17 research outputs found
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