Chromodomain Ligand Optimization via Target-Class Directed Combinatorial Repurposing

Efforts to develop strategies for small molecule chemical probe discovery against the readers of the methyl-lysine (Kme) post-translational modification have been met with limited success. Targeted disruption of these protein-protein interactions via peptidomimetic inhibitor optimization is a promising alternative to small molecule hit discovery; however, recognition of identical peptide motifs by multiple Kme reader proteins presents a unique challenge in the development of selective Kme reader chemical probes. These selectivity challenges are exemplified by the Polycomb repressive complex 1 (PRC1) chemical probe, UNC3866, which demonstrates sub-micromolar off-target affinity toward the non-PRC1 chromodomains CDYL2 and CDYL. Moreover, since peptidomimetics are challenging subjects for structure-activity relationship (SAR) studies, traditional optimization of UNC3866 would prove costly and time-consuming. Herein, we report a broadly applicable strategy for the affinity-based, target-class screening of chromodomains via the repurposing of UNC3866 in an efficient, combinatorial peptide library. A first-generation library yielded UNC4991, a UNC3866 analog that exhibits a distinct selectivity profile while maintaining sub-micromolar affinity toward the CDYL chromodomains. Additionally, in vitro pull-down experiments from HeLa nuclear lysates further demonstrate the selectivity and utility of this compound for future elucidation of CDYL protein function

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>_d = 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 techniquesIsothermal Titration Calorimetry, X-ray Crystallography, Circular Dichroism, Size Exclusion Chromatography-Multi-Angle Light Scattering, and Surface Plasmon Resonance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