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Crystal structure of A-485 bound to Δp300 HAT (a) Superposition of the Δp300 HAT-A-485 complex (green) with the inactive Δp300 HAT Y1467F mutant complexed with the bi-substrate inhibitor Lys-CoA (PDB ID: 3BIY,gray). (b) Zoomed view of the A-485 binding site. Key interacting residues are shown with hydrogen bonds indicated by red dashes (some residues are omitted for viewing clarity). The 2Fo-Fc electron density map calculated at 1.95Å for A-485 is contoured at 1σ. (c) Comparison of the Δp300 HAT-A-485 complex (green) with the Acetyl-CoA bound structure (PDB ID: 4PZS) (gray) illustrates how a subtle shift in helix 3 accommodates the fluorophenyl ring in a hydrophobic pocket. (d) A-485 is a competitive acetyl-CoA-site p300 inhibitor. The IC 50 for A-485 were calculated as per Fig. 1b. Error bars represent S.D. of 3 independent technical replicates (source data are provided).
Source publication
The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the developmen...
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... delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are ...
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... internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent ...
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... inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a ...
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... are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As ...
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... ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive ...
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... definitively establish the binding mode and delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As shown in Extended Data Fig. 4a, the specificity of A-485 likely stems primarily from the L1 loop which is absent in other HATs. Superposition of the HAT domains of hPCAF, hMYST3, hHAT1, hTIP60 and hGCN5 with A-485 reveals that each would be predicted to clash with A-485, making it unlikely that other HAT family members would be able to make the necessary structural accommodations to provide potent binding. For example, the indane ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive catalytic inhibitor of ...
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... definitively establish the binding mode and delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As shown in Extended Data Fig. 4a, the specificity of A-485 likely stems primarily from the L1 loop which is absent in other HATs. Superposition of the HAT domains of hPCAF, hMYST3, hHAT1, hTIP60 and hGCN5 with A-485 reveals that each would be predicted to clash with A-485, making it unlikely that other HAT family members would be able to make the necessary structural accommodations to provide potent binding. For example, the indane ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive catalytic inhibitor of ...
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... definitively establish the binding mode and delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As shown in Extended Data Fig. 4a, the specificity of A-485 likely stems primarily from the L1 loop which is absent in other HATs. Superposition of the HAT domains of hPCAF, hMYST3, hHAT1, hTIP60 and hGCN5 with A-485 reveals that each would be predicted to clash with A-485, making it unlikely that other HAT family members would be able to make the necessary structural accommodations to provide potent binding. For example, the indane ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive catalytic inhibitor of ...
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... definitively establish the binding mode and delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As shown in Extended Data Fig. 4a, the specificity of A-485 likely stems primarily from the L1 loop which is absent in other HATs. Superposition of the HAT domains of hPCAF, hMYST3, hHAT1, hTIP60 and hGCN5 with A-485 reveals that each would be predicted to clash with A-485, making it unlikely that other HAT family members would be able to make the necessary structural accommodations to provide potent binding. For example, the indane ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive catalytic inhibitor of ...
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... definitively establish the binding mode and delineate specific interactions of A-485 with p300, we determined the X-ray crystal structure of the fully active human p300 HAT domain (1287 to 1666 with auto-inhibitory internal loop deletion of amino acids 1523-1554 plus K1637R and M1652G mutations (Δp300 HAT), in complex with A-485 at 1.95Å resolution (data collection shown in Extended Data Fig. 2). Comparison with structures of the inactive Δp300 HAT Y1467F mutant in complex with acetyl-CoA 9 (Extended Data Fig. 1c) or the bi-substrate inhibitor Lys-CoA 10 (Fig. 2a) confirms that the A-485 binding site overlaps with that for acetyl-CoA, but not the peptide substrate binding site. Additionally, two molecules of Δp300 HAT are oriented such that Lys-1558 of one molecule inserts into the lysine substrate tunnel of a symmetry related molecule (Extended Data Fig. 3), highlighting the inherent accessibility of the inhibitor-bound p300 peptide site for lysine presented in the KXXK sequence motif 10,11 . The methyl-urea of A-485 is inserted through the L1 loop where it makes two equivalent hydrogen bonds to Gln-1455 (Fig. 2b). Two other notable hydrogen bonds are made between the 4' carbonyl of the oxazolidinedione to Ser-1400 and from the amide carbonyl to a coordinated water molecule. The majority of the remaining molecular interactions are hydrophobic in nature including the fluorophenyl ring which sits in a hydrophobic pocket that expands by means of a shift in helix 3 to accommodate its size (Fig. 2c). Consistent with its lack of activity, the urea moiety of A-486 would be expected to clash with Gln-1455 and Lys-1456 on the side of the L1 loop. Since substantial sequence divergence between HATs has been reported 12 , we further utilized the structure to gain insight into the selectivity of A-485 for p300/CBP over other HATs. As shown in Extended Data Fig. 4a, the specificity of A-485 likely stems primarily from the L1 loop which is absent in other HATs. Superposition of the HAT domains of hPCAF, hMYST3, hHAT1, hTIP60 and hGCN5 with A-485 reveals that each would be predicted to clash with A-485, making it unlikely that other HAT family members would be able to make the necessary structural accommodations to provide potent binding. For example, the indane ring of A-485 would interfere with the alpha backbone of the motif A helix of PCAF (Extended Data Fig. 4b). These structural superpositions are consistent with the HAT inhibitor specificity that we observe for A-485 (Supplementary Table 3). The activity assay IC 50 for inhibition increased linearly with increasing concentrations of acetyl-CoA ( Fig. 2d) confirming acetyl-CoA competitive inhibition. An AlphaLISA-based peptide substrate binding assay demonstrated that unlabeled peptide but not A-485 competed with binding, confirming that A-485 is not competitive with the peptide substrate (Extended Data Fig. 4c). Together, these results indicate that A-485 is an acetyl-CoA competitive catalytic inhibitor of ...
Citations
... Based on the observation that EP300 is part of ETO2 complexes, we next investigated the molecular consequences of HAT inhibition on ETO2 activity using a CBP/EP300 catalytic inhibitor (A485) or a PROTAC-mediated selective degradation of EP300/CBP (dCBP-1). 39,40 Both A485 and dCBP1 strongly reduced ETO2 and MYB protein expression ( Figure 7C). Notably, while dCBP-1 showed the strongest effect and was associated with the expected loss of EP300 expression, short-term treatment with A485 (6-12 h) showed a significantly decreased ETO2 expression without affecting EP300 or MYB. ...
Transcriptional cofactors of the ETO family are recurrent fusion partners in acute leukemia. We characterized the ETO2 regulome by integrating transcriptomic and chromatin binding analyses in human erythroleukemia xenografts and controlled ETO2 depletion models. We demonstrate that beyond its well‐established repressive activity, ETO2 directly activates transcription of MYB, among other genes. The ETO2‐activated signature is associated with a poorer prognosis in erythroleukemia but also in other acute myeloid and lymphoid leukemia subtypes. Mechanistically, ETO2 colocalizes with EP300 and MYB at enhancers supporting the existence of an ETO2/MYB feedforward transcription activation loop (e.g., on MYB itself). Both small‐molecule and PROTAC‐mediated inhibition of EP300 acetyltransferases strongly reduced ETO2 protein, chromatin binding, and ETO2‐activated transcripts. Taken together, our data show that ETO2 positively enforces a leukemia maintenance program that is mediated in part by the MYB transcription factor and that relies on acetyltransferase cofactors to stabilize ETO2 scaffolding activity.
... Furthermore, we demonstrated that the p300-PRMT1 interaction resulted in acetylation of PRMT1 through the increase of acetyl-lysine signal on an immunoprecipitated FLAG-PRMT1 following co-expression of Myc-p300 and FLAG-PRMT1. Further, this acetylation can be abrogated by inhibition of p300 acetyltransferase activity using A-485 (Lasko et al., 2017) (Fig. 1D). Having demonstrated p300-catalyzed acetylation of PRMT1, we performed a detailed characterization of the acetylation and ubiquitination sites of PRMT1 (Fig. 1E) in order to build a more complete picture on the impact of lysine acetylation on PRMT1 homeostasis. ...
PRMT1 plays many important roles in both normal and disease biology, thus understanding it’s regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.
... To test the hypothesis that P300-mediated acetylation modulates endothelial mechanotransduction of hemodynamic stresses, we treated human umbilical vein endothelial cells (HUVEC) with A-485, a small molecule catalytic inhibitor of the acetyltransferase activity of P300 [24]. In a study comparing the acetylomes of P300 knockout cells and A-485 treated cells, 93% similarity was observed, demonstrating that A-485 effectively reduces the acetyltransferase activity of P300 [19]. ...
... We evaluated the role of P300 in endothelial cell adaptation to flow using a small molecule catalytic inhibitor, A-485 [24], and siRNA-mediated knockdown. Recent studies of endothelial cells exposed to fluid shear stress have revealed significant alterations to H3K27ac signatures at enhancer elements [8]. ...
... Acetylation of histone tails is typically associated with weakened interactions between histones and DNA, which can alter chromatin compaction and therefore can cause changes in nuclear stiffness. In the publication that originally described A-485, the authors observed that inhibition of P300 via A-485 does not drive global reductions in histone acetylation; rather, depletion of acetylation was largely specific to the H3K27 residue [24]. For instance, H3K9 acetylation, another acetylation mark deposited by P300, was relatively stable in the presence of A-485 [24], which we also observed (Fig. S2). ...
P300 is a lysine acetyltransferase that plays a significant role in regulating transcription and the nuclear acetylome. While P300 has been shown to be required for the transcription of certain early flow responsive genes, relatively little is known about its role in the endothelial response to hemodynamic fluid stress. Here we sought to define the role of P300 in mechanotransduction of fluid shear stress in the vascular endothelium.
To characterize cellular mechanotransduction and physical properties after perturbation of P300, we performed bulk RNA sequencing, confocal and Brillouin microscopy, and functional assays on HUVEC.
Inhibition of P300 in HUVEC triggers a hyper-alignment phenotype, with cells aligning to flow sooner and more uniformly in the presence of the P300 inhibitor A-485 compared to load controls. Bulk transcriptomics revealed differential expression of genes related to the actin cytoskeleton and migration in cells exposed to A-485. Scratch wound and bead sprouting assays demonstrated that treatment with A-485 increased 2D and 3D migration of HUVEC. Closer examination of filamentous actin revealed the presence of a perinuclear actin cap in both P300 knockdown HUVEC and HUVEC treated with A-485. Interrogation of cell mechanical properties via Brillouin microscopy demonstrated that HUVEC treated with A-485 had lower Brillouin shifts in both the cell body and the nucleus, suggesting that P300 inhibition triggers an increase in cellular and nuclear compliance.
Together, these results point to a novel role of P300 in modulating endothelial cell mechanics and mechanotransduction of hemodynamic shear stress.
... Therefore, development of small-molecule inhibitors of CBP/p300 is an active area of drug discovery for diverse human diseases, including cancer. The HAT inhibitor A-485 and the BRD inhibitor inobrodib (CCS1477) are potent and promising inhibitors of CBP/p300 14,15 . The CBP and p300 proteins are a paralog pair that share high sequence homology and functional similarity. ...
... The CBP and p300 proteins are a paralog pair that share high sequence homology and functional similarity. Basically, existing inhibitors of CBP/p300, including A-485 and inobrodib, selectively inhibit the function of CBP and p300 simultaneously 14,15 . ...
... Drug susceptibility screening using these inhibitors revealed that the CBP/p300 inhibitor A-485 was the most selective for SMARCB1deficient cancers ( Supplementary Fig. 1m). A-485 acts as a dual inhibitor of CBP and p300 by targeting the HAT domain (Fig. 1h) 14 . ...
SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the “simultaneous inhibition of a paralog pair” concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.
... In addition, we have investigated the combination of HAT catalytic inhibitors and AURKA inhibitors. A485, a catalytic inhibitor targeting both CREBBP/EP300 with enhanced potency and selectivity [20], showed significant synergistic effect in conjunction with AURKA inhibitors ( Supplementary Fig. 4A-D). However, another CREBBP/ EP300 catalytic inhibitor C646, failed to show synergistic effect with AURKA inhibitors (Supplementary Fig. 4E, F) due to its low inhibitory effect on H3K27ac. ...
... However, the combination of CREBBP/EP300 inhibitor C646 and AURKA inhibitors shows no synergistic effect. The cell-free dissociation constants (Kds) of C646 (400 nM) [40] is considerably higher than those of SGC-CBP30 (CREBBP: 21 nM; EP300: 32 nM) [41] and A485 (CREBBP: 2.6 nM; EP300: 9.8 nM) [20], which results Fig. 4 MYC target genes were deregulated with the treatment of SGC-CBP30 and alisertib. A Volcano plot showing differentiated genes with the combined treatment of SGC-CBP30 (10 μM) and alisertib (100 nM) for 24 h. ...
Loss-of-function mutations in CREBBP, which encodes for a histone acetyltransferase, occur frequently in B-cell malignancies, highlighting CREBBP deficiency as an attractive therapeutic target. Using established isogenic cell models, we demonstrated that CREBBP-deficient cells are selectively vulnerable to AURKA inhibition. Mechanistically, we found that co-targeting CREBBP and AURKA suppressed MYC transcriptionally and post-translationally to induce replication stress and apoptosis. Inhibition of AURKA dramatically decreased MYC protein level in CREBBP-deficient cells, implying a dependency on AURKA to sustain MYC stability. Furthermore, in vivo studies showed that pharmacological inhibition of AURKA was efficacious in delaying tumor progression in CREBBP-deficient cells and was synergistic with CREBBP inhibitors in CREBBP-proficient cells. Our study sheds light on a novel synthetic lethal interaction between CREBBP and AURKA, indicating that targeting AURKA represents a potential therapeutic strategy for high-risk B-cell malignancies harboring CREBBP inactivating mutations.
... From a small molecule perspective, significant efforts have led to the development of potent and selective inhibitors of the EP300 and CREBBP HAT domain. 7,8 One of the most potent of these compounds is CPI-1612. 9,10 In biochemical assays CPI-1612 disrupts EP300 and CREBBP catalytic activity at half-maximal inhibitor concentrations of <0.5 nM and 2.9 nM, respectively (Table S1). ...
The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit non-redundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the non-catalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.
... EP300 and its homologue CREBBP encode highly related acetyltransferases that act as transcriptional coactivators in multiple signaling pathways [11,12], suggesting the potential involvement of EP300/CREBBP in cancers [13]. Notably, EP300/ CREBBP participate in transcriptional regulation and chromatin modifications for the establishment of transcriptional regulation system, with a major role in maintaining normal hematopoiesis as well as promoting leukemogenesis [14,15]. ...
... EP300/CREBBP inhibitor exhibited high potential in MLL-r AML Our KO results identified EP300/CREBBP as a potential therapeutic target in MLL-r AML. A recent study reported A-485, a new inhibitor selectively targeting histone acetyltransferase (HAT) domain of EP300/CREBBP, showed antitumor effects in several hematologic malignancies and prostate cancer [13]. We subsequently investigated the therapeutic potential of A-485 in MLL-r AML cells. ...
Acute myeloid leukemia (AML) with mixed-lineage leukemia ( MLL ) gene rearrangements (MLL-r) is an aggressive subtype of blood cancer with dismal prognosis, underscoring the urgent need for novel therapeutic strategies. E1A-binding protein (EP300) and CREB-binding protein (CREBBP) function as essential transcriptional coactivators and acetyltransferases, governing leukemogenesis through diverse mechanisms. Targeting EP300/CREBBP holds great promise for treating leukemia with some certain cytogenetic abnormalities. Here, we demonstrated that EP300 and CREBBP are core epigenetic regulators in the pathogenesis of MLL-r AML through assaying the transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Knocking-out EP300/CREBBP and inhibitor (A-485) treatment depressed the MLL-r cells proliferation, while the MLL wild-type cells remained uninfluenced. We found that the CDK4/RB/E2F axis was downregulated specifically in MLL-r AML cell after A-485 treatment by RNA-seq, western blot and cut-tag analyses. EP300/CREBBP inhibitor selectively exerted potent anti-leukemia activity through blocking the MLL-r-BET complex binding to H3K27Ac modification on critical genes loci, distinct from global histone acetylation. Collectively, our study identified EP300/CREBBP as a critical epigenetic driver of MLL-r leukemia and validated their therapeutic potential through targeting inhibition, offering a promising avenue for improving clinical outcomes in this aggressive leukemia.
... The imbalance of H3K27ac modification created by HDAC exclusion may be corrected by CBP/p300 inhibition. We tested this hypothesis with the compound A-485, a highly selective and drug-like CBP/p300 catalytic inhibitor [24], to decrease the over-deposited H3K27ac modification in SS18-SSX condensates (Supplementary Fig. 13a). We show that A-485 dramatically impede the activation of SS18-SSX downstream genes (Fig. 5a). ...
Nuclear condensates have been shown to regulate cell fate control, but its role in oncogenic transformation remains largely unknown. Here we show acquisition of oncogenic potential by nuclear condensate remodeling. The proto-oncogene SS18 and its oncogenic fusion SS18-SSX1 can both form condensates, but with drastically different properties and impact on 3D genome architecture. The oncogenic condensates, not wild type ones, readily exclude HDAC1 and 2 complexes, thus, allowing aberrant accumulation of H3K27ac on chromatin loci, leading to oncogenic expression of key target genes. These results provide the first case for condensate remodeling as a transforming event to generate oncogene and such condensates can be targeted for therapy.
One sentence summary: Expulsion of HDACs complexes leads to oncogenic transformation.
... Evidence from knockout studies in the mouse [12][13][14][15] , and studies in cancer cells 5,[16][17][18] have implicated these proteins as critical to the development of normal tissues and indeed, as potential targets for therapeutic development in disease states. As a result, significant efforts by many have led to a host of small molecule inhibitors and degraders useful to interrogate disease biology driven by EP300/CBP 5,10,[19][20][21][22][23][24][25][26][27] . ...
... This implies that a broad-based search for tumor types with exceptional responses to domain-specific EP300/CBP-targeted therapeutics would be a high-yield approach to identifying tumors for specific inhibition. Given observed on-target toxicities associated with inhibition of both EP300 and CBP, therefore, this approach would be predicted to maximize a potential therapeutic window for clinical translation 5,22 . ...
... Cancer cell lines are broadly sensitive to EP300/CBP inhibitors Several small molecule inhibitors of distinct protein domains of EP300/CBP are available, though the majority target the histone acetyltransferase domain (HAT) or bromodomain (BRD) 5,10,21,22 . Recently, nearly equipotent inhibitors of each of the HAT and BRD have been developed: the spirooxazolidinedione A485 21,22 and the dimethylisoxazol-benzimidazole CCS1477 5 . ...
Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.
... In addition to the androgen receptor, other transcription factors are involved in regulating various cancer properties associated with prostate tumorigenesis. Among these, the cyclic AMP response element (CRE)-binding protein (CREB), the SRY-box transcription factors (SOX), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) have been characterized as regulators of gene expression influencing prostate cancer progressions [16][17][18]. ...
Although much less common than anthocyanins, 3-Deoxyanthocyanidins (3-DAs) and their glucosides can be found in cereals such as red sorghum. It is speculated that their bioavailability is higher than that of anthocyanins. Thus far, little is known regarding the therapeutic effects of 3-DAs and their O-β-D-glucosides on cancer, including prostate cancer. Thus, we evaluated their potential to decrease cell viability, to modulate the activity of transcription factors such as NFκB, CREB, and SOX, and to regulate the expression of the gene CDH1, encoding E-Cadherin. We found that 4′,7-dihydroxyflavylium chloride (P7) and the natural apigeninidin can reduce cell viability, whereas 4′,7-dihydroxyflavylium chloride (P7) and 4′-hydroxy-7-O-β-D-glucopyranosyloxyflavylium chloride (P3) increase the activities of NFkB, CREB, and SOX transcription factors, leading to the upregulation of CDH1 promoter activity in PC-3 prostate cancer cells. Thus, these compounds may contribute to the inhibition of the epithelial-to-mesenchymal transition in cancer cells and prevent the metastatic activity of more aggressive forms of androgen-resistant prostate cancer.
