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9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain.

Picaud S, Strocchia M, Terracciano S, Lauro G, Mendez J, Daniels DL, Riccio R, Bifulco G, Bruno I, Filippakopoulos P - J. Med. Chem. (2015)

Bottom Line: The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4.Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket.The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems.

View Article: PubMed Central - PubMed

Affiliation: †Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4. Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket. The compound does not exhibit any cytotoxic effect in HEK293 cells and displaces the BRD9 bromodomain from chromatin in bioluminescence proximity assays without affecting the BRD4/histone complex. The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems.

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Complex of compound 11 and 9H-purineactivity in cells. (A) Overview of the complex of compound 11 with the first bromodomain of BRD4. The ligand retained the acetyllysinemimetic pose that was observed in the case of 7d. (B)Compound 11FcFo omit map from the BRD4(1)/11 complex contouredat 2σ. (C) Detail of compound 11 biding to BRD4(1)demonstrating the acetyllysine mimetic binding mode, initiating interactionswith the conserved asparagine (N140), and packing between the ZA-loopL92 and the ZA-channel W81 while retaining the network of conservedwater interactions. The model and structure factors of the BRD4(1)/11 complex shown in panels A, B, and C have been depositedto the PDB with ascension code 4XYA. (D) Titration of compounds 7d and 11 into HEK293 cells transfected with NanoLuc-fusedbromodomain of BRD9 and Halo-tagged histone H3.3. The ligands disruptthe histone/BRD9 interaction with an apparent IC50 of 3.5μM (7d) and 480 nM (11), resultingin loss of signal due to separation of the BRD9-histone complex. (E)Titration of compounds 7d and 11 into HEK293cells transfected with NanoLuc-fused full length BRD4 (UniProt code O60885) and Halo-taggedhistone H3.3. Although the ligands gradually disrupt the BRD4-FL/H3.3interaction, they fail to elicit the same effect as in the case ofBRD9 in the concentration range tested.
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fig5: Complex of compound 11 and 9H-purineactivity in cells. (A) Overview of the complex of compound 11 with the first bromodomain of BRD4. The ligand retained the acetyllysinemimetic pose that was observed in the case of 7d. (B)Compound 11FcFo omit map from the BRD4(1)/11 complex contouredat 2σ. (C) Detail of compound 11 biding to BRD4(1)demonstrating the acetyllysine mimetic binding mode, initiating interactionswith the conserved asparagine (N140), and packing between the ZA-loopL92 and the ZA-channel W81 while retaining the network of conservedwater interactions. The model and structure factors of the BRD4(1)/11 complex shown in panels A, B, and C have been depositedto the PDB with ascension code 4XYA. (D) Titration of compounds 7d and 11 into HEK293 cells transfected with NanoLuc-fusedbromodomain of BRD9 and Halo-tagged histone H3.3. The ligands disruptthe histone/BRD9 interaction with an apparent IC50 of 3.5μM (7d) and 480 nM (11), resultingin loss of signal due to separation of the BRD9-histone complex. (E)Titration of compounds 7d and 11 into HEK293cells transfected with NanoLuc-fused full length BRD4 (UniProt code O60885) and Halo-taggedhistone H3.3. Although the ligands gradually disrupt the BRD4-FL/H3.3interaction, they fail to elicit the same effect as in the case ofBRD9 in the concentration range tested.

Mentions: In order to testour hypothesis that compound 11 bindingwould also result in structural rearrangement of the bromodomain bindingcavity of BRD9, in a similar way to compound 7d, whilenot affecting the cavity of BRD4(1), we attempted to determine thecrystal structures of this compound in complex with these two bromodomains.Compound 11 readily crystallized with BRD4(1) and wasfound to occupy the acetyllysine binding cavity of the bromodomain(Figure 5A) and was very well-defined in thedensity (Figure 5B) despite its weaker bindingaffinity of 1.37 μM toward BRD4(1) (Table 1). The ligand was found to directly interact with the conserved asparagine(Asn140) as well as with the conserved network of water moleculeswhile packing between the ZA-channel tryptophan (Trp81) and the ZA-loopleucine (Leu92) (Figure 5C). Our efforts howeverto obtain a BRD9/11 complex did not yield diffractingquality crystals suitable for structure determination, and as such,we employed computational methods to account for its binding to BRD9.Rigid docking into the BRD9/7d complex structure resultedin a conformation similar to that observed with compound 7d, with the ligand engaging the conserved asparagine via its primaryamine function and the 6-aryl-substituted ring packing betweent theZA-loop Ile53 and Phe44 (Supporting Information Figure 3A). We then performed induced-fit docking employing thealgorithms previously described for purine fragments using the complexof BRD9/7d as starting point and obtained a pose wherebythe 2-amine function inverted and inserted in the BRD pocket, withoutany changes in the surrounding side chains of Phe44, Phe47, Ile53,and Tyr106 (Supporting Information Figure3B). Intrigued by this finding, we performed another induced fit dockingexperiment, starting with the BRD9 apo structure and allowing residuesto freely move in the presence of the ligand. We observed a similarset of side chain rearrangement within the BRD9 acetyllysine cavity,including a rotation of Phe47 resulting in capping of the bindinggroove, accompanied by repositioning of Phe44 from helix C and Ile53from the ZA-loop (Supporting Information Figure 3C). We therefore concluded that the 2-amine-9H-purine scaffolds that we developed can induce a closed pocket withinthe bromodomain of BRD9 resulting in tight binding, without at thesame time exhibiting high affinity for BRD4(1) or other BET familymembers. Indeed, we determined the dissociation constants for bindingto BET domains employing ITC and found that both compounds 7d and 11 exhibited low micromolar affinities (1.4–4.6μM) correlating well with the higher thermal shifts observed(Supporting Information Table S1), whileretaining higher affinity against the bromodomain of BRD9 (Supporting Information Figure 4 and Tables 2 and 3).


9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain.

Picaud S, Strocchia M, Terracciano S, Lauro G, Mendez J, Daniels DL, Riccio R, Bifulco G, Bruno I, Filippakopoulos P - J. Med. Chem. (2015)

Complex of compound 11 and 9H-purineactivity in cells. (A) Overview of the complex of compound 11 with the first bromodomain of BRD4. The ligand retained the acetyllysinemimetic pose that was observed in the case of 7d. (B)Compound 11FcFo omit map from the BRD4(1)/11 complex contouredat 2σ. (C) Detail of compound 11 biding to BRD4(1)demonstrating the acetyllysine mimetic binding mode, initiating interactionswith the conserved asparagine (N140), and packing between the ZA-loopL92 and the ZA-channel W81 while retaining the network of conservedwater interactions. The model and structure factors of the BRD4(1)/11 complex shown in panels A, B, and C have been depositedto the PDB with ascension code 4XYA. (D) Titration of compounds 7d and 11 into HEK293 cells transfected with NanoLuc-fusedbromodomain of BRD9 and Halo-tagged histone H3.3. The ligands disruptthe histone/BRD9 interaction with an apparent IC50 of 3.5μM (7d) and 480 nM (11), resultingin loss of signal due to separation of the BRD9-histone complex. (E)Titration of compounds 7d and 11 into HEK293cells transfected with NanoLuc-fused full length BRD4 (UniProt code O60885) and Halo-taggedhistone H3.3. Although the ligands gradually disrupt the BRD4-FL/H3.3interaction, they fail to elicit the same effect as in the case ofBRD9 in the concentration range tested.
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fig5: Complex of compound 11 and 9H-purineactivity in cells. (A) Overview of the complex of compound 11 with the first bromodomain of BRD4. The ligand retained the acetyllysinemimetic pose that was observed in the case of 7d. (B)Compound 11FcFo omit map from the BRD4(1)/11 complex contouredat 2σ. (C) Detail of compound 11 biding to BRD4(1)demonstrating the acetyllysine mimetic binding mode, initiating interactionswith the conserved asparagine (N140), and packing between the ZA-loopL92 and the ZA-channel W81 while retaining the network of conservedwater interactions. The model and structure factors of the BRD4(1)/11 complex shown in panels A, B, and C have been depositedto the PDB with ascension code 4XYA. (D) Titration of compounds 7d and 11 into HEK293 cells transfected with NanoLuc-fusedbromodomain of BRD9 and Halo-tagged histone H3.3. The ligands disruptthe histone/BRD9 interaction with an apparent IC50 of 3.5μM (7d) and 480 nM (11), resultingin loss of signal due to separation of the BRD9-histone complex. (E)Titration of compounds 7d and 11 into HEK293cells transfected with NanoLuc-fused full length BRD4 (UniProt code O60885) and Halo-taggedhistone H3.3. Although the ligands gradually disrupt the BRD4-FL/H3.3interaction, they fail to elicit the same effect as in the case ofBRD9 in the concentration range tested.
Mentions: In order to testour hypothesis that compound 11 bindingwould also result in structural rearrangement of the bromodomain bindingcavity of BRD9, in a similar way to compound 7d, whilenot affecting the cavity of BRD4(1), we attempted to determine thecrystal structures of this compound in complex with these two bromodomains.Compound 11 readily crystallized with BRD4(1) and wasfound to occupy the acetyllysine binding cavity of the bromodomain(Figure 5A) and was very well-defined in thedensity (Figure 5B) despite its weaker bindingaffinity of 1.37 μM toward BRD4(1) (Table 1). The ligand was found to directly interact with the conserved asparagine(Asn140) as well as with the conserved network of water moleculeswhile packing between the ZA-channel tryptophan (Trp81) and the ZA-loopleucine (Leu92) (Figure 5C). Our efforts howeverto obtain a BRD9/11 complex did not yield diffractingquality crystals suitable for structure determination, and as such,we employed computational methods to account for its binding to BRD9.Rigid docking into the BRD9/7d complex structure resultedin a conformation similar to that observed with compound 7d, with the ligand engaging the conserved asparagine via its primaryamine function and the 6-aryl-substituted ring packing betweent theZA-loop Ile53 and Phe44 (Supporting Information Figure 3A). We then performed induced-fit docking employing thealgorithms previously described for purine fragments using the complexof BRD9/7d as starting point and obtained a pose wherebythe 2-amine function inverted and inserted in the BRD pocket, withoutany changes in the surrounding side chains of Phe44, Phe47, Ile53,and Tyr106 (Supporting Information Figure3B). Intrigued by this finding, we performed another induced fit dockingexperiment, starting with the BRD9 apo structure and allowing residuesto freely move in the presence of the ligand. We observed a similarset of side chain rearrangement within the BRD9 acetyllysine cavity,including a rotation of Phe47 resulting in capping of the bindinggroove, accompanied by repositioning of Phe44 from helix C and Ile53from the ZA-loop (Supporting Information Figure 3C). We therefore concluded that the 2-amine-9H-purine scaffolds that we developed can induce a closed pocket withinthe bromodomain of BRD9 resulting in tight binding, without at thesame time exhibiting high affinity for BRD4(1) or other BET familymembers. Indeed, we determined the dissociation constants for bindingto BET domains employing ITC and found that both compounds 7d and 11 exhibited low micromolar affinities (1.4–4.6μM) correlating well with the higher thermal shifts observed(Supporting Information Table S1), whileretaining higher affinity against the bromodomain of BRD9 (Supporting Information Figure 4 and Tables 2 and 3).

Bottom Line: The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4.Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket.The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems.

View Article: PubMed Central - PubMed

Affiliation: †Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, U.K.

ABSTRACT
The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4. Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket. The compound does not exhibit any cytotoxic effect in HEK293 cells and displaces the BRD9 bromodomain from chromatin in bioluminescence proximity assays without affecting the BRD4/histone complex. The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems.

Show MeSH
Related in: MedlinePlus