Novel Bisubstrate Inhibitors for Protein N-Terminal Acetyltransferase D

Abstract

Protein N-terminal acetyltransferase D (NatD, NAA40) that specifically acetylates the alpha-N-terminus of histone H4 and H2A has been implicated in various diseases, but no inhibitor has been reported for this important enzyme. Based on the acetyl transfer mechanism of NatD, we designed and prepared a series of highly potent NatD bisubstrate inhibitors by covalently linking coenzyme A to different peptide substrates via an acetyl or propionyl spacer. The most potent bisubstrate inhibitor displayed an apparent K i value of 1.0 nM. Biochemical studies indicated that bisubstrate inhibitors are competitive to the peptide substrate and noncompetitive to the cofactor, suggesting that NatD undergoes an ordered Bi−Bi mechanism. We also demonstrated that these inhibitors are highly specific toward NatD, displaying about 1000-fold selectivity over other closely related acetyltransferases. High-resolution crystal structures of NatD bound to two of these inhibitors revealed the molecular basis for their selectivity and inhibition mechanism, providing a rational path for future inhibitor development.

Optimized bisubstrate inhibitors for the actin N-terminal acetyltransferase NAA80

“…The level of product formation for actylation of MDEL 24 , DDDI 24 and EEEI 24 was determined to be 119 ± 5.61 μM, 50.5 ± 1.0 μM, and 42.4 ± 0.95 μM, respectively ( Goris et al, 2018 ). In our inhibitor design, we decided to continue using a tetrapeptide for the protein-mimicking part as previous studies have shown that these are the most important residues for inhibitory activity ( Liszczak et al, 2011 ; Foyn et al, 2013 ; Liszczak et al, 2013 ; Støve et al, 2016 ; Hong et al, 2017 ; Goris et al, 2018 ; Deng et al, 2020 ; Deng et al, 2021 ; Deng et al, 2023 ). Co-crystal structures of NAT-bisubstrate inhibitor complexes typically show that these four amino acids are most important for protein-inhibitor binding interactions.…”

“…The first catalytic mechanism of a human NAT ( Evjenth et al, 2012 ), as well as the first structures of a NAT and NAT-complex bound to its peptide substrate, have been presented ( Liszczak et al, 2011 ; Liszczak et al, 2013 ; Støve et al, 2016 ). Taking advantage of this information, we and others have developed peptidic NAT inhibitors that are based on covalently linking mimics of the two substrates of the biochemical reaction, i.e., coenzyme A (CoA) and a short peptide carrying an N-terminal bromoacetyl group ( Figure 1 ) giving a thioether-linked acetyl moiety ( Foyn et al, 2013 ; Støve et al, 2016 ; Goris et al, 2018 ; Deng et al, 2021 ). In these studies it has been found that it is sufficient to use the four amino acids from the N-terminus of the protein substrates to obtain potent bisubstrate inhibitors.…”

“…Such bisubstrate inhibitors demonstrate specificity and significant inhibitory potential with IC 50 and K i in the low μM/nM range. For NatD, increasing the length of the acetyl linker to propionyl has proven beneficial for the design of highly potent inhibitors ( Deng et al, 2021 ). Further, for most NATs, access to bisubstrate inhibitors has led to significant advances in the field by enabling the determination of protein crystal structures ( Liszczak et al, 2013 ; Støve et al, 2016 ; Hong et al, 2017 ; Goris et al, 2018 ; Deng et al, 2020 ; Deng et al, 2023 ).…”

NatD epigenetically activates FOXA2 expression to promote breast cancer progression by facilitating MMP14 expression

“…Recently, some inhibitors have been reported for this important acetyltransferase. 42 , 43 Given that NatD promotes breast cancer cell metastasis, NatD enzymatic inhibition may provide an epigenetic therapeutic strategy for breast cancer. In the current study, we show that FOXA2 acts as a key factor mediating the function of NatD and MMP14 in breast cancer cells.…”

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

“…The competitive inhibition study was performed using a previously described fluorescence assay. 38 hNatD activity was measured at 25 °C under the following conditions in a final reaction volume of 40 μL: 50 nM hNatD, 15 μM ThioGlo4, 0.6 μM AcCoA, and 6 μM H4-5 in reaction buffer (25 mM HEPES, 150 mM NaCl, 0.01% (v/v) Triton, pH 7.5). The 10X concentration of mutant or modified peptides was diluted with reaction buffer and then 3-fold serially diluted with reaction buffer.…”