Substrate-dependent inhibition of purified p300 HAT
Biochemical assay · Purified semisynthetic p300 histone acetyltransferase domain with H4-15 peptide and acetyl-CoA substrates [1]
Direct radiometric assays supported competitive inhibition with respect to acetyl-CoA and noncompetitive inhibition with respect to H4-15 peptide. These are purified-enzyme kinetic findings; they do not establish a unique cellular target.
| Endpoint | Reported result | Reference |
|---|---|---|
| p300 inhibition with acetyl-CoA varied and H4-15 fixed at 100 μM | Ki 400 ± 60 nM | [1] |
| p300 inhibition with H4-15 peptide varied and acetyl-CoA fixed at 10 μM | Ki 530 ± 40 nM | [1] |
Literature-Reported Treatment Conditions
- Treatment concentration
- Three inhibitor conditions: 0, 0.5×IC50 and IC50; exact molar inhibitor concentrations for these kinetic series are not supplied in the retrieved method text.
- Exposure duration
- Enzyme concentration and reaction time were varied to keep substrate turnover below 10%; no single duration assigned to all kinetic series.
- Method
- Radiolabeled acetyl transfer to H4-15, electrophoretic separation and phosphorimage quantification; acetyl-CoA varied from 5–120 μM or peptide from 25–500 μM. Assays performed in duplicate.
- Vehicle
- DMSO, held constant (<5%) in the referenced direct-assay procedure.
- Reported limits
- Meaning of caption ± values and number of independent enzyme preparations not specified in the read locations. Figure images and SI not retrieved; no curve-derived values or SI selectivity results adopted.
Methods and source locations
Compound reported in the paper: C646; Compound label in the paper: ChemBridge #5838646
Applies to the reported C646 preparation at parent-compound level, not an isolated stereoisomer, salt conversion, or validation of an Adooq lot.
Results: Mechanism of p300 inhibition; Figure 1B–C caption; Experimental Procedures: Kinetic characterization of verified inhibitors and analogs (BioC passages 15, 57–58, 77)
p300 inhibition with acetyl-CoA varied and H4-15 fixed at 100 μM: Direct 14C-acetyl transfer assay; global fitting of substrate-dependent initial rates. Competitive pattern versus acetyl-CoA. The Figure 1 caption does not define the meaning of ±. — Figure 1B caption; Results: Mechanism of p300 inhibition; Experimental Procedures: Kinetic characterization (passages 15, 58, 77)
p300 inhibition with H4-15 peptide varied and acetyl-CoA fixed at 10 μM: Direct 14C-acetyl transfer assay; noncompetitive pattern versus H4-15. Figure 1 caption reports ± without defining its meaning. — Figure 1C caption; Experimental Procedures: Kinetic characterization (passages 58, 77)