The overall goal of this method is to determine the HDAC inhibitory potential of compounds and to identify selectivity towards HDAC6 or HDAC1 in cells using a single ultra high performance liquid chromatography mass spectrometry assay. This method can help answer key questions in the drug discovery field such as determining whether an HDAC inhibitor is selective for HDAC6 over Class I HDACs in living cells. The main advantage of this technique is that it requires a single incubation step with cells, inhibitors, and selective substrates in order to determine potency against two HDAC isoforms simultaneously.
The implication of this technique extend toward early drug discovery of selective HDAC inhibitors because it detects whether HDAC6 selectivity is maintained in living cells avoiding endogenous protein acetylation assays. Though this method assesses selectivity of HDAC inhibitors in HeLa cells, it can also be applied to other cell lines such as primary and other tumor cells including clinical specimens. Culture HeLa cells to 80%confluency in a T75 cell culture flask in minimal essential media supplemented with 10%fetal bovine serum, penicillin G, and streptomycin.
Wash the cells twice with five milliliters of Dulbecco's Phosphate-Buffered Saline. Then, aspirate the buffer and add one milliliter of cell dissociation reagent. Incubate at 37 degrees Celsius for five minutes to detach the cells.
After five minutes, add nine milliliters of MEM culture medium to the cells and thoroughly resuspend the cell suspension. Transfer the cells to a new 15 milliliter tube and then count the cells using a hemocytometer. Adjust the density of the cell suspension to 60, 000 cells per milliliter in MEM.
Next, add 100 microliters of the cell suspension to the control and test wells of a sterile, flat bottom, tissue culture treated 96 well plate. Then, add 100 microliters of MEM culture medium to the blank wells of the 96 well plate. Incubate the 96 well plate at 37 degrees Celsius for 24 hours.
Aspirate the medium of the wells of the 96 well plate. While aspirating the medium, avoid detaching and removing cells from the well. This can be achieved by using a low aspiration rate and carefully placing the aspirating tips at the bottom corner of the well surface.
Then, add 25 microliters of the two times test compounds in MEM to the test wells, including Trichostatin A, MS275, and Tubastatin A.Add 25 microliters of MEM to the control and blank wells. Next, add 25 microliters of the substrate mixture to the control and test wells. Then, add 25 microliters of MEM to the blank wells.
Incubate the 96 well plate at 37 degrees Celsius for eight hours in a humidified 5%carbon dioxide incubator. To perform cell lysis, first add 10 microliters of six times RIPA buffer supplemented with protease inhibitor to each well of the 96 well plate. Stop the reaction by adding 160 microliters of cold acetonitrile to each well and mix by pipetting up and down.
Place the plate in a minus 80 degrees Celsius freezer for 10 minutes. Remove the plate from the freezer and transfer 220 microliters of the content of each well to a non-sterile, conical bottom 96 well plate. Centrifuge the plate at 5, 000 times g and four degrees Celsius for 10 minutes.
Following centrifugation, transfer 200 microliters of supernatant into a 96 well plate compatible with the UHPLC system. Then, seal the plate with peelable heat-sealing foil using a plate sealer. Prepare the ultra high performance liquid chromatography mass spectrometry system by filling it with the mobile phase system.
Place the 96 well plate into a sample manager containing a plate holder. Run the samples according to the UHPLC ESI MS/MS conditions found in the text. To perform data analysis, integrate the peak area of each substrate and its deacetylated product with the appropriate UHPLC integration software.
For each substrate, calculate the peak area ratio of deacetylated to acetylated in each chromatogram. Include the test and control samples. Finally, calculate the percent HDAC inhibition of each test sample according to the equation in the text protocol.
The UHPLC-MS chromatogram of a sample is presented here. The peaks corresponding to the substrates added to the cells are detected according to their mass transitions as described in table one of the written protocol. MOCPAC is deacetylated by HDAC1 to provide the deacetylated MOCPAC peak.
Similarly, BATCP is deacetylated by HDAC6 to provide the deacetylated BATCP peak. Finally, MAL provides the deacetylated MAL peak which reflects the deacetylation of several endogenous HDACs unspecifically. Shown here, the figure with the chromatogram obtained with different concentrations of the HDAC inhibitor Trichostatin A.A dose response evaluation of standard HDAC inhibitors was performed.
The integrated values obtained from MOCPAC and dMOCPAC were used to calculate the potency of standard inhibitors against HDAC1. Following IC 50 calculations, MS275, a selective Class I HDAC inhibitor, showed lower IC 50 values for HDAC1 than Tubastatin A, a known HDAC6 inhibitor. On the other hand, the isolated values from BATCP and dBATCP were used to calculate the potency of the standards against HDAC6.
This time, Tubastatin A provided a lower IC 50 value than MS275, confirming their isoform selectivity profile. Trichostatin A is a potent, nonselective inhibitor. Once mastered, this technique can be done in 72 hours if it's performed properly including the cell treatment time and the analysis of the 96 well plate.
While attempting this procedure, it's important to remember that the incubation times indicated here are for HeLa cells. If another cell line is used, this needs to be optimized. Following this procedure, selective HDAC inhibitory activity of other isoforms could be measured as long as specific substrates exist.
Don't forget that working with organic solvents and other chemicals can be extremely hazardous and protection glasses, gloves, and a lab coat should always be worn while performing this procedure.
