Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**

Huarui Cui; Anand Divakaran; Anil K. Pandey; Jorden A. Johnson; Huda Zahid; Zachariah J. Hoell; Mikael O. Ellingson; Ke Shi; Hideki Aihara; Daniel A. Harki; William C.K. Pomerantz

doi:10.1002/anie.202008625

Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**

Huarui Cui, Anand Divakaran, Anil K. Pandey, Jorden A. Johnson, Huda Zahid, Zachariah J. Hoell, Mikael O. Ellingson, Ke Shi, Hideki Aihara, Daniel A. Harki, William C.K. Pomerantz

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28 Scopus citations

Abstract

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9–33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.

Original language	English (US)
Pages (from-to)	1220-1226
Number of pages	7
Journal	Angewandte Chemie - International Edition
Volume	60
Issue number	3
DOIs	https://doi.org/10.1002/anie.202008625
State	Published - Nov 18 2020

Bibliographical note

Funding Information:
We gratefully acknowledge support for this research from the Masonic Cancer Center at the University of Minnesota, (W.C.K.P and D.A.H) the NIH (R35-GM118047 to H.A. and R01-GM110129 to D.A.H), the American Heart Association (15SDG25710427 to W.C.K.P.). J.A.J. was supported by a NIH Biotechnology training grant (T32-GM008347-23) and A.D. was supported by the NIH chemistry-biology interface training grant (T32-GM008700). This work is based on research conducted at the NE-CAT beamlines at the Advanced Photon Source, which are supported by the NIH (P30-GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10-RR029205). We thank staff at the NE-CAT beamlines for assistance in data collection, Dr. Peter Ycas for help preparing the His9-BRD4 D1 plasmid, and Dr. Brian Van Ness for providing MM.1S cells.

Funding Information:
We gratefully acknowledge support for this research from the Masonic Cancer Center at the University of Minnesota, (W.C.K.P and D.A.H) the NIH (R35‐GM118047 to H.A. and R01‐GM110129 to D.A.H), the American Heart Association (15SDG25710427 to W.C.K.P.). J.A.J. was supported by a NIH Biotechnology training grant (T32‐GM008347‐23) and A.D. was supported by the NIH chemistry‐biology interface training grant (T32‐GM008700). This work is based on research conducted at the NE‐CAT beamlines at the Advanced Photon Source, which are supported by the NIH (P30‐GM124165). The Pilatus 6M detector on 24‐ID‐C beamline is funded by a NIH‐ORIP HEI grant (S10‐RR029205). We thank staff at the NE‐CAT beamlines for assistance in data collection, Dr. Peter Ycas for help preparing the His9‐BRD4 D1 plasmid, and Dr. Brian Van Ness for providing MM.1S cells.

Publisher Copyright:
© 2020 Wiley-VCH GmbH

Keywords

BET bromodomains
BRD4 D1 selectivity
epigenetics
inhibitors
structure-activity relationships

PubMed: MeSH publication types

Journal Article
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

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10.1002/anie.202008625

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Cui, H., Divakaran, A., Pandey, A. K., Johnson, J. A., Zahid, H., Hoell, Z. J., Ellingson, M. O., Shi, K., Aihara, H., Harki, D. A., & Pomerantz, W. C. K. (2020). Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**. Angewandte Chemie - International Edition, 60(3), 1220-1226. https://doi.org/10.1002/anie.202008625

Cui, H, Divakaran, A, Pandey, AK, Johnson, JA, Zahid, H, Hoell, ZJ, Ellingson, MO, Shi, K , Aihara, H , Harki, DA & Pomerantz, WCK 2020, 'Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**', Angewandte Chemie - International Edition, vol. 60, no. 3, pp. 1220-1226. https://doi.org/10.1002/anie.202008625

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abstract = "Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9–33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.",

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author = "Huarui Cui and Anand Divakaran and Pandey, {Anil K.} and Johnson, {Jorden A.} and Huda Zahid and Hoell, {Zachariah J.} and Ellingson, {Mikael O.} and Ke Shi and Hideki Aihara and Harki, {Daniel A.} and Pomerantz, {William C.K.}",

note = "Funding Information: We gratefully acknowledge support for this research from the Masonic Cancer Center at the University of Minnesota, (W.C.K.P and D.A.H) the NIH (R35-GM118047 to H.A. and R01-GM110129 to D.A.H), the American Heart Association (15SDG25710427 to W.C.K.P.). J.A.J. was supported by a NIH Biotechnology training grant (T32-GM008347-23) and A.D. was supported by the NIH chemistry-biology interface training grant (T32-GM008700). This work is based on research conducted at the NE-CAT beamlines at the Advanced Photon Source, which are supported by the NIH (P30-GM124165). The Pilatus 6M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10-RR029205). We thank staff at the NE-CAT beamlines for assistance in data collection, Dr. Peter Ycas for help preparing the His9-BRD4 D1 plasmid, and Dr. Brian Van Ness for providing MM.1S cells. Funding Information: We gratefully acknowledge support for this research from the Masonic Cancer Center at the University of Minnesota, (W.C.K.P and D.A.H) the NIH (R35‐GM118047 to H.A. and R01‐GM110129 to D.A.H), the American Heart Association (15SDG25710427 to W.C.K.P.). J.A.J. was supported by a NIH Biotechnology training grant (T32‐GM008347‐23) and A.D. was supported by the NIH chemistry‐biology interface training grant (T32‐GM008700). This work is based on research conducted at the NE‐CAT beamlines at the Advanced Photon Source, which are supported by the NIH (P30‐GM124165). The Pilatus 6M detector on 24‐ID‐C beamline is funded by a NIH‐ORIP HEI grant (S10‐RR029205). We thank staff at the NE‐CAT beamlines for assistance in data collection, Dr. Peter Ycas for help preparing the His9‐BRD4 D1 plasmid, and Dr. Brian Van Ness for providing MM.1S cells. Publisher Copyright: {\textcopyright} 2020 Wiley-VCH GmbH",

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doi = "10.1002/anie.202008625",

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AU - Divakaran, Anand

AU - Pandey, Anil K.

AU - Johnson, Jorden A.

AU - Zahid, Huda

AU - Hoell, Zachariah J.

AU - Ellingson, Mikael O.

AU - Shi, Ke

AU - Aihara, Hideki

AU - Harki, Daniel A.

AU - Pomerantz, William C.K.

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N2 - Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9–33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.

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KW - structure-activity relationships

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Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement**

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