Name: Forfatter Spotlight: Utvikling av acetyl-klikk-analyse for screening av HAT1-hemmere
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 å gi H4K12CoA. Vi takker medlemmer av Gruberlaben for nyttige diskusjoner og tilbakemeldinger. Vi takker støtte fra NIH/NCI (1K08CA245024), CPRIT (RR200090) og V Foundation (V2022-022).

1. Metode 1: Produsere og rense rekombinant HAT1 / Rbap46 kompleks
<ol><li>Tine, gjenopprette og utvide HEK293f-celler<ol><li>Tine 1-10 millioner HEK293f pattedyrceller26 i 30 ml freestyle 293 uttrykksmedier i en kolbe på 100 ml. Inkuber i 8% CO2 ved 37 ° C mens du roterer ved 60 o / min.</li>
		<li>Neste dag, tell og kontroller cellens levedyktighet, og juster deretter rotasjonshastigheten til 120 o / min. Utvid til 300 ml kultur i en 1 L kolbe, oppretthold såtett...

Utvikling av en klikkbasert analyse for screening av HAT1-enzymaktivitet

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I løpet av det siste tiåret har klikkkjemi blitt fremtredende20, noe som muliggjør presis utforming av vekselvirkende kjemiske strukturer. Innenfor denne konteksten har ulike bioortogonale kovalente forbindelser21 dukket opp som lovende alternativer for å danne komplekser i sitt naturlige miljø. Klikkkjemi benytter par av funksjonelle grupper som viser raske og selektive reaksjoner, kjent som "klikkreaksjoner". Disse reaksjonene forekommer effektivt under miljøvennlig...

Blant de mange eukaryote acetyltransferasene var HAT1 den første histonacetyltransferasen som ble isolert 1,2,3. Etterfølgende undersøkelser har fast etablert sin sentrale rolle i kromatinreplikasjon, spesielt i syntesen av nye nukleosomer under S-fase4. Våre forskningsbestrebelser førte til erkjennelsen av at HAT1 er sterkt stimulert av epidermal vekstfaktor (EGF) behandling i brystceller5. Videre har det kommet frem at HAT1 er nødvendig for rask celleproliferasjon og tumordannelse in vivo 6,7,8,9. Data indikerer at HAT1 er kritisk for å koordinere anabole og epigenetiske prosesser for celledeling, som driver tumorvekst.
HAT1 di-acetylerer den aminoterminale halen av histon H4 på lysiner 5 og 12 i kompleks med chaperonproteinet Rbap46, som binder histonen og presenterer aminoterminalen til HAT1. Histontetramere eller disomer10, sammen med HAT1/Rbap46 og andre histonchaperoner11, importeres deretter til kjernen. Histoner frigjøres deretter for å bli deponert ved replikasjonsgaffelen eller andre steder for å støtte genaktivering eller undertrykkelse. Funksjonen til HAT1-di-acetyleringsmerket på histon H4 er ikke fullt ut forstått. Det er sannsynligvis raskt fjernet i løpet av et tidsrom på 15-30 min ved virkningen av histondeacetylaser 12,13,14,15 etter at H4 er satt inn i kromatin. Dermed er HAT1-di-acetyleringsmerket ikke forplantet i kromatin og kan ikke tjene en ekte epigenetisk rolle, selv om en rolle i rekrutteringen av kromatinmodifiserende enzymer til begynnende kromatin har blitt postulert12. HAT1 acetylat kromatin direkte; Dens aktivitet er begrenset til løselige histoner.
Utviklingen av småmolekylære histonacetyltransferasehemmere har blitt hemmet av ikke-spesifikke og lavkapasitetsanalyser, noe som ofte resulterer i generering av biologisk reaktive forbindelser16,17. Gullstandardanalysen for å måle acetyltransferaseaktiviteter krever bruk av 3H-acetyl-coA, som begrenser gjennomstrømningen og krever stråling. Ikke desto mindre har nylig spesifikke og svært potente småmolekylære acetyltransferasehemmere rettet mot CBP / p30018 og KAT6A / B19,20 blitt beskrevet og bekreftet ved bruk av 3H-acetyl-CoA. Fremover blir forbedrede analyser for å oppnå bedre gjennomstrømning og unngå laboratoriefarer utarbeidet.
Nylige fremskritt innen acetyleringsovervåking21 har brukt klikkkjemi for å muliggjøre enzymatisk reaksjonsovervåking. Det finnes en rekke klikkaktiverte forløpere som er tilgjengelige via enkle syntetiske ruter eller tilgjengelig for kjøp som kan inkorporeres i enzymreaksjoner. Disse reaksjonene utføres vanligvis i rekombinante systemer, selv om cellebaserte analyser også er mulige22. Fordelen med klikkaktiverte kofaktorer og substrater er at screening direkte kan måle enzymaktivitet uten behov for koblede avlesningssystemer som ofte forstyrres av screeningforbindelser og krever ytterligere håndteringstrinn. Dette tillater inhibitorbehandlinger bare under det enzymatiske trinnet, mens alle nedstrøms funksjonaliserings- og deteksjonstrinn utføres etter omfattende vask for å fjerne forbindelser, og dermed begrense potensialet for at analyseinterferens oppstår. Disse fordelene gjør utformingen av klikkaktiverte analyser å foretrekke fremfor koblede analyser som vanligvis er avhengige av påvisning av fritt koenzym A.
En viktig faktor er aksept av klikkaktiverte kofaktorer i det enzymaktive nettstedet. Eksisterende klikkaktiverte kofaktorer er kanskje ikke helt kompatible med det aktive nettstedet som er optimalisert for den opprinnelige kofaktoren. Strukturell informasjon og modellering kan brukes til å designe aminosyresubstitusjoner for å forstørre det aktive stedet for å innlemme endrede substrater23. Dette kan muliggjøre screening med forbedret enzymkinetikk og lavere substrat- og enzymnivåer. Ulempen med denne tilnærmingen er at endrede katalytiske lommer kanskje ikke identifiserer hemmere som interagerer sterkt med det opprinnelige enzymet. Til syvende og sist er det nødvendig med en kombinasjon av tilnærminger for å identifisere og validere potensielle enzymhemmere.
Her beskriver vi en metode utviklet for å rense og analysere HAT1-enzymaktivitet ved bruk av klikk-kofaktor 4-pentynoyl-CoA24. Denne analysen (figur 1) bruker den opprinnelige enzymsekvensen i kompleks med det nødvendige partnerproteinet Rbap46, som har vist seg å øke enzymaktiviteten. Rensing av enzymet fra humane celler muliggjør enzymaktivering i cellulo, noe som kan bevare stimulerende posttranslasjonelle modifikasjoner som er viktige for full enzymaktivitet. Design og optimalisering av rekombinante enzymanalyser for kjemiske skjermer med høy gjennomstrømning har med hell blitt brukt til å identifisere og karakterisere HAT1 småmolekylhemmere.

<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 kjent som histonacetyltransferase 1, spiller en avgjørende rolle i kromatinsyntese ved å stabilisere og acetylere begynnende H4 før nukleosommontering. Det er nødvendig for tumorvekst i ulike systemer, noe som gjør det til et potensielt mål for kreftbehandling. For å lette identifiseringen av forbindelser som kan hemme HAT1 enzymatisk aktivitet, har vi utviklet en acetyl-klikk-analyse for rask screening. I denne enkle analysen bruker vi rekombinant HAT1 / Rbap46, som er renset fra aktiverte humane celler. Metoden benytter acetyl-CoA analog 4-pentynoyl-CoA (4P) i en klikk-kjemi tilnærming. Dette innebærer enzymatisk overføring av et alkynhåndtak gjennom en HAT1-avhengig acyleringsreaksjon til et biotinylert H4N-terminalt peptid. Det fangede peptidet immobiliseres deretter på nøytravidinplater, etterfulgt av klikk-kjemisk funksjonalisering med biotin-azid. Deretter brukes streptapavidin-peroksidase rekruttering for å oksidere amplex rødt, noe som resulterer i en kvantitativ fluorescerende utgang. Ved å introdusere kjemiske hemmere under acyleringsreaksjonen, kan vi kvantifisere enzymatisk inhibering basert på en reduksjon av fluorescenssignalet. Det er viktig at denne reaksjonen er skalerbar, noe som muliggjør høy gjennomstrømningsscreening av potensielle hemmere 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: Utvikling av acetyl-klikk-analyse for screening av HAT1-hemmere

En acetyl-klikk-kjemianalyse for å måle histonacetyltransferase 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.

Standard kurver i dupliserte (16 brønner) bør inkluderes på hver plate for å sikre riktig analyseytelse. Standard kurvedata bør settes opp i tabellform, med et område på 100% til 0% i henhold til forholdet mellom Pra-inneholdende peptid og native H4-peptid i oppløsning (tabell 1). Amplex rødt signal vil være det høyeste i 100% pra/0% native H4 peptidbrønner, og lavest i 0% pra/100% native H4 peptidbrønner. Etter at fluorescens er påvist og brønner er gjennomsnittet, bør den resulterende s...

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

Raske og nøyaktige kjemiske analyser for å skjerme for spesifikke hemmere er et viktig verktøy i stoffutviklingsarsenalet. Her presenterer vi en skalerbar acetyl-klikk-kjemianalyse for å måle inhiberingen av HAT1-acetyleringsaktivitet.

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

Vår utviklede acetyl-klikkanalyse fungerer som et verktøy for å vurdere HAT1-acetyltransferaseaktivitet, som er nøkkelen til å isolere H4-histoner før nukleosominkorporering. Vi tar sikte på å bruke denne metoden for høy gjennomstrømningsscreening av småmolekylære hemmere mot HAT1-enzymet. Denne metoden måler enzymatisk aktivitet direkte på peptidsubstratet.Dette gir fordeler sammenlignet med andre enzymatiske analyser ved å unngå biologisk variabilitet og antistoffrelaterte kostnader. Dette er også høy gjennomstrømning og kan tilpasses til bruk sammen med andre acetyltransferaser. Vi har nylig publisert en liten molekylær HAT1-hemmer basert på denne analysen som har aktivitet i celler og dyr.Vi fortsetter å bruke denne analysen til å screene flere kjemiske biblioteker og utføre strukturaktivitetsrelasjoner. Vi fremmer metoder for å spore histonproduksjon, folding, chaperoning og kromatinintegrasjon. Dette understreker behovet for forbedrede kjemiske verktøy for å utforske biologiske mekanismer og stimulere narkotikaforskning.Vårt mål er å demonstrere at målretting av histonproduksjon kan ha terapeutisk fordel for sykdommer som kreft.

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.