click assay for the measurement of histone acetyltransferase 1 acetylation

Udvikling af et klikbaseret assay til screening af HAT1-enzymaktivitet

Among the numerous eukaryotic acetyltransferases, HAT1 was the initial histone acetyltransferase to be isolated1,2,3. Subsequent investigations have firmly established its pivotal role in chromatin replication, particularly in the synthesis of new nucleosomes during S-phase4. Our research endeavors led to the recognition that HAT1 is highly stimulated by epidermal growth factor (EGF) treatment in mammary cells5. Furthermore, it has come to light that HAT1 is required for rapid cell proliferation and tumor formation in vivo6,7,8,9. Data indicate that HAT1 is critical in coordinating anabolic and epigenetic processes for cell division, driving tumor growth.
HAT1 di-acetylates the amino-terminal tail of histone H4 on lysines 5 and 12 in complex with the chaperone protein Rbap46, which binds the histone and presents the amino-terminus to HAT1. Histone tetramers or disomes10, together with HAT1/Rbap46 and other histone chaperones11, are then imported to the nucleus. Histones are then released to be deposited at the replication fork, or other sites to support gene activation or repression. The function of the HAT1 di-acetylation mark on histone H4 is not fully understood. It is likely quickly removed within a span of 15-30 min by the action of histone deacetylases12,13,14,15 after H4 is inserted into chromatin. Thus, the HAT1 di-acetylation mark is not propagated in chromatin and may not serve a true epigenetic role, although a role in the recruitment of chromatin-modifying enzymes to nascent chromatin has been postulated12. Also, HAT1 does not directly acetylate chromatin; its activity is restricted to soluble histones.
The development of small-molecule histone acetyltransferase inhibitors has been hampered by nonspecific and low-throughput assays, often resulting in the generation of biologically reactive compounds16,17.&#160;The gold-standard assay to measure acetyltransferase activities requires the use of 3H-acetyl-coA, which limits throughput and requires radiation. Nonetheless, recently, specific and highly potent small molecule acetyltransferase inhibitors targeting CBP/p30018 and KAT6A/B19,20 have been described and confirmed through the use of 3H-acetyl-CoA. Moving forward, improved assays to achieve better throughput and avoid laboratory hazards are being devised.
Recent advances in acetylation monitoring21 have used click chemistry to enable enzymatic reaction monitoring. There are a variety of click-enabled precursors that are accessible by simple synthetic routes or available for purchase that can be incorporated into enzyme reactions. These reactions are typically carried out in recombinant systems, although cell-based assays are also feasible22. The advantage of click-enabled co-factors and substrates is that screening can directly measure enzyme activity without the need for coupled read-out systems that are often perturbed by screening compounds and require additional handling steps. This allows for inhibitor treatments only during the enzymatic step, whereas all downstream functionalization and detection steps are carried out after extensive washing to remove compounds, thus limiting the potential for assay interference to occur. These advantages make the design of click-enabled assays preferable to coupled assays that commonly rely on the detection of free coenzyme A.
One important consideration is the acceptance of click-enabled co-factors into the enzyme active site. Existing click-enabled co-factors may not be fully compatible with the active site optimized for the native co-factor. Structural information and modeling can be used to design amino acid substitutions to enlarge the active site to incorporate altered substrates23. This may enable screening with improved enzyme kinetics and lower substrate and enzyme levels. The drawback of this approach is that altered catalytic pockets may not identify inhibitors that interact strongly with the native enzyme. Ultimately, a combination of approaches is required to identify and validate potential enzyme inhibitors.
Here, we describe a method developed to purify and assay HAT1 enzyme activity using the click co-factor 4-pentynoyl-CoA24. This assay (Figure 1) uses the native enzyme sequence in complex with its required partner protein Rbap46, which has been shown to boost enzyme activity. Purification of the enzyme from human cells allows for enzyme activation in cellulo, which may preserve stimulating post-translational modifications important for full enzyme activity. Design and optimization of recombinant enzyme assays for high-throughput chemical screens have successfully been used to identify and characterize HAT1 small molecule inhibitors.

Forfatter Spotlight: Udvikling af acetyl-klik-analyse til screening af HAT1-hæmmere

En acetyl-klik kemi assay til måling af histon acetyltransferase 1 acetylering

Our developed acetyl-click assay serves as a tool to assess HAT1 acetyl transferase activity, which is key in isolating H4 histones before nucleosome incorporation. We aim to use this method for high throughput screening of small molecule inhibitors against the HAT1 enzyme. This method measures enzymatic activity directly on the peptide substrate.This offers advantages compared to other enzymatic assays by avoiding biologic variability and antibody related costs. This is also high throughput and can be adapted to use with other acetyl transferases. We have recently published a small molecule HAT1 inhibitor based on this assay that has activity in cells and animals.We continue to use this assay to screen additional chemical libraries and perform structure activity relationships. We are advancing methods to track histone production, folding, chaperoning, and chromatin integration. This highlights the need for improved chemical tools to explore biological mechanisms and stimulate drug discovery.Our goal is to demonstrate that targeting histone production can have therapeutic benefit for diseases such as cancer.

Klik assay til måling af histonacetyltransferase 1-acetylering

Til at begynde med resuspenderes det syntetiserede peptid, der virker som en positiv kontrol i en koncentration på 0, 1 milligram pr. Milliliter i DMSO. Bland det resuspenderede peptid med biotinyleret histon H4-peptid for at skabe en standardkurve. Derefter samles acetyleringsreaktionerne i to eksemplarer i 96-brønds PCR-plader.En 20-mikroliter reaktion bør omfatte 10 mikroliter af enzymet, en mikroliter H4-peptid, en mikroliter 20X buffer og en mikroliter DTT. Tilsæt en mikroliter DMSO eller testforbindelsen i hvert hul. Rør forsigtigt blandingen for at blande.Derefter inkuberes blandingen på is i 10 minutter for at muliggøre enzyminhibitorkompleksdannelse. Til fortsættelse af acetyleringsreaktionen kombineres to mikroliter 4-Penneolie-coenzym A med fire mikroliter vand. Tilsæt seks mikroliter af denne blanding til brøndene.Efter forsigtig blanding centrifugeres blandingen ved 300 G i 30 sekunder ved stuetemperatur. Inkuber indholdet ved 37 grader Celsius i en time i lukkede rør. Sluk indholdet med 20 mikroliter otte-molært urinstof og opbevar ved minus 20 grader Celsius indtil videre behandling.Derefter pipetteres 80 mikroliter PBST i hver brønd i BSA-blokerede sorte NeutrAvidin-belagte 96-brøndplader. Tilsæt 40 mikroliter reaktionsindhold i pladens brønde. Derefter pipetteres standardkurvepeptiderne i to eksemplarer i de resterende huller.Omrør forsigtigt peptiderne på en orbitalryster i en time ved stuetemperatur for at lette bindingen. Når bindingen er afsluttet, aspireres væsken fra brøndene. Brug en pladevasker til at vaske brøndene med 200 mikroliter PBST.Til klikreaktionen tilsættes THPTA kobberblandingen til natriumascorbat i vand og biotinazid i DMSO. Dispenser 19 mikroliter af de friskblandede reagenser i hvert hul. Forsegl pladen med aluminiumsfolie.Inkuber det derefter ved 37 grader Celsius i en time. Opsug opløsningen fra brøndene inden vask som før. Derefter tilsættes 100 mikroliter fortyndet Streptavidin peberrodsperoxidaseblanding i hver brønd.Inkuber ved stuetemperatur i en time med blid omrøring på en orbitalryster. For at kombinere Amplex Red-detektionsreagenserne pipetteres 500 mikroliter fortyndet hydrogenperoxid til Amplex Red-reagenset. Tilsæt 50 mikroliter af denne blanding til 4,5 ml natriumphosphat.Pipette 100 mikroliter af Amplex Red reaktionsblandingen til de vaskede brønde. Inkuber derefter pladen ved stuetemperatur i 30 minutter væk fra lyset. Start SmartControl-softwaren, og klik på Plate Out.Når pladen er overført til instrumentet, skal du klikke på Amplex Red-ikonet og ændre pladelayoutet. Tryk på ikonet Amplex Red igen for at se det ændrede layout. Vælg derefter standardbrøndene øverst til venstre fra layoutet.Skift filnavnet, og tryk derefter på Start justering. Når forstærkningen er indstillet, skal du klikke på Start måling. Tryk på Aktuel tilstand for at observere målingen i realtid.Når målingen er afsluttet, skal du lukke fanen. Klik derefter på Åbn sidste kørsel på toppanelet. Naviger til fanen Mars.Tryk derefter på Eksporter til Excel fra startmenuen for at eksportere dataresultaterne. Forbindelser A og C blev observeret at hæmme HAT1-aktivitet med næsten 100% i flere fortyndinger.

Research

JoVE Journal

Biochemistry

Click Assay for the Measurement of Histone Acetyltransferase 1 Acetylation

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...