β‐Actin Peptide‐Based Inhibitors of Histidine Methyltransferase SETD3

Abstract

SETD3 was recently identified as the histidine methyltransferase responsible for N 3 -methylation of His73 of β-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected β-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing β-actin peptide was found to be the most potent SETD3 inhibitor (IC 50 = 161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for β-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.

Histidine methyltransferase SETD3 methylates structurally diverse histidine mimics in actin

“…We carried out enzymatic assays with the synthetic 16‐mer βA peptides using MALDI‐TOF MS assays 30 . A βA peptide (10 μM) was incubated in the presence of the recombinantly expressed human SETD3 (1 μM) and SAM cosubstrate (100 μM) at 37°C (standard conditions).…”

“…We carried out enzymatic assays with the synthetic 16-mer βA peptides using MALDI-TOF MS assays. 30 A βA peptide (10 μM) was incubated in the presence of the recombinantly expressed human SETD3 (1 μM) and SAM cosubstrate (100 μM) at 37 C (standard conditions). All peptides possessing histidine and its analogs were studied at pH 7.2, pH 9.0, and pH 10.5; enzymatic mixtures were quenched after 1 and 3 hr.…”

“…26 βA peptides possessing methionine analogs at the His73 position were shown to inhibit SETD3 with nanomolar potency. 30 Owing to SETD3's unique reactivity toward histidine over lysine and methionine, we sought to explore the substrate specificity of SETD3 to gain a deeper understanding of the underlying mechanism and biocatalytic scope of the SETD3 histidine methyltransferase. Here, we report biomolecular studies on human SETD3-catalyzed methylation of histidine and its simplest mimics incorporated into βA peptides.…”

Structure-function conservation between the methyltransferases SETD3 and SETD6

“…DOT1L inhibitor EPZ-5676 is also undergoing phase I/II against Relapsed/Refractory Leukemia [44]. Recently, a β-actin based-peptide was designed as an inhibitor for SETD3 [45]. However, there is still no available data regarding its effect on cells.…”

The Structure, Activity, and Function of the SETD3 Protein Histidine Methyltransferase

“…Although the M73-containing peptide is a poor substrate for SETD3, it has been found to exhibit strong affinity to the enzyme and inhibit the methylation of the H73 peptide. Based on this observation, actin based peptidomimetics that act as effective substrate competitive inhibitors of human SETD3 were developed [50]. These are 16-residue-long analogs of the actin peptide (66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81), in which the H73 residue is substituted by a simple natural or non-natural amino acid.…”