Name: Forfatter Spotlight: Udvikling af acetyl-klik-analyse til screening af HAT1-hæmmere
Uploaded: 2024-01-26T14:20:00
Description: Watch this Scientific Journal Video about Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening at JoVE.com

Vi takker George Zheng for at levere H4K12CoA. Vi takker medlemmer af Gruber Lab for nyttige diskussioner og feedback. Vi takker støtten fra NIH/NCI (1K08CA245024), CPRIT (RR200090) og V Foundation (V2022-022).

1. Metode 1: Fremstilling og rensning af rekombinant HAT1/Rbap46-kompleks
<ol><li>Optøning, gendannelse og udvidelse af HEK293f-celler<ol><li>Optø 1-10 millioner HEK293f pattedyrceller26 i 30 ml freestyle 293 ekspressionsmedier i en 100 ml kolbe. Der inkuberes i 8 % CO2 ved 37 °C, mens der roteres ved 60 omdr./min.</li>
		<li>Den næste dag skal du tælle og kontrollere cellelevedygtigheden og derefter justere rotationshastigheden til 120 o / min. Udvid til 300 ml ...

Udvikling af et klikbaseret assay til screening af HAT1-enzymaktivitet

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I det sidste årti blev klikkemi fremtrædende20, hvilket muliggør præcis design af interagerende kemiske strukturer. Inden for denne sammenhæng har forskellige bioortogonale kovalente forbindelser21 vist sig som lovende muligheder for dannelse af komplekser i deres naturlige miljø. Klikkemi anvender par af funktionelle grupper, der udviser hurtige og selektive reaktioner, almindeligvis kendt som "klikreaktioner". Disse reaktioner forekommer effektivt under miljøvenlig...

Blandt de mange eukaryote acetyltransferaser var HAT1 den oprindelige histonacetyltransferase, der skulle isoleres 1,2,3. Efterfølgende undersøgelser har fast etableret sin centrale rolle i kromatinreplikation, især i syntesen af nye nukleosomer under S-fase4. Vores forskningsbestræbelser førte til erkendelsen af, at HAT1 i høj grad stimuleres af epidermal vækstfaktor (EGF) behandling i brystceller5. Desuden er det kommet frem, at HAT1 er nødvendig for hurtig celleproliferation og tumordannelse in vivo 6,7,8,9. Data indikerer, at HAT1 er afgørende for at koordinere anabolske og epigenetiske processer til celledeling, hvilket driver tumorvækst.
HAT1 di-acetylerer den aminoterminale hale af histon H4 på lysiner 5 og 12 i kompleks med chaperonproteinet Rbap46, som binder histonen og præsenterer aminoterminalen til HAT1. Histontetramerer eller disomer10 sammen med HAT1/Rbap46 og andre histonchaperoner11 importeres derefter til kernen. Histoner frigives derefter for at blive deponeret ved replikationsgaflen eller andre steder for at understøtte genaktivering eller undertrykkelse. Funktionen af HAT1-di-acetyleringsmærket på histon H4 forstås ikke fuldt ud. Det fjernes sandsynligvis hurtigt inden for et tidsrum på 15-30 minutter ved virkningen af histondeacetylaser 12,13,14,15 efter H4 er indsat i kromatin. Således formeres HAT1-di-acetyleringsmærket ikke i kromatin og tjener muligvis ikke en ægte epigenetisk rolle, selvom en rolle i rekrutteringen af kromatinmodificerende enzymer til spirende kromatin er blevet postuleret12. HAT1 acetylerer heller ikke direkte kromatin; Dens aktivitet er begrænset til opløselige histoner.
Udviklingen af småmolekylære histonacetyltransferasehæmmere er blevet hæmmet af uspecifikke analyser med lav kapacitet, hvilket ofte resulterer i dannelsen af biologisk reaktive forbindelser16,17. Guldstandardanalysen til måling af acetyltransferaseaktiviteter kræver anvendelse af 3H-acetyl-coA, hvilket begrænser gennemstrømningen og kræver stråling. Ikke desto mindre er specifikke og højpotente acetyltransferasehæmmere med små molekyler rettet mod CBP/p30018 og KAT6A/B19,20 for nylig blevet beskrevet og bekræftet ved anvendelse af 3H-acetyl-CoA. Fremadrettet udtænkes forbedrede analyser for at opnå bedre gennemstrømning og undgå laboratoriefarer.
Nylige fremskridt inden for acetyleringsovervågning21 har brugt klikkemi til at muliggøre enzymatisk reaktionsovervågning. Der er en række klikaktiverede forløbere, der er tilgængelige via enkle syntetiske ruter eller kan købes, der kan inkorporeres i enzymreaktioner. Disse reaktioner udføres typisk i rekombinante systemer, selvom cellebaserede assays også er mulige22. Fordelen ved klikaktiverede co-faktorer og substrater er, at screening direkte kan måle enzymaktivitet uden behov for koblede udlæsningssystemer, der ofte forstyrres af screeningsforbindelser og kræver yderligere håndteringstrin. Dette tillader kun inhibitorbehandlinger under det enzymatiske trin, mens alle nedstrøms funktionaliserings- og detektionstrin udføres efter omfattende vask for at fjerne forbindelser, hvilket begrænser potentialet for assayinterferens. Disse fordele gør designet af klikaktiverede assays at foretrække frem for koblede assays, der almindeligvis er afhængige af påvisning af frit coenzym A.
En vigtig overvejelse er accepten af klikaktiverede co-faktorer i enzymets aktive site. Eksisterende klikaktiverede cofaktorer er muligvis ikke fuldt kompatible med det aktive website, der er optimeret til den oprindelige cofaktor. Strukturel information og modellering kan bruges til at designe aminosyresubstitutioner for at forstørre det aktive sted for at inkorporere ændrede substrater23. Dette kan muliggøre screening med forbedret enzymkinetik og lavere substrat- og enzymniveauer. Ulempen ved denne fremgangsmåde er, at ændrede katalytiske lommer muligvis ikke identificerer hæmmere, der interagerer stærkt med det oprindelige enzym. I sidste ende kræves en kombination af tilgange for at identificere og validere potentielle enzymhæmmere.
Her beskriver vi en metode udviklet til at oprense og assay HAT1-enzymaktivitet ved hjælp af klik-co-faktor 4-pentynoyl-CoA24. Dette assay (figur 1) bruger den oprindelige enzymsekvens i kompleks med dets krævede partnerprotein Rbap46, som har vist sig at øge enzymaktiviteten. Oprensning af enzymet fra humane celler muliggør enzymaktivering i cellulo, hvilket kan bevare stimulerende posttranslationelle modifikationer, der er vigtige for fuld enzymaktivitet. Design og optimering af rekombinante enzymassays til kemiske skærme med høj kapacitet er med succes blevet brugt til at identificere og karakterisere HAT1 små molekylehæmmere.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4P CoA</td>
			<td>Cayman Chemical</td>
			<td>10547</td>
			<td>Click chemistry co-factor</td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Fisher Sci</td>
			<td>A12222</td>
			<td>Fluorescence substrate</td>
		</tr>
		<tr>
			<td>Biotin-PEG-Azide</td>
			<td>Alfa Aesar</td>
			<td>J64996MC</td>
			<td>Click chemistry</td>
		</tr>
		<tr>
			<td>Copper Sulfate</td>
			<td>Sigma-aldrich</td>
			<td>&#160;7758-98-7</td>
			<td>Click chemistry</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>&#160;67-68-5</td>
			<td>diluent</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Acros Organics</td>
			<td>03-12-3483</td>
			<td>reducting agent</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>VWR</td>
			<td>102987-310</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>Freestyle 293 Expression Medium</td>
			<td>Thermo Fisher</td>
			<td>12338018</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>Freestyle 293-F cells</td>
			<td>Thermo Fisher</td>
			<td>R790-07</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>H4-peptide/1-23-GGK-biotin</td>
			<td>Anaspec</td>
			<td>AS65097</td>
			<td>peptide substrate</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-aldrich</td>
			<td>&#160;7365-45-9</td>
			<td>EB buffer</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30% solution</td>
			<td>Sigma-aldrich</td>
			<td>&#160;Z00183-99-0</td>
			<td>initiator</td>
		</tr>
		<tr>
			<td>M2 FLAG antibody slurry</td>
			<td>Millipore-Sigma</td>
			<td>A2220</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>Macrosep 10K Filter (Pall Lab)</td>
			<td>VWR</td>
			<td>89131-980</td>
			<td>Protein purification</td>
		</tr>
		<tr>
			<td>Neutravidin Plate</td>
			<td>Thermo Sci</td>
			<td>15127</td>
			<td>BSA-pre-blocked</td>
		</tr>
		<tr>
			<td>NP40 (IGEPAL)</td>
			<td>MP Biomedical</td>
			<td>198596</td>
			<td>20x buffer</td>
		</tr>
		<tr>
			<td>pHEK-293 plasmid</td>
			<td>Takara Bio</td>
			<td>3390</td>
			<td>Protein expression</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 10x</td>
			<td>Alfa Aesar</td>
			<td>&#160;Z00082-33-6</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>Pra peptide</td>
			<td>Genscript</td>
			<td>Custom synthesis</td>
			<td>biotinylated</td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma-aldrich</td>
			<td>&#160;134-03-2</td>
			<td>Click chemistry</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-aldrich</td>
			<td>&#160;7647-14-5</td>
			<td>EB buffer</td>
		</tr>
		<tr>
			<td>Sodium phosphate</td>
			<td>VWR International</td>
			<td>7558-80-7</td>
			<td>buffer</td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>EMD Millipore</td>
			<td>189730</td>
			<td>competitor</td>
		</tr>
		<tr>
			<td>Streptavidin-HRP</td>
			<td>Cell Signaling</td>
			<td>3999S</td>
			<td>enzyme</td>
		</tr>
		<tr>
			<td>THPTA ligand</td>
			<td>Fisher Sci</td>
			<td>1010-500</td>
			<td>Click chemistry</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-aldrich</td>
			<td>&#160;77-86-1</td>
			<td>20x buffer</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>VWR International</td>
			<td>&#160;9002-93-1</td>
			<td>EB buffer</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-aldrich</td>
			<td>&#160;9005-64-5</td>
			<td>Wash buffer</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>57-13-6</td>
			<td>quencher</td>
		</tr>
	</tbody>
</table>

an acetyl-click chemistry assay to measure histone acetyltransferase 1 acetylation

HAT1, også kendt som histonacetyltransferase 1, spiller en afgørende rolle i kromatinsyntese ved at stabilisere og acetylere spirende H4 før nukleosomsamling. Det er nødvendigt for tumorvækst i forskellige systemer, hvilket gør det til et potentielt mål for kræftbehandling. For at lette identifikationen af forbindelser, der kan hæmme HAT1 enzymatisk aktivitet, har vi udtænkt et acetyl-klik-assay til hurtig screening. I denne enkle analyse anvender vi rekombinant HAT1 / Rbap46, som renses fra aktiverede humane celler. Metoden anvender acetyl-CoA analog 4-pentynoyl-CoA (4P) i en klik-kemi tilgang. Dette involverer enzymatisk overførsel af et alkynhåndtag gennem en HAT1-afhængig acyleringsreaktion til et biotinyleret H4 N-terminalt peptid. Det fangede peptid immobiliseres derefter på neutravidinplader efterfulgt af klikkemifunktionalisering med biotinazid. Derefter anvendes streptavidin-peroxidaserekruttering til at oxidere amplex rød, hvilket resulterer i en kvantitativ fluorescerende udgang. Ved at indføre kemiske hæmmere under acyleringsreaktionen kan vi kvantificere enzymatisk hæmning baseret på en reduktion af fluorescenssignalet. Det er vigtigt, at denne reaktion er skalerbar, hvilket muliggør screening med høj gennemstrømning af potentielle hæmmere for HAT1 enzymatisk aktivitet.

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.

Standardkurver i to eksemplarer (16 huller) skal medtages på hver plade for at sikre korrekt analyseydelse. Standardkurvedata bør opstilles i tabelform med et interval på 100% til 0% i henhold til forholdet mellem Pra-holdigt peptid og naturligt H4-peptid i opløsning (tabel 1). Amplex rødt signal vil være det højeste i 100% pra/0% native H4 peptidbrønde, og lavest i 0% pra/100% native H4 peptidbrønde. Efter fluorescens er detekteret, og brønde er gennemsnitlige, skal den resulterende standardgr...

Watch this Scientific Journal Video about Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening at JoVE.com

Hurtige og præcise kemiske assays til screening for specifikke hæmmere er et vigtigt værktøj i arsenalet af lægemiddeludvikling. Her præsenterer vi et skalerbart acetyl-klik-kemiassay til måling af hæmningen af HAT1-acetyleringsaktivitet.

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

Vores udviklede acetyl-klik-assay fungerer som et værktøj til at vurdere HAT1-acetyltransferaseaktivitet, hvilket er nøglen til isolering af H4-histoner før nukleosominkorporering. Vi sigter mod at bruge denne metode til high throughput screening af små molekylehæmmere mod HAT1-enzymet. Denne metode måler enzymatisk aktivitet direkte på peptidsubstratet.Dette giver fordele sammenlignet med andre enzymatiske assays ved at undgå biologisk variabilitet og antistofrelaterede omkostninger. Dette er også høj gennemstrømning og kan tilpasses til brug med andre acetyltransferaser. Vi har for nylig offentliggjort en lille molekyle HAT1-hæmmer baseret på dette assay, der har aktivitet i celler og dyr.Vi fortsætter med at bruge dette assay til at screene yderligere kemiske biblioteker og udføre strukturaktivitetsrelationer. Vi udvikler metoder til at spore histonproduktion, foldning, chaperoning og kromatinintegration. Dette understreger behovet for forbedrede kemiske værktøjer til at udforske biologiske mekanismer og stimulere lægemiddelopdagelse.Vores mål er at demonstrere, at målretning af histonproduktion kan have terapeutisk fordel for sygdomme som kræft.

an-acetyl-click-chemistry-assay-to-measure-histone-acetyltransferase

Research

JoVE Journal

Biochemistry

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 assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.