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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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BRD pocketSAR. (A) Compounds designed to probe the acetyllysinemimetic character of the purine scaffold, disrupting interactions(N9-methyl analogues) or reaching deeper into the acetyllysine cavity(8-methyl analogues). (B) Thermal shift assay against human bromodomains.Compound 10 was heavily colored and interfered with theassay. Thermal shifts are color-coded as indicated in the inset. Compoundshighlighted with a colored star were further validated as shown in(C)/(D). (C) Substitution of the primary amine group to a hydroxyl(compound 8a) impairs binding toward BRD9 as demonstratedby ITC experiments, while cyclization of the aromatic substituentresults in enhanced potency (compound 11). The insetshows the normalized binding enthalpies corrected for the heat ofprotein dilution as a function of binding site saturation (symbolsas indicated in the figure). Solid lines represent a nonlinear least-squaresfit using a single-site binding model. (D) Isothermal titration calorimetryvalidation of compound 11 binding to BRD4(1) showingraw injection heats for titrations of protein into compound. Datahave been corrected and displayed as described in (C). All ITC titrationswere carried out in 50 mM HEPES, pH 7.5 (at 25 °C), 150 mM NaCl,and 15 °C while stirring at 1000 rpm.
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fig4: BRD pocketSAR. (A) Compounds designed to probe the acetyllysinemimetic character of the purine scaffold, disrupting interactions(N9-methyl analogues) or reaching deeper into the acetyllysine cavity(8-methyl analogues). (B) Thermal shift assay against human bromodomains.Compound 10 was heavily colored and interfered with theassay. Thermal shifts are color-coded as indicated in the inset. Compoundshighlighted with a colored star were further validated as shown in(C)/(D). (C) Substitution of the primary amine group to a hydroxyl(compound 8a) impairs binding toward BRD9 as demonstratedby ITC experiments, while cyclization of the aromatic substituentresults in enhanced potency (compound 11). The insetshows the normalized binding enthalpies corrected for the heat ofprotein dilution as a function of binding site saturation (symbolsas indicated in the figure). Solid lines represent a nonlinear least-squaresfit using a single-site binding model. (D) Isothermal titration calorimetryvalidation of compound 11 binding to BRD4(1) showingraw injection heats for titrations of protein into compound. Datahave been corrected and displayed as described in (C). All ITC titrationswere carried out in 50 mM HEPES, pH 7.5 (at 25 °C), 150 mM NaCl,and 15 °C while stirring at 1000 rpm.

Mentions: We next questioned whether the primary amine functionat position2 of the 9H-purine core scaffold is necessary forbinding to bromodomains. First we substituted the amine with a chlorinegroup (compound 8a, Figure 4A)while retaining the 6-(5-bromo-2-methoxyphenyl) substitution, resultingin loss of affinity toward all BRDs in our panel (Figure 4B). In the case of BRD9 we confirmed this observationby performing an isothermal titration calorimetry measurement whichyielded a Kd of 7.9 μM (Figure 4C). As with compounds from previous series, methylsubstitution at position 9 completely abolished bromodomain binding(compound 8b, Figure 4A), measuredby both thermal melt (Figure 4B) and ITC assaysin the case of BRD9 (Figure 4C); larger substituents(compound 8c) could not be tolerated suggesting thatthe core scaffold retained its pose within the bromodomain bindingcavity. Hydroxy substitution at position 2 while retaining a 6-(5-halide-2-methoxyphenyl)substituent (compounds 8d–f) hadvariable effects on the 9H-purine affinity towardBRDs. Moreover, fluoro (8d) and bromo (8f) substituted compounds lost affinity across the panel, while thechloro-substituted compound (8e) promiscuously boundto most bromodomains in the ΔTm assay,albeit weaker than its primary amine analogue 7c (Figure 4A,B), thus suggesting that the interactions initiatedby the hydroxyl group and the conserved asparagine (Asn100 in BRD9;Asn140 in BRD4(1)) are not favored over the primary amine.


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)

BRD pocketSAR. (A) Compounds designed to probe the acetyllysinemimetic character of the purine scaffold, disrupting interactions(N9-methyl analogues) or reaching deeper into the acetyllysine cavity(8-methyl analogues). (B) Thermal shift assay against human bromodomains.Compound 10 was heavily colored and interfered with theassay. Thermal shifts are color-coded as indicated in the inset. Compoundshighlighted with a colored star were further validated as shown in(C)/(D). (C) Substitution of the primary amine group to a hydroxyl(compound 8a) impairs binding toward BRD9 as demonstratedby ITC experiments, while cyclization of the aromatic substituentresults in enhanced potency (compound 11). The insetshows the normalized binding enthalpies corrected for the heat ofprotein dilution as a function of binding site saturation (symbolsas indicated in the figure). Solid lines represent a nonlinear least-squaresfit using a single-site binding model. (D) Isothermal titration calorimetryvalidation of compound 11 binding to BRD4(1) showingraw injection heats for titrations of protein into compound. Datahave been corrected and displayed as described in (C). All ITC titrationswere carried out in 50 mM HEPES, pH 7.5 (at 25 °C), 150 mM NaCl,and 15 °C while stirring at 1000 rpm.
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fig4: BRD pocketSAR. (A) Compounds designed to probe the acetyllysinemimetic character of the purine scaffold, disrupting interactions(N9-methyl analogues) or reaching deeper into the acetyllysine cavity(8-methyl analogues). (B) Thermal shift assay against human bromodomains.Compound 10 was heavily colored and interfered with theassay. Thermal shifts are color-coded as indicated in the inset. Compoundshighlighted with a colored star were further validated as shown in(C)/(D). (C) Substitution of the primary amine group to a hydroxyl(compound 8a) impairs binding toward BRD9 as demonstratedby ITC experiments, while cyclization of the aromatic substituentresults in enhanced potency (compound 11). The insetshows the normalized binding enthalpies corrected for the heat ofprotein dilution as a function of binding site saturation (symbolsas indicated in the figure). Solid lines represent a nonlinear least-squaresfit using a single-site binding model. (D) Isothermal titration calorimetryvalidation of compound 11 binding to BRD4(1) showingraw injection heats for titrations of protein into compound. Datahave been corrected and displayed as described in (C). All ITC titrationswere carried out in 50 mM HEPES, pH 7.5 (at 25 °C), 150 mM NaCl,and 15 °C while stirring at 1000 rpm.
Mentions: We next questioned whether the primary amine functionat position2 of the 9H-purine core scaffold is necessary forbinding to bromodomains. First we substituted the amine with a chlorinegroup (compound 8a, Figure 4A)while retaining the 6-(5-bromo-2-methoxyphenyl) substitution, resultingin loss of affinity toward all BRDs in our panel (Figure 4B). In the case of BRD9 we confirmed this observationby performing an isothermal titration calorimetry measurement whichyielded a Kd of 7.9 μM (Figure 4C). As with compounds from previous series, methylsubstitution at position 9 completely abolished bromodomain binding(compound 8b, Figure 4A), measuredby both thermal melt (Figure 4B) and ITC assaysin the case of BRD9 (Figure 4C); larger substituents(compound 8c) could not be tolerated suggesting thatthe core scaffold retained its pose within the bromodomain bindingcavity. Hydroxy substitution at position 2 while retaining a 6-(5-halide-2-methoxyphenyl)substituent (compounds 8d–f) hadvariable effects on the 9H-purine affinity towardBRDs. Moreover, fluoro (8d) and bromo (8f) substituted compounds lost affinity across the panel, while thechloro-substituted compound (8e) promiscuously boundto most bromodomains in the ΔTm assay,albeit weaker than its primary amine analogue 7c (Figure 4A,B), thus suggesting that the interactions initiatedby the hydroxyl group and the conserved asparagine (Asn100 in BRD9;Asn140 in BRD4(1)) are not favored over the primary amine.

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