Design, Synthesis, and Kinetic Characterization of Protein N-Terminal Acetyltransferase Inhibitors

Abstract

The N-termini of 80-90% of human proteins are acetylated by the N-terminal acetyltransferases (NATs), NatA-NatF. The major NAT complex, NatA, and particularly the catalytic subunit hNaa10 (ARD1) has been implicated in cancer development. For example, knockdown of hNaa10 results in cancer cell death and the arrest of cell proliferation. It also sensitized cancer cells to drug-induced cytotoxicity. Human NatE has a distinct substrate specificity and is essential for normal chromosome segregation. Thus, NAT inhibitors may potentially be valuable anticancer therapeutics, either directly or as adjuvants. Herein, we report the design and synthesis of the first inhibitors targeting these enzymes. Using the substrate specificity of the enzymes as a guide, we synthesized three bisubstrate analogues that potently and selectively inhibit the NatA complex (CoA-Ac-SES4; IC50 = 15.1 μM), hNaa10, the catalytic subunit of NatA (CoA-Ac-EEE4; Ki = 1.6 μM), and NatE/hNaa50 (CoA-Ac-MLG7; Ki* = 8 nM); CoA-Ac-EEE4 is a reversible competitive inhibitor of hNaa10, and CoA-Ac-MLG7 is a slow tight binding inhibitor of hNaa50. Our demonstration that it is possible to develop NAT selective inhibitors should assist future efforts to develop NAT inhibitors with more drug-like properties that can be used to chemically interrogate in vivo NAT function.

Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

“…43,44 The identification of Naa50 as a target of Lys-CoA-BPyne 1 initially struck us as paradoxical, as this enzyme belongs to the N-terminal acetyltransferase (NAT) family and has been shown to favor protein acetylation of N-terminal Met residues. 45,46 However, literature investigation revealed that, of the 7 NAT catalytic subunits encoded in the human genome, Naa50 is the only member to have biochemically characterized ε-lysine acetyltransferase activity, providing a molecular rationale for its targeting by 1 . 47 The selective identification of Naa50 by 1 suggests that chemoproteomic profiling may have applications in identifying new KAT activities present in acetyltransferase families that are distinct in sequence from canonical KATs.…”

The biological functions of Naa10 — From amino-terminal acetylation to human disease

“…The recent design and synthesis of the first 3 bisubstrate inhibitors that potently and selectively inhibit the NatA/Naa10 complex, monomeric Naa10, and hNaa50 (Foyn et al, 2013a), further increases the toolset to discriminate between NAT-dependent and independent function. Additionally, it would be interesting to analyze the mechanism and the signal pathways involved in the nuclear import or a potential nucleo-cytoplasmic shuttling of Naa10.…”

“…Besides acting in a complex, it has been shown that a fraction of human Naa10 exists independent of Naa15 in the cytoplasm and is able to acetylate acidic side chains like aspartate and glutamate in γ- and β-actin (Van Damme et al, 2011b; Foyn et al, 2013a). These Type I actins are natural NatB substrates (iMet followed by an amino acid with an acidic side chain) and are therefore initially acetylated by NatB in yeast and humans (Van Damme et al, 2012).…”

The world of protein acetylation

“…As the NATs are essential for cell cycle progression and have several anti-apoptotic roles in cancer, they are potential therapeutic targets in cancer therapy. Indeed, NAT-inhibitors are under development with the ultimate goal of utilization in clinical treatment as well as further studying the cellular roles of NATs[456].…”