This work was supported by grants from James and Esther King Biomedical Research Program (6JK03 and 20K07), and Bankhead-Coley Cancer Research Program (4BF02 and 6BC03), Florida Department of Health, Florida Breast Cancer Foundation, and UF Health Cancer Center. Additionally, we would like to thank Dr. Zachary Osking and Dr. Andrea Lin for their support during the publication process.

The authors have no conflicts of interest or disclosures to make.

Lysine acetyltransferases (KATs) acetylate several lysine residues on histone tails and transcription factors to regulate gene transcription2,3. Work in the last two decades has revealed that KATs, such as CBP/p300, PCAF and GCN5, interact with oncogenic transcription factors and help drive tumor growth in several solid tumor types4,5,9,15</s...

Lysine acetyltransferases (KATs) catalyze the acetylation of lysine residues on both histone and non-histone proteins1,2,3,4. Recent research reveals that KATs and their acetyltransferase function can promote solid tumor growth4,5,6,7,8,9. For example, CREB-binding protein (CBP)/p300 are two paralogous KATs that regulate numerous signaling pathways in cancer2,3. CBP/p300 have a well characterized histone acetyltransferase (HAT) function and catalyze Histone 3 Lysine 27 acetylation (H3K27ac)2,4,5,10,11, an important marker for active enhancers, promoter regions and active gene transcription12,13,14. CBP/p300 serve as critical co-activators for pro-growth signaling pathways in solid tumors by activating transcription of oncogenes through acetylation of histones and other transcription factors4,9,15,16,17,18. Due to their role in tumor progression, CBP/p300 and other KATs are under investigation for the development of novel inhibitors that block their oncogenic function4,5,6,7,8,9,18,19,20. A-485 and GNE-049 represent two successful attempts to develop potent and specific inhibitors for CBP/p3004,9. Additional inhibitors are currently under investigation for CBP/p300 and other KATs.
The quality of previously described KAT inhibitors (KATi) is being called into question, with many inhibitors showing off target effects and poor characterization21. Therefore, rigorous characterization and validation of novel drug candidates is essential for the development of high-quality chemical probes. Outlined here are three protocols that form a pipeline for screening and rigorously validating the potency and specificity of novel KATi, with a specific focus on inhibiting the HAT function (HATi) of KATs. CBP/p300 and their inhibitors are used as examples, but these protocols can be adapted for other KATs that have a HAT function7.
The first protocol is an in vitro histone acetyltransferase (HAT) assay that utilizes purified recombinant p300 and histones in a controlled test tube reaction. This assay is simple to perform, is cost-effective, can be used to screen compounds in a low throughput setting, and does not require radioactive materials. In this protocol, recombinant p300 catalyzes lysine acetylation on histone tails during a brief incubation period and the levels of histone acetylation are measured using standard immunoblotting procedures. The enzymatic reaction can be performed in the presence or absence of CBP/p300 inhibitors to screen for compounds that reduce histone acetylation. Additionally, the HAT assay can be used to verify whether novel compounds are selective for CBP/p300 by assessing their activity against other purified KATs, such as PCAF. The HAT assay is an excellent starting point for investigating novel inhibitors due to its simplicity, low cost, and the ability to determine the potency/selectivity of an inhibitor. Indeed, this protocol is often used in the literature as an in vitro screen5,10. However, inhibitors identified in the HAT assay are not always effective in cell culture because a test tube reaction is much simpler than a living cell system. Therefore, it is essential to further characterize inhibitors in cell culture experiments22,23.
The second protocol in the pipeline is the Chromatin Hyperacetylation Inhibition (ChHAI) assay. This cell based assay utilizes histone deacetylase inhibitors (HDACi) as a tool to hyperacetylate histones in chromatin before co-incubation with a HATi24. Basal histone acetylation can be low in cell culture, making it difficult to probe for via immunoblotting without the addition of an HDACi to increase acetylation. The purpose of the ChHAI assay is to identify novel HATi that can attenuate the increase in histone acetylation caused by HDAC inhibition. The advantages of this assay include its low cost, relative ease to perform, and the use of cells in culture, which provides more physiological relevance than the test tube HAT assay. Similar to the HAT assay, this protocol uses standard immunoblotting for data collection.
The HAT and ChHAI assays provide data about the potency of novel compounds for inhibiting global histone acetylation, but do not provide insight into how these compounds affect modifications at specific genomic regions. Therefore, the final protocol, Chromatin Immunoprecipitation-quantitative Polymerase Chain Reaction (ChIP-qPCR) is a cell culture experiment that investigates DNA-protein interactions at specific regions of the genome. In the ChIP protocol, chromatin is crosslinked to preserve DNA-protein interactions. The chromatin is then extracted from cells and the DNA-protein complex undergoes selective immunoprecipitation for the protein of interest (e.g., using an antibody specific for H3K27ac). The DNA is then purified and analyzed using qPCR. For example, ChIP-qPCR can be used to determine if a novel HATi downregulates histone acetylation at individual oncogenes, such as Cyclin D125. While ChIP-qPCR is a common technique used in the field, it can be difficult to optimize4,10,26. This protocol provides tips for avoiding potential pitfalls that can occur while performing the ChIP-qPCR procedure and includes quality control checks that should be performed on the data.
When used together, these three protocols allow for the rigorous characterization and validation of novel HATi. Additionally, these methods offer many advantages because they are easy to perform, relatively cheap and provide data on global as well as regional histone acetylation.

<table>
	<tbody>
		<tr>
			<td>1.5 ml tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td>For all methods</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Sarstedt AG &#38; Co.</td>
			<td>83.3902</td>
			<td>For cell culture of MCF-7 cells</td>
		</tr>
		<tr>
			<td>10 ul tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-454</td>
			<td>For all Methods</td>
		</tr>
		<tr>
			<td>1000 ul tips</td>
			<td>Corning</td>
			<td>4846</td>
			<td>For all Methods</td>
		</tr>
		<tr>
			<td>10X Glycine buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>10X Running Buffer</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>10X TBST</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>12 well plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td>For Method 2</td>
		</tr>
		<tr>
			<td>15 cm dish</td>
			<td>Sarstedt AG &#38; Co.</td>
			<td>83.3903</td>
			<td>For Method 3</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-200249</td>
			<td>For Methods 2 and 3</td>
		</tr>
		<tr>
			<td>1X TBST with 5% milk and 0.02% Sodium Azide</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. Can be used to dilute primary antibodies that will be used more than once. Allows for short-term storage of primary antibody dilutions. Do not use for secondary antibody diluton. CAUTION: Sodium Azide is toxic.</td>
		</tr>
		<tr>
			<td>1X TBST with 5% milk</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. Used to block PVDF membrane and for antibody diltions. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>200 ul tips</td>
			<td>Corning</td>
			<td>4844</td>
			<td>For all Methods</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>for SDS sample buffer preparation</td>
		</tr>
		<tr>
			<td>4-20% polyacrylamide gel</td>
			<td>Thermo Fisher: Invitrogen</td>
			<td>XP04205BOX</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>5X Assay buffer</td>
			<td></td>
			<td></td>
			<td>For Method 1. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>5X Passive lysis buffer</td>
			<td></td>
			<td></td>
			<td>For Method 2. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>6X Sodium Dodecyl Sulfate (SDS)</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>A-485</td>
			<td>MedChemExpress</td>
			<td>HY-107455</td>
			<td>CBP/p300 Inhbitor for use in Methods 2 and 3. Dissolved in DMSO.</td>
		</tr>
		<tr>
			<td>Acetyl-CBP(K1535)/p300(K1499) antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4771</td>
			<td>For Method 1</td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Sigma-Aldrich</td>
			<td>A2056</td>
			<td>for use in Method 1</td>
		</tr>
		<tr>
			<td>Acetyl-Histone H3 (Lys 27) antibody (H3K27ac)</td>
			<td>Cell Signaling Technology</td>
			<td>CST 8173</td>
			<td>antoibodies for H3K27ac for immunoblots and ChIP</td>
		</tr>
		<tr>
			<td>Acetyl-Histone H3 (Lys18) antibody (H3K18ac)</td>
			<td>Cell Signaling Technology</td>
			<td>CST 9675</td>
			<td>antoibodies for H3K18ac for immunoblots and ChIP</td>
		</tr>
		<tr>
			<td>alpha tubulin antibody</td>
			<td>Millipore Sigma</td>
			<td>T5168</td>
			<td>For Method 2. Dilute 1:20,000</td>
		</tr>
		<tr>
			<td>Anacardic acid</td>
			<td>Cayman Chemical</td>
			<td>13144</td>
			<td>For Method 1</td>
		</tr>
		<tr>
			<td>anti-mouse IgG HRP linked secondary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076</td>
			<td>For Methods 1 and 2. Dilute 1:10,000</td>
		</tr>
		<tr>
			<td>anti-rabbit IgG secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-035-152</td>
			<td>For Methods 1 and 2. Dilute 1:10,000 to 1:20,000</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>MIDSCI</td>
			<td>BX810</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Belly Dancer Rotating Platform</td>
			<td>Stovall Life Science Incorporated</td>
			<td>not available</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Bovine Calf Serum (BCS)</td>
			<td>HyClone</td>
			<td>SH30072.03</td>
			<td>cell culture media</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126</td>
			<td>for SDS sample buffer preparation</td>
		</tr>
		<tr>
			<td>CDTA</td>
			<td>Spectrum Chemical</td>
			<td>125572-95-4</td>
			<td>For buffer preparation</td>
		</tr>
		<tr>
			<td>cell scraper</td>
			<td>Millipore Sigma</td>
			<td>CLS3010</td>
			<td>For Method 3</td>
		</tr>
		<tr>
			<td>ChIP dilution buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>ChIP Elution Buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Complete DMEM for MCF-7 Cells</td>
			<td></td>
			<td></td>
			<td>For Methods 2 and 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Covaris 130 &#181;l microTUBE</td>
			<td>Covaris</td>
			<td>520045</td>
			<td>Sonication tube for use with Covaris S220 in Method 3</td>
		</tr>
		<tr>
			<td>Covaris S220 Focused-ultrasonicator</td>
			<td>Covaris</td>
			<td>S220</td>
			<td>DNA sonicator for use in Method 3</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>41639</td>
			<td>for drug dilution and vehicle control treatment</td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>43815</td>
			<td>for SDS sample buffer preparation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>cell culture media</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP120-1</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Example transfer tank and transfer apparatus</td>
			<td>Bio-rad</td>
			<td>1704070</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit</td>
			<td>Millipore Sigma</td>
			<td>17-10086</td>
			<td>For Method 3</td>
		</tr>
		<tr>
			<td>FK228 (Romidepsin)</td>
			<td>Cayman Chemical</td>
			<td>128517-07-7</td>
			<td>HDAC Inhibitor for use in Method 2</td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>for cell fixation</td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td>For buffer preparation</td>
		</tr>
		<tr>
			<td>glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>54457</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>High salt wash buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3</td>
		</tr>
		<tr>
			<td>IGEPAL (NP-40)</td>
			<td>Sigma-Aldrich</td>
			<td>I3021</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Immobilon Chemiluminescent HRP Substrate</td>
			<td>Millipore Sigma</td>
			<td>WBKLS0500</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sigma-Aldrich</td>
			<td>L9650</td>
			<td>For buffer preparation</td>
		</tr>
		<tr>
			<td>LiCl wash buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Low salt wash buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Magnetic Separator</td>
			<td>Promega</td>
			<td>Z5341</td>
			<td>For use in Method 3</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>494437</td>
			<td>For buffer preparation</td>
		</tr>
		<tr>
			<td>Mini gel tank</td>
			<td>Invitrogen</td>
			<td>A25977</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>MS-275 (Entinostat)</td>
			<td>Cayman Chemical</td>
			<td>209783-80-2</td>
			<td>HDAC Inhibitor for use in Method 2. Dissolved in DMSO.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td>for buffer preparation in Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG</td>
			<td>Bethyl Laboratories</td>
			<td>P120-101</td>
			<td>Control rabbit antibody for use in Method 3</td>
		</tr>
		<tr>
			<td>Nuclei swelling buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>PCR Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>For use in Method 3</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>cell culture media</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>For Methods 2 and 3</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>80635</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>powdered milk</td>
			<td>Nestle Carnation</td>
			<td></td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Power Pac 200 for western blot transfer</td>
			<td>Bio-rad</td>
			<td></td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Power Pac 3000 for SDS gel running</td>
			<td>Bio-rad</td>
			<td></td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Prestained Protein Ladder</td>
			<td>Thermo Fisher</td>
			<td>26616</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>PI8340</td>
			<td>for use in Method 3</td>
		</tr>
		<tr>
			<td>Protein A Magentic Beads</td>
			<td>New England BioLabs</td>
			<td>S1425S</td>
			<td>For use in Method 3</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>New England BioLabs</td>
			<td>P8107S</td>
			<td>For use in Method 3</td>
		</tr>
		<tr>
			<td>PTC-100 Programmable Thermal Controller</td>
			<td>MJ Research Inc.</td>
			<td>PTC-100</td>
			<td>For Method 1</td>
		</tr>
		<tr>
			<td>PVDF Transfer Membrane</td>
			<td>Millipore Sigma</td>
			<td>IEVH00005</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Recombinant H3.1</td>
			<td>New England BioLabs</td>
			<td>M2503S</td>
			<td>for use in Method 1</td>
		</tr>
		<tr>
			<td>Recombinant p300</td>
			<td>ENZO Life Sciences</td>
			<td>BML-SE451-0100</td>
			<td>for use in Method 1</td>
		</tr>
		<tr>
			<td>SAHA (Vorinostat)</td>
			<td>Cayman Chemical</td>
			<td>149647-78-9</td>
			<td>HDAC Inhibitor for use in Method 2</td>
		</tr>
		<tr>
			<td>SDS lysis buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Fisher Scientific</td>
			<td>26628-22-8</td>
			<td>For Methods 1 and 2. CAUTION: Sodium Azide is toxic. See SDS for proper handling.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>71725</td>
			<td>for SDS sample buffer preparation</td>
		</tr>
		<tr>
			<td>Standard Heatblock</td>
			<td>VWR Scientific Products</td>
			<td>MPN: 949030</td>
			<td>For Methods 1 and 2</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td>For all methods</td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td></td>
			<td></td>
			<td>For Method 3. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td></td>
			<td></td>
			<td>For Methods 1 and 2. See Table 1 for recipe.</td>
		</tr>
		<tr>
			<td>Trichostatin A</td>
			<td>Cayman Chemical</td>
			<td>58880-19-6</td>
			<td>HDAC Inhibitor for use in Method 2</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>for buffer preparation</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>9005-64-5</td>
			<td>for buffer preparation in Methods 1 and 2</td>
		</tr>
		<tr>
			<td>X-ray film processor</td>
			<td>Konica Minolta Medical &#38; Graphic, Inc.</td>
			<td>SRX-101A</td>
			<td>For Methods 1 and 2</td>
		</tr>
	</tbody>
</table>

assays for validating histone acetyltransferase inhibitors

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.
This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.
The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.
The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

The in vitro histone acetyltransferase (HAT) assay can be used to probe for compounds that inhibit p300 HAT activity towards a histone substrate. Figure 1A provides an experimental schematic for the HAT assay. Anacardic acid, a known HATi3,38, was utilized in this assay in a concentration range from 12.5-100 &#181;M. At 100 &#181;M, anacardic acid downregulates p300 catalyzed histone acetylation at Histone 3, Lysines 9 and 18 versus ...

Watch this Scientific Journal Video about Assays for Validating Histone Acetyltransferase Inhibitors at JoVE.com

Assays for Validating Histone Acetyltransferase Inhibitors

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

assays-for-validating-histone-acetyltransferase-inhibitors

Research

JoVE Journal

Cancer Research

6.4K Views.  University of Florida College of Medicine. Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Video: Assays for Validating Histone Acetyltransferase Inhibitors

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Characterization of the Effects of Migrastatic Inhibitors on 3D Tumor Spheroid Invasion by High-resolution Confocal Microscopy

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and invasive potential of cancer cells, and consequently the metastatic spread of disease, are being discovered and considered as complementary treatments in highly invasive cancers such as gliomas. Thus, generating data enabling the detailed analyses of cells in a 3D environment following the addition of a drug is required. The methodology described here, combining spheroid invasion assays with high-resolution image capture and data analysis by confocal laser scanning microscopy (CLSM), enabled detailed characterization of the effects of the potential anti-migratory inhibitor MI-192 on glioma cells. Spheroids were generated from cell lines for invasion assays in low adherent 96-well plates and then prepared for CLSM analysis. The described workflow was preferred over other commonly used spheroid-generating techniques due to both ease and reproducibility. This, combined with the enhanced image resolution attained by confocal microscopy compared to conventional wide-field approaches, allowed the identification and analysis of distinct morphological changes in migratory cells in a 3D environment following treatment with the migrastatic drug MI-192.

The effects of migrastatic inhibitors on glioma cancer cell migration in three-dimensional (3D) invasion assays using a histone deacetylase (HDAC) inhibitor are characterized by high-resolution confocal microscopy.

Drug discovery and development in cancer research is increasingly being based on drug screens in a 3D format. Novel inhibitors targeting the migratory and ...

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...