3 research outputs found
Structure–Activity Relationships and Kinetic Studies of Peptidic Antagonists of CBX Chromodomains
To better understand the contribution
of methyl-lysine (Kme) binding proteins to various disease states,
we recently developed and reported the discovery of <b>1</b> (UNC3866), a chemical probe that targets two families of Kme binding
proteins, CBX and CDY chromodomains, with selectivity for CBX4 and
-7. The discovery of <b>1</b> was enabled in part by the use
of molecular dynamics simulations performed with CBX7 and its endogenous
substrate. Herein, we describe the design, synthesis, and structure–activity
relationship studies that led to the development of <b>1</b> and provide support for our model of CBX7–ligand recognition
by examining the binding kinetics of our antagonists with CBX7 as
determined by surface-plasmon resonance
Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2
The
function of EED within polycomb repressive complex 2 (PRC2)
is mediated by a complex network of protein–protein interactions.
Allosteric activation of PRC2 by binding of methylated proteins to
the embryonic ectoderm development (EED) aromatic cage is essential
for full catalytic activity, but details of this regulation are not
fully understood. EED’s recognition of the product of PRC2
activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates
PRC2 methyltransferase activity at adjacent nucleosomes leading to
H3K27me3 propagation and, ultimately, gene repression. By coupling
combinatorial chemistry and structure-based design, we optimized a
low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2)
peptide to a smaller, more potent peptidomimetic ligand (<i>K</i><sub>d</sub> = 1.14 ± 0.14 μM) of the aromatic cage of
EED. Our strategy illustrates the effectiveness of applying combinatorial
chemistry to achieve both ligand potency and property optimization.
Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate
that targeted disruption of EED’s reader function can lead
to allosteric inhibition of PRC2 catalytic activity
Quantitative Characterization of Bivalent Probes for a Dual Bromodomain Protein, Transcription Initiation Factor TFIID Subunit 1
Multivalent binding is an efficient
means to enhance the affinity
and specificity of chemical probes targeting multidomain proteins
in order to study their function and role in disease. While the theory
of multivalent binding is straightforward, physical and structural
characterization of bivalent binding encounters multiple technical
difficulties. We present a case study where a combination of experimental
techniques and computational simulations was used to comprehensively
characterize the binding and structure–affinity relationships
for a series of Bromosporine-based bivalent bromodomain ligands with
a bivalent protein, Transcription Initiation Factor TFIID subunit
1 (<b>TAF1</b>). Experimental techniquesî—¸Isothermal Titration
Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion
Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonanceî—¸were
used to determine structures, binding affinities, and kinetics of
monovalent ligands and bivalent ligands with varying linker lengths.
The experimental data for monomeric ligands were fed into explicit
computational simulations, in which both ligand and protein species
were present in a broad range of concentrations, and in up to a 100
s time regime, to match experimental conditions. These simulations
provided accurate estimates for apparent affinities (in good agreement
with experimental data), individual dissociation microconstants and
other microscopic details for each type of protein–ligand complex.
We conclude that the expected efficiency of bivalent ligands in a
cellular context is difficult to estimate by a single technique <i>in vitro</i>, due to higher order associations favored at the
concentrations used, and other complicating processes. Rather, a combination
of structural, biophysical, and computational approaches should be
utilized to estimate and characterize multivalent interactions