Protein concentrations were determined using the Bradford assay
Protein concentrations were determined using the Bradford assay. between 50100 uM for p300 . In our earlier characterization having a different assay, the IC50 of Ketorolac 16 (AA) is about 1000 M for PCAF . The last Ketorolac finding is definitely well in line with our current finding that 16 (AA) inhibits about 20% of the HAT activity of p300 and PCAF at 200 M. The reported variations demonstrate the inhibitory potency of 16 (AA) depends strongly within the assay conditions and the enzyme resource. Of interest, some of the 16 (AA) analogs showed activation Ketorolac of the acetyltransferase activity of p300 and PCAF in contrast with the observed inhibitory effect on Tip60. Under the applied assay conditions, 21 and 24 enhance the PCAF activity significantly at a concentration of 200 M. Our observations are not only: Ketorolac the activation of HAT activity by several AA analogs was also observed previously.[7,31,32,33] However, the exact mechanism, the concentration dependence and time dependence of the activation remain to be investigated in order to validate small molecule HAT activation as a relevant trend. Among the effective Tip60 inhibitors, you will find three salicylates having a phenethyl substitution in the 6-position, 6a, 6c and 20. These inhibitors have determined logD ideals at pH 7.4 of respectively 4.53, 3.07 and 4.19, which are lower than the calculated value 5.21 for 16 (AA) (determined using MarvinSketch 5.4). The high logD value for AA is generally considered to be a disadvantage for its bioactivity  and therefore the inhibitors with the 6-phenethyl salicylate type can be considered improved prospects for development of Tip60 inhibitors. Inhibitor 20 inhibited the Tip60 activity by 88% at 200 M and experienced no observable inhibitory effect on p300 and PCAF, in contrast to AA that showed about 20% inhibition of these enzymes at 200 M. We consequently selected this inhibitor for further evaluation in order to understand the potency and selectivity of this AA analog for HAT inhibition. We identified the IC50 value for inhibition of the HAT activity of Tip60 and MOF for 20 and compared that with 16 (AA). MOF is definitely another MYST family HAT that is relevant in eukaryotic gene transcription. The IC50 was identified as the concentration of the inhibitor at which half of the enzyme activity was inhibited. The results are demonstrated in Table 2. Under these experimental conditions, the IC50s of 16 (AA) and 20 are 64 M and 74 M for Tip60, and 43 M and 47 M for MOF. In contrast, IC50s of both 16 (AA) and 20 are higher than 200 M for p300 and PCAF (the exact IC50 values cannot be determined due to compound insolubility at high PI4K2A concentrations). These data show that 16 (AA) and the additional tested analogs tend to become specific inhibitors for the MYST family HATs. This is exemplified by 20, which at 200 M inhibited about 90% of Tip60 activity but experienced no inhibitory impact on p300 and PCAF (Number 1). The similar inhibitory potency for the MYST family HATs Tip60 and MOF can be explained from the 67% sequence similarity in their catalytic region (Blast sequence alignment). Table 2 IC50 data of AA and 20 for the inhibition of HATs is the maximal velocity (in the absence of inhibitor), [S] is the concentration of the varied substrate, versus 1/[Ac-CoA] a series of right lines intersect at a point very close to the ordinate, which further helps the competitive inhibition mode. The measurable versus 1/[H4-20] is clearly situated to the left of the ordinate. The kinetic data fitted yielded BL21(DE3). The His-tagged proteins were purified on Ni-NTA beads. Protein concentrations were identified using the Bradford assay. Proteins were flash freezing in a storage buffer comprising 25 mM HEPES pH=7.0, 500 mM NaCl, 10 mM.