Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

Abstract

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.

Effects of Oncohistone Mutations and PTM Crosstalk on the N-Terminal Acetylation Activities of NatD

“…In contrast, hNatD displayed a comparable catalytic efficiency for H4 synthetic peptides consisting of the first 5, 8, and 19 residues in a biochemical radioactive study . To systematically understand how substrate length affects hNatD catalysis, we determined the steady-state kinetic parameters for both full-length H4 protein and a panel of truncated H4/H2A synthetic peptides using recombinant hNatD and a continuous fluorescence assay . The steady-state parameters are comparable for H4 protein and the cofactor acetyl coenzyme A (AcCoA) (Figure A).…”

Novel Bisubstrate Inhibitors for Protein N-Terminal Acetyltransferase D

“…All synthesized bisubstrate analogues were first evaluated using an established fluorescence assay. 22 Initial testing was performed in the presence of both AcCoA and an H4-8 peptide (SGRGKGGK-CONH 2 ) at their respective Michaelis constant (K m ) values. However, the resulting IC 50 values of compounds 1−8 were close to or lower than the concentration of NatD (45 nM) in the assay (Table 1 and Figure S3a,b), suggesting that they are tight-binding inhibitors.…”

“…All synthesized bisubstrate analogues were first evaluated using an established fluorescence assay . Initial testing was performed in the presence of both AcCoA and an H4-8 peptide (SGRGKGGK-CONH 2 ) at their respective Michaelis constant ( K m ) values.…”

Novel bisubstrate inhibitors for protein N-terminal acetyltransferase D

“…All synthesized bisubstrate analogues were evaluated with an established fluorescence assay. 22 Initial testing was performed in the presence of both AcCoA and peptide substrate SGRGK at their respective Michaelis constant (Km) values, but resulting IC50 values were close to the enzyme concentration. Thus, we optimized the condition to characterize IC50 values in the presence of both concentrations of AcCoA and SGRGK peptide at 4xKm values.…”