Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (<b>1</b>), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (<b>3</b>) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin <b>3</b> inhibits DNA methyltransferase activity in the major groove at its target site more effectively than <b>1</b>, providing a molecular basis for design of sequence-specific antagonists of CpG methylation

Modular Synthesis of Cell-Permeating 2‑Ketoglutarate Esters

Cell-permeating esters of 2-ketoglutarate (2-KG) have been synthesized through a convergent sequence from two modules in two and three steps, respectively. This route provides access to a full series of mono- and disubstituted 2-KG esters, enabling us to define the effect of regioisomeric masking on metabolite release and antihypoxic activity in cell-based assays. In addition to providing insight into the biological activity of cell permeable 2-KG esters, the straightforward and modular nature of this synthetic route may prove useful for the development of next-generation 2-KG analogues for diagnostic and therapeutic applications

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

Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC–MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease

Characterization and Solubilization of Pyrrole–Imidazole Polyamide Aggregates

To optimize the biological activity of pyrrole–imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied polyamides were found to form measurable particles 50–500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-β-cyclodextrin (HpβCD). In mice, the use of HpβCD allowed for improved injection conditions and subsequent investigations of the availability of polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im polyamides and facilitate their study in animal models

Chemical Control of a CRISPR-Cas9 Acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci

Chemical Control of a CRISPR-Cas9 Acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci

Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300

C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC–MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks

Animal Toxicity of Hairpin Pyrrole-Imidazole Polyamides Varies with the Turn Unit

A hairpin pyrrole-imidazole polyamide (<b>1</b>) targeted to the androgen receptor consensus half-site was found to exert antitumor effects against prostate cancer xenografts. A previous animal study showed that <b>1</b>, which has a chiral amine at the α-position of the γ-aminobutyric acid turn (γ-turn), did not exhibit toxicity at doses less than 10 mg/kg. In the same study, a polyamide with an acetamide at the β-position of the γ-turn resulted in animal morbidity at 2.3 mg/kg. To identify structural motifs that cause animal toxicity, we synthesized polyamides <b>1</b>–<b>4</b> with variations at the α- and β-positions in the γ-turn. Weight loss, histopathology, and serum chemistry were analyzed in mice post-treatment. While serum concentration was similar for all four polyamides after injection, dose-limiting liver toxicity was only observed for three polyamides. Polyamide <b>3</b>, with an α-acetamide, caused no significant evidence of rodent toxicity and retains activity against LNCaP xenografts

Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors

Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts

Defining Metabolic and Nonmetabolic Regulation of Histone Acetylation by NSAID Chemotypes

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for their effects on inflammatory gene expression. Although NSAIDs are known to impact multiple cellular signaling mechanisms, a recent finding is that the NSAID salicylate can disrupt histone acetylation, in part through direct inhibition of the lysine acetyltransferase (KAT) p300/CBP. While salicylate is a relatively weak KAT inhibitor, its CoA-linked metabolite is more potent; however, the ability of NSAID metabolites to inhibit KAT enzymes biochemically and in cells remains relatively unexplored. Here we define the role of metabolic and nonmetabolic mechanisms in inhibition of KAT activity by NSAID chemotypes. First, we screen a small panel of NSAIDs for biochemical inhibition of the prototypical KAT p300, leading to the finding that many carboxylate-containing NSAIDs, including ibuprofen, are able to function as weak inhibitors. Assessing the inhibition of p300 by ibuprofen-CoA, a known NSAID metabolite, reveals that linkage of ibuprofen to CoA increases its biochemical potency toward p300 and other KAT enzymes. In cellular studies, we find that carboxylate-containing NSAIDs inhibit histone acetylation. Finally, we exploit the stereoselective metabolism of ibuprofen to assess the role of its acyl-CoA metabolite in regulation of histone acetylation. This unique strategy reveals that formation of ibuprofen-CoA and histone acetylation are poorly correlated, suggesting metabolism may not be required for ibuprofen to inhibit histone acetylation. Overall, these studies provide new insights into the ability of NSAIDs to alter histone acetylation, and illustrate how selective metabolism may be leveraged as a tool to explore the influence of metabolic acyl-CoAs on cellular enzyme activity