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Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia

[Image: see text] Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CRE...

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Autores principales: Zhang, Kaiming, Horikoshi, Naoki, Li, Shanshan, Powers, Alexander S., Hameedi, Mikhail A., Pintilie, Grigore D., Chae, Hee-Don, Khan, Yousuf A., Suomivuori, Carl-Mikael, Dror, Ron O., Sakamoto, Kathleen M., Chiu, Wah, Wakatsuki, Soichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2022
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8875425/
https://www.ncbi.nlm.nih.gov/pubmed/35233453
http://dx.doi.org/10.1021/acscentsci.1c01090
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author Zhang, Kaiming
Horikoshi, Naoki
Li, Shanshan
Powers, Alexander S.
Hameedi, Mikhail A.
Pintilie, Grigore D.
Chae, Hee-Don
Khan, Yousuf A.
Suomivuori, Carl-Mikael
Dror, Ron O.
Sakamoto, Kathleen M.
Chiu, Wah
Wakatsuki, Soichi
author_facet Zhang, Kaiming
Horikoshi, Naoki
Li, Shanshan
Powers, Alexander S.
Hameedi, Mikhail A.
Pintilie, Grigore D.
Chae, Hee-Don
Khan, Yousuf A.
Suomivuori, Carl-Mikael
Dror, Ron O.
Sakamoto, Kathleen M.
Chiu, Wah
Wakatsuki, Soichi
author_sort Zhang, Kaiming
collection PubMed
description [Image: see text] Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target–apoferritin interface, resulting in a cryo-EM structure at 2.6 Å resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix–helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.
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spelling pubmed-88754252022-02-28 Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia Zhang, Kaiming Horikoshi, Naoki Li, Shanshan Powers, Alexander S. Hameedi, Mikhail A. Pintilie, Grigore D. Chae, Hee-Don Khan, Yousuf A. Suomivuori, Carl-Mikael Dror, Ron O. Sakamoto, Kathleen M. Chiu, Wah Wakatsuki, Soichi ACS Cent Sci [Image: see text] Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target–apoferritin interface, resulting in a cryo-EM structure at 2.6 Å resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix–helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors. American Chemical Society 2022-02-07 2022-02-23 /pmc/articles/PMC8875425/ /pubmed/35233453 http://dx.doi.org/10.1021/acscentsci.1c01090 Text en © 2022 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Zhang, Kaiming
Horikoshi, Naoki
Li, Shanshan
Powers, Alexander S.
Hameedi, Mikhail A.
Pintilie, Grigore D.
Chae, Hee-Don
Khan, Yousuf A.
Suomivuori, Carl-Mikael
Dror, Ron O.
Sakamoto, Kathleen M.
Chiu, Wah
Wakatsuki, Soichi
Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title_full Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title_fullStr Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title_full_unstemmed Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title_short Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia
title_sort cryo-em, protein engineering, and simulation enable the development of peptide therapeutics against acute myeloid leukemia
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8875425/
https://www.ncbi.nlm.nih.gov/pubmed/35233453
http://dx.doi.org/10.1021/acscentsci.1c01090
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