Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain

Abstract

AmideÀπ interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amideÀπ interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amideÀπ interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostereÀπ interaction. We show that compared to the amideÀπ interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.

Trimethyllysine Reader Proteins Exhibit Widespread Charge-Agnostic Binding via Different Mechanisms to Cationic and Neutral Ligands

“…48−52 Previously, we developed a genetic code expansion (GCE) methodology incorporating para-substituted Phe derivatives 45 to systematically tune the electrostatic potentials (ESPs) of aromatic residues within cages of readers, which provides information on the electrostatic contribution to binding at the individual residue level (Figure 2). This approach has elucidated the driving forces of various reader proteins binding to different PTMs, 53,54 including cation−π interactions with Kme3. 45,46 Using this GCE method, we investigated the DIDO1 PHD and the SGF29 TTD (Figure 2).…”

“…To gain further insight into the mechanisms of binding of the unselective readers, we turned to tuning the electrostatics of aromatic residues, which is an established strategy to evaluate cation−π interactions. − Previously, we developed a genetic code expansion (GCE) methodology incorporating para -substituted Phe derivatives to systematically tune the electrostatic potentials (ESPs) of aromatic residues within cages of readers, which provides information on the electrostatic contribution to binding at the individual residue level (Figure ). This approach has elucidated the driving forces of various reader proteins binding to different PTMs, , including cation−π interactions with Kme3. , Using this GCE method, we investigated the DIDO1 PHD and the SGF29 TTD (Figure ). We prepared a series of DIDO1 PHD Tyr8 mutants and SGF29 TTD Tyr238 mutants via GCE and measured binding to H3K4me3 and H3K4tBuNle peptides via ITC (Figures and S5).…”

Trimethyllysine reader proteins exhibit widespread charge-agnostic binding via different mechanisms to cationic and neutral ligands

“…[44][45][46][47][48] Previously, we developed a genetic code expansion (GCE) methodology incorporating of para-substituted Phe derivatives 41 to systematically tune the electrostatic potentials (ESP) of aromatic residues within cages of readers, which provides information on the electrostatic contribution to binding at the individual residue level (Figure 2). This approach has elucidated the driving forces of various reader proteins binding to different PTMs, 49,50 including cation−π interactions with Kme3. 41,42 Using this GCE method, we investigated the DIDO1 PHD and the SGF29 TTD (Figure 2).…”

Contribution of Electrostatic CH3–π Interactions to Recognition of Histone Asymmetric Dimethylarginine by the SPIN1 Triple Tudor Domain

“…Additionally, Rme2a CH 3 −π interactions are approximately as tunable as activated CH 3 −π interactions between the methyl of an acetyl group and an aromatic ring (slope = −0.03) 47 as well as amide−π interactions between acyllysines and an aromatic ring (slope = −0.02 to −0.03). 47,48 Computational Calculations Support Electrostatic CH 3 −π Interaction Contributing to SPIN1 TTD Binding of Rme2a. To interrogate this Rme recognition event in greater depth, we used computational methods to calculate interaction energies (ΔE Int ) between Rme2a and the SPIN1 TTD aromatic box residues using the previously reported crystal structure in three different model environments (Figure S8).…”