1753.e6
Journal	Cell Chemical Biology
Volume	29
Issue number	12
DOIs	https://doi.org/10.1016/j.chembiol.2022.10.001
Publication status	Published - 2022 Dec 15

Bibliographical note

Funding Information:
This work was supported by the National Research Foundation of Korea ( NRF-2022R1A2B5B03001658 , NRF-2022M3E5E8081185 , and NRF-2022M3H9A2096199 to H.W.R., NRF-2021R1F1A1047853 to J.K.S., NRF-2018M3A9G4078528 , NRF-2019R1F1A1060487 , and NRF-2020R1A5A2017323 to E.H., NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center ( NRF-2017R1A5A1015366 ), and the Korea Health Industry Development Institute ( KHIDI ) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology ( ICT ), Republic of Korea (grant HU20C0326 ). E.H. was supported by a Korea Evaluation Institute of Industrial Technology ( KEIT ) grant funded by the Korea Government ( MOTIE ) ( 20020237 ). H.J.K. was supported by grants from the National Research Foundation of Korea ( MSIP ; 2021R1A3B1077371 and 2015K1A1A2028365 ) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation ( SSTF-BA2201-08 ). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work.

Funding Information:
This work was supported by the National Research Foundation of Korea (NRF-2022R1A2B5B03001658, NRF-2022M3E5E8081185, and NRF-2022M3H9A2096199 to H.W.R. NRF-2021R1F1A1047853 to J.K.S. NRF-2018M3A9G4078528, NRF-2019R1F1A1060487, and NRF-2020R1A5A2017323 to E.H. NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center (NRF-2017R1A5A1015366), and the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology (ICT), Republic of Korea (grant HU20C0326). E.H. was supported by a Korea Evaluation Institute of Industrial Technology (KEIT) grant funded by the Korea Government (MOTIE) (20020237). H.J.K. was supported by grants from the National Research Foundation of Korea (MSIP; 2021R1A3B1077371 and 2015K1A1A2028365) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation (SSTF-BA2201-08). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work. H.W.R. conceptualized the idea; E.H. H.G.M. Y.H.P. S.Y.K. S.L. and C.Y.L. prepared the chemical probes and performed the cytotoxicity assay; C.K. M.G.K. and C.P. conducted the cloning, imaging, western blot, stable cell line generation, and mass sampling experiments; M.K. and H.J.K. conducted PLA experiments; J.K.S. conducted the mass analysis; C.K. C.P. H.J.K. E.H. J.K.S. and H.W.R. wrote the manuscript. The authors have no competing interests. A patent application has been filed by Seoul National University relating to this work. We support inclusive, diverse, and equitable conduct of research.

Publisher Copyright:
© 2022 Elsevier Ltd

All Science Journal Classification (ASJC) codes

Biochemistry
Molecular Medicine
Molecular Biology
Pharmacology
Drug Discovery
Clinical Biochemistry

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10.1016/j.chembiol.2022.10.001

Cite this

@article{56679f7d1d904a4fb6ba5a37b3442765,

title = "Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID",

abstract = "Direct identification of the proteins targeted by small molecules can provide clues for disease diagnosis, prevention, and drug development. Despite concentrated attempts, there are still technical limitations associated with the elucidation of direct interactors. Herein, we report a target-ID system called proximity-based compound-binding protein identification (PROCID), which combines our direct analysis workflow of proximity-labeled proteins (Spot-ID) with the HaloTag system to efficiently identify the dynamic proteomic landscape of drug-binding proteins. We successfully identified well-known dasatinib-binding proteins (ABL1, ABL2) and confirmed the unapproved dasatinib-binding kinases (e.g., BTK and CSK) in a live chronic myeloid leukemia cell line. PROCID also identified the DNA helicase protein SMARCA2 as a dasatinib-binding protein, and the ATPase domain was confirmed to be the binding site of dasatinib using a proximity ligation assay (PLA) and in cellulo biotinylation assay. PROCID thus provides a robust method to identify unknown drug-interacting proteins in live cells that expedites the mode of action of the drug.",

author = "Chulhwan Kwak and Cheolhun Park and Minjeong Ko and Im, {Chun Young} and Heegyum Moon and Park, {Young Hoon} and Kim, {So Young} and Seungyeon Lee and Kang, {Myeong Gyun} and Kwon, {Ho Jeong} and Eunmi Hong and Seo, {Jeong Kon} and Rhee, {Hyun Woo}",

note = "Funding Information: This work was supported by the National Research Foundation of Korea ( NRF-2022R1A2B5B03001658 , NRF-2022M3E5E8081185 , and NRF-2022M3H9A2096199 to H.W.R., NRF-2021R1F1A1047853 to J.K.S., NRF-2018M3A9G4078528 , NRF-2019R1F1A1060487 , and NRF-2020R1A5A2017323 to E.H., NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center ( NRF-2017R1A5A1015366 ), and the Korea Health Industry Development Institute ( KHIDI ) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology ( ICT ), Republic of Korea (grant HU20C0326 ). E.H. was supported by a Korea Evaluation Institute of Industrial Technology ( KEIT ) grant funded by the Korea Government ( MOTIE ) ( 20020237 ). H.J.K. was supported by grants from the National Research Foundation of Korea ( MSIP ; 2021R1A3B1077371 and 2015K1A1A2028365 ) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation ( SSTF-BA2201-08 ). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work. Funding Information: This work was supported by the National Research Foundation of Korea (NRF-2022R1A2B5B03001658, NRF-2022M3E5E8081185, and NRF-2022M3H9A2096199 to H.W.R. NRF-2021R1F1A1047853 to J.K.S. NRF-2018M3A9G4078528, NRF-2019R1F1A1060487, and NRF-2020R1A5A2017323 to E.H. NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center (NRF-2017R1A5A1015366), and the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology (ICT), Republic of Korea (grant HU20C0326). E.H. was supported by a Korea Evaluation Institute of Industrial Technology (KEIT) grant funded by the Korea Government (MOTIE) (20020237). H.J.K. was supported by grants from the National Research Foundation of Korea (MSIP; 2021R1A3B1077371 and 2015K1A1A2028365) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation (SSTF-BA2201-08). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work. H.W.R. conceptualized the idea; E.H. H.G.M. Y.H.P. S.Y.K. S.L. and C.Y.L. prepared the chemical probes and performed the cytotoxicity assay; C.K. M.G.K. and C.P. conducted the cloning, imaging, western blot, stable cell line generation, and mass sampling experiments; M.K. and H.J.K. conducted PLA experiments; J.K.S. conducted the mass analysis; C.K. C.P. H.J.K. E.H. J.K.S. and H.W.R. wrote the manuscript. The authors have no competing interests. A patent application has been filed by Seoul National University relating to this work. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: {\textcopyright} 2022 Elsevier Ltd",

year = "2022",

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day = "15",

doi = "10.1016/j.chembiol.2022.10.001",

language = "English",

volume = "29",

pages = "1739--1753.e6",

journal = "Cell Chemical Biology",

issn = "2451-9448",

publisher = "Elsevier Inc.",

number = "12",

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T1 - Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID

AU - Kwak, Chulhwan

AU - Park, Cheolhun

AU - Ko, Minjeong

AU - Im, Chun Young

AU - Moon, Heegyum

AU - Park, Young Hoon

AU - Kim, So Young

AU - Lee, Seungyeon

AU - Kang, Myeong Gyun

AU - Kwon, Ho Jeong

AU - Hong, Eunmi

AU - Seo, Jeong Kon

AU - Rhee, Hyun Woo

N1 - Funding Information: This work was supported by the National Research Foundation of Korea ( NRF-2022R1A2B5B03001658 , NRF-2022M3E5E8081185 , and NRF-2022M3H9A2096199 to H.W.R., NRF-2021R1F1A1047853 to J.K.S., NRF-2018M3A9G4078528 , NRF-2019R1F1A1060487 , and NRF-2020R1A5A2017323 to E.H., NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center ( NRF-2017R1A5A1015366 ), and the Korea Health Industry Development Institute ( KHIDI ) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology ( ICT ), Republic of Korea (grant HU20C0326 ). E.H. was supported by a Korea Evaluation Institute of Industrial Technology ( KEIT ) grant funded by the Korea Government ( MOTIE ) ( 20020237 ). H.J.K. was supported by grants from the National Research Foundation of Korea ( MSIP ; 2021R1A3B1077371 and 2015K1A1A2028365 ) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation ( SSTF-BA2201-08 ). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work. Funding Information: This work was supported by the National Research Foundation of Korea (NRF-2022R1A2B5B03001658, NRF-2022M3E5E8081185, and NRF-2022M3H9A2096199 to H.W.R. NRF-2021R1F1A1047853 to J.K.S. NRF-2018M3A9G4078528, NRF-2019R1F1A1060487, and NRF-2020R1A5A2017323 to E.H. NRF-2022R1C1C2006983 to C.K.), the Organelle Network Research Center (NRF-2017R1A5A1015366), and the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare and Ministry of Science and Information & Communication Technology (ICT), Republic of Korea (grant HU20C0326). E.H. was supported by a Korea Evaluation Institute of Industrial Technology (KEIT) grant funded by the Korea Government (MOTIE) (20020237). H.J.K. was supported by grants from the National Research Foundation of Korea (MSIP; 2021R1A3B1077371 and 2015K1A1A2028365) and the Brain Korea 21 Yonsei Education & Research Center for Biosystems, Republic of Korea. H.W.R. was supported by the Samsung Science & Technology Foundation (SSTF-BA2201-08). The authors thank Dr. Jae Hyun Bae (ERASCA, San Diego) for helpful discussion on this work. H.W.R. conceptualized the idea; E.H. H.G.M. Y.H.P. S.Y.K. S.L. and C.Y.L. prepared the chemical probes and performed the cytotoxicity assay; C.K. M.G.K. and C.P. conducted the cloning, imaging, western blot, stable cell line generation, and mass sampling experiments; M.K. and H.J.K. conducted PLA experiments; J.K.S. conducted the mass analysis; C.K. C.P. H.J.K. E.H. J.K.S. and H.W.R. wrote the manuscript. The authors have no competing interests. A patent application has been filed by Seoul National University relating to this work. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: © 2022 Elsevier Ltd

PY - 2022/12/15

Y1 - 2022/12/15

N2 - Direct identification of the proteins targeted by small molecules can provide clues for disease diagnosis, prevention, and drug development. Despite concentrated attempts, there are still technical limitations associated with the elucidation of direct interactors. Herein, we report a target-ID system called proximity-based compound-binding protein identification (PROCID), which combines our direct analysis workflow of proximity-labeled proteins (Spot-ID) with the HaloTag system to efficiently identify the dynamic proteomic landscape of drug-binding proteins. We successfully identified well-known dasatinib-binding proteins (ABL1, ABL2) and confirmed the unapproved dasatinib-binding kinases (e.g., BTK and CSK) in a live chronic myeloid leukemia cell line. PROCID also identified the DNA helicase protein SMARCA2 as a dasatinib-binding protein, and the ATPase domain was confirmed to be the binding site of dasatinib using a proximity ligation assay (PLA) and in cellulo biotinylation assay. PROCID thus provides a robust method to identify unknown drug-interacting proteins in live cells that expedites the mode of action of the drug.

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Identification of proteomic landscape of drug-binding proteins in live cells by proximity-dependent target ID

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