Christopher R. Travis

Inhibition of histone trimethyllysine (Kme3) reader proteins is a promising therapeutic strategy due to their roles in gene regulation and disease, particularly cancer. However, developing effective inhibitors has proven challenging because of the extended, solvent-exposed binding interface and the cooperative reliance on interactions with multiple positively charged residues in the histone tail in addition to Kme3. While these cationic groups are often necessary for strong affinity binding, they reduce cell penetration of peptidic inhibitors, increasing the challenge of inhibitor development for this class of proteins. Herein we report the evaluation of three new imidazolium amino acids (IAAs) as novel lipophilic cationic bioisosteres for Kme3, dimethylarginine (Rme2), and Arg against a panel of histone reader proteins. We find that these biomimetics can replace Kme3, Rme2, and Arg, with similar or even improved binding in some cases, while also providing unique selectivity patterns relative to the parent amino acid. Moreover, we find that these imidazolium amino acids promote improved cytosolic penetration due to their increased lipophilicity and lack of hydrogen bond donors. These new lipophilic cationic amino acids provide promising new chemotypes for inhibition of histone reader proteins, as well as other protein–protein interactions that rely on cationic residues.

Fetal hemoglobin (HbF) expression is silenced postnatally in adult erythroid cells. Sufficiently increased expression of HbF has been shown to overcome the pathophysiologic sequelae of both sickle cell disease and beta-thalassemia. As the MBD2a-NuRD chromatin remodeling complex is required for silencing of HbF, the present studies were aimed at exploring a potential therapeutic approach for disrupting this complex. AlphaFold 3 and a recent crystal structure were employed to predict the critical interaction domains linking GATAD2A in the histone deacetylase core subcomplex (HDCC) of NuRD and the CHD4 ATPase which has been shown to be required for silencing of the fetal gamma-globin ( HBG ) genes. The two predicted critical domains, the CR2 helical domain of GATAD2A and the C-terminal domains 1 and 2 (C1b and C2ab) of CHD4, were validated by in vitro biophysical studies. Mutation of two amino acids in the CR2 helical domain of the endogenous GATAD2A gene in HUDEP-2 cells resulted in dissociation of CHD4, loss of repressive chromatin over the HBG promoter and ~40% HbF levels compared to < 1% in control cells. Strikingly, enforced expression of a peptide containing the helical portion of the CR2 domain of GATAD2A in both HUDEP-2 cells and primary adult erythroid cells resulted in high levels of HbF, with up to ~75% HbF compared to mutant peptide control level of ~9% in the latter without perturbing erythroid differentiation. These results suggest that targeting the critical interaction domains of GATAD2A and CHD4 with a macrocyclic peptide or small molecule may lead to much needed small molecule therapeutics for sickle cell disease. Key Points: Association of CHD4 with the HDCC core of the MBD2-NuRD chromatin remodeling complex is required for silencing of HbF expression in adult human erythroid cells Genetic alteration or enforced peptide expression of a critical helical domain of GATAD2A results in dissociation of CHD4 from the MBD2-NuRD complex and high-level expression of HbF.

Inhibition of histone trimethyllysine (Kme3) reader proteins is a promising therapeutic strategy due to their roles in gene regulation and disease, particularly cancer. However, developing effective inhibitors has proven challenging because of the extended, solvent-exposed binding interface and the cooperative reliance on interactions with multiple positively charged residues in the histone tail in addition to Kme3. While these cationic groups are often necessary for strong affinity binding, they reduce cell penetration of peptidic inhibitors, increasing the challenge of inhibitor development for this class of proteins. Herein we report the evaluation of three new imidazolium amino acids as novel lipophilic cationic isosteres for Kme3, dimethylarginine (Rme2), and Arg against a panel of histone reader proteins. We find that these isosteres can replace Kme3, Rme2, and Arg, with equivalent or even improved binding in some cases, while also providing unique selectivity patterns relative to the parent amino acid. Moreover, we find that these imidazolium amino acids promote improved cytosolic penetration due to their increased lipophilicity and lack of hydrogen bond donors. These new isosteres provide promising new chemotypes for inhibition of histone reader proteins, as well as other protein-protein interactions which rely on cationic residues.

Reader proteins that bind histone trimethyllysine (Kme3) have become active therapeutic targets in recent years. Binding of Kme3 in the conserved aromatic cages of these readers via cation-π interactions is a key contributor to these protein-protein interactions. We explored whether the replacement of one methyl group of Kme3 with an electron-withdrawing group (EWG) could improve binding by increasing the electrostatic component of the cation-π interaction. We examined these Kme3 analogs in binding studies with histone reader proteins, nuclear magnetic resonance (NMR) studies in a β-hairpin peptide model system, and computational studies to gain mechanistic insight into their binding. Kme3 analogs with both alkyl and benzyl substitution with EWGs were examined. We found that EWGs on the ligand differentially affected binding to a series of Kme3 readers but did not improve binding. This is likely due to sterics of the different analogs that preclude optimal cation-π interactions. The unique selectivity patterns of these ligands nonetheless offer promise toward selective inhibitor development within this protein class. NMR studies of the alkyl-substituted analogs in the β-hairpin model system show that EWGs strengthen the cation-π interaction enthalpically but with an entropic penalty. NMR studies demonstrate that the benzyl group makes direct contacts with Trp in the β-hairpin model, providing aromatic interactions in addition to cation-π interactions. Computational energy decomposition analysis confirms the more favorable electrostatic interactions compared to Kme3, but other factors counter the enhanced electrostatic component. This mechanistic investigation demonstrates how EWG-containing Kme3 analogs contribute to cation-π interactions and provide unique selectivity patterns relative to Kme3.

Histone trimethyllysine (Kme3) reader proteins are emerging therapeutic targets. However, development of selective inhibitors has proven challenging given the conserved nature of the aromatic cage which binds Kme3 as well as the myriad reader proteins which bind Kme3 at the same position on histone tails. These readers rely on both the presence of Kme3 as well as the appropriate surrounding histone tail sequence to bind, suggesting that binding is highly cooperative. We recently found that a small subset of Kme3 readers bind with equal or tighter affinity to histone tail peptides which replace Kme3 with its neutral isostere, tBuNle. This unexpected result offers promise for therapeutic design. Herein, we utilize histone 3 tail peptides containing Kme3 or the unnatural tBuNle to probe cooperativity in reader protein binding. Through three case studies, we quantitatively determine that the degree of cooperativity in a reader protein binding histone Kme3 influences the degree of its aromatic cage preference for cationic versus neutral ligands. Moreover, we find that the degree of cooperativity differs for each reader, suggesting that such differences in cooperativity could be utilized strategically for selective inhibitor design and that mutation to either histones or readers to alter cooperativity could significantly affect a reader protein's selectivity for a specific post-translational modification.

Over the past 50 years, research has uncovered the co-regulatory proteins and complexes that silence the expression of the γ-globin gene in a developmental stage-specific manner. Recent research expanded the list of these regulatory factors by showing that the eight twenty-one protein 2 (ETO2) helps recruit the nucleosome remodeling and deacetylase (NuRD) complex to the globin locus. Furthermore, ETO2 regulates hematopoietic differentiation and is a potential therapeutic target for acute leukemia. In this work, we identify critical interactions between ETO2 and the GATA Zn finger domain containing the 2A (GATAD2A) component of NuRD. The ETO2 nervy homology region 4 (NHR4) domain interacts with multiple polyproline-leucine motifs within GATAD2A. We demonstrate that oligomerization of the ETO2 nervy homology region 3 (NHR3) enhances its affinity for peptides containing at least two polyproline-leucine motifs. Replacing the native motifs from GATAD2A with a higher-affinity sequence from known-binder N-CoR markedly enhances binding affinity, yielding a peptide that disrupts the interaction between ETO2 and target proteins. Enforced peptide expression elevates γ-globin expression levels and induces differentiation of HUDEP-2 and K562 cells. These findings provide insight into ETO2-mediated recruitment of co-regulatory proteins and yield a novel approach for ETO2 inhibition through multivalent binding of the NHR4 domain.

Histone serotonylation has emerged as a key post-translational modification. WDR5 preferentially binds to serotonylated histone 3 (H3), and this binding event has been associated with tumorigenesis. Herein, we utilize genetic code expansion, structure-activity relationship studies, and computation to study an edge-face aromatic interaction between WDR5 Phe149 and serotonin on H3 that is key to this protein-protein interaction. We find experimentally that this edge-face aromatic interaction is unaffected by modulating the electrostatics of the face component but is weakened by electron-withdrawing substituents on the edge component. Overall, these results elucidate that this interaction is governed by van der Waals forces as well as electrostatics of the edge ring, a result that clarifies discrepancies among previous theoretical models and model system studies of this interaction type. This is the first evaluation of the driving force of an edge-face aromatic interaction at a protein-protein interface and provides a key benchmark for the nature of these understudied interactions that are abundant in the proteome.

Per-residue analysis of the thermodynamics of unfolding of β-hairpins provides insight into the non-covalent interactions between residues, and the individual contributions of residues and secondary structure type to the Δ H , Δ S , and Δ C _p of unfolding.

Methylation of arginine (Arg) residues on histones creates a new binding epitope, enabling recognition by aromatic cage binding pockets in Tudor domains; these protein-protein interactions (PPIs) govern gene expression. Despite their biological importance, the molecular details of methylated Arg recognition are poorly understood. While the desolvation, hydrogen bonding, and guanidinium stacking of methylated Arg have been explored in model systems and proposed to contribute to binding, direct interactions between the methyl groups and the aromatic residues in the binding pocket have not previously been investigated. Herein, we mechanistically study the CH₃-π interactions between the SPIN1 triple Tudor domain and histone asymmetric dimethylarginine. We find that these CH₃-π interactions are electrostatically tunable, exhibiting cation-π character, albeit attenuated relative to cation-π interactions with quaternary ammonium ions, offering key insight into how methylation of Arg alters its binding epitope to enable new PPIs.