The polycomb paralogs CBX2, CBX4, CBX6, CBX7, and CBX8 are epigenetic readers that rely on "aromatic cage" motifs to engage their partners' methyllysine side chains. Each CBX carries out distinct functions, yet each includes a highly similar methyllysine-reading chromodomain as a key element. CBX7 is the only chromodomain that has yet been targeted by chemical inhibition. We report a small set of peptidomimetic agents in which a simple chemical modification switches the ligands from one with promiscuity across all polycomb paralogs to one that provides selective inhibition of CBX6. The structural basis for this selectivity, which involves occupancy of a small hydrophobic pocket adjacent to the aromatic cage, was confirmed through molecular dynamics simulations. Our results demonstrate the increases in affinity and selectivity generated by ligands that engage extended regions of chromodomain binding surfaces.

X-ray crystallography reveals how a calixarene can bind to dimethyllysine to form a complex with features similar to the aromatic cage motif of a chromodomain bound to a histone tail.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2, CBX4, CBX6, CBX7, and CBX8, are incorporated into PRC1 complexes, where they mediate targeting to trimethylated lysine 27 of histone H3 (H3K27me3) via the N-terminal chromodomain (ChD). Individual CBX paralogs have been implicated as drug targets in cancer; however, high similarities in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here we report the selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs to identify modifications to a parental ligand that confer both selectivity and potency for the ChD of CBX8. This on-DNA, medicinal chemistry approach enabled the development of SW2_110A, a selective, cell-permeable inhibitor of the CBX8 ChD. SW2_110A binds CBX8 ChD with a K d of 800 nM, with minimal 5-fold selectivity for CBX8 ChD over all other CBX paralogs in vitro. SW2_110A specifically inhibits the association of CBX8 with chromatin in cells and inhibits the proliferation of THP1 leukemia cells driven by the MLL-AF9 translocation. In THP1 cells, SW2_110A treatment results in a significant decrease in the expression of MLL-AF9 target genes, including HOXA9, validating the previously established role for CBX8 in MLL-AF9 transcriptional activation, and defining the ChD as necessary for this function. The success of SW2_110A provides great promise for the development of highly selective and cell-permeable probes for the full CBX family. In addition, the approach taken provides a proof-of-principle demonstration of how DELs can be used iteratively for optimization of both ligand potency and selectivity.

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Catalytic reactions are limited in their turnover by certain steps in the cycle. We present a free, web-based interface where the rate constants of various steps in a cycle can be visualized. Population of a web form using known data will generate a highly customizable graphic for annotation by the user to represent their chemistry.

Using existing and academically available software, we present a new method for the structural prediction of binding events containing flexible protein targets. SLICE (Selective Ligand-Induced Conformational Ensemble) combines opportunistic stochastic jumps of ligand position with standard molecular dynamics to model the induced-fit binding of ligands starting with unbound host coordinates. To induce the structural adaptations of the complex at the binding site, conformational jumps in ligand position are selected in SLICE from structures generated by a docking software. Multiple binding trajectories from the docking set are followed using molecular dynamics for a set time to relax the host structure and generate new host poses. A new configurational jump is made on the set of newly generated host poses. The process is then repeated. The method was implemented with AutoDock Vina as the docking method, Vina scores as the selection criterion, and Amber code for molecular dynamics and applied to several test systems. A system consisting of Chromobox protein homologue 8 (CBX8) and its small peptide ligand, H3K9Me3, for which the final (bound) configuration is known, is used for verifying SLICE in the present setup. The setup was also applied to several nonpeptide molecules on known difficult flexible targets exhibiting a large disparity between apo and holo host states. The SLICE simulations provide a promising approach to generate induced-fit configurations compared to existing long (microsecond) classical and accelerated dynamics approaches in all the test systems considered here. However, further optimization of SLICE parameters is required for replicating crystal structure coordinates for some systems. We discuss in the following pages the various SLICE parameters and how they can be optimized for the system at hand.

<p>Catalytic reactions are limited in their turnover by certain steps in the cycle. We present a free, open-source, web-based interface to generate visualizations of the rate constants of various steps in the cycle. Population of a web form using known data will generate a highly customizable graphic for annotation by the user to represent their chemistry.</p>

We present a new methodology for accelerated dynamics in simulations of binding events of large flexible ligands at flexible protein binding sites. SLICE (Selective Ligand-Induced Conformational Ensemble) combines opportunistic stochastic jumps within the conformational space with standard molecular dynamics, to model the complex conformational evolution in the induced-fit binding of a peptide ligand, starting from the unbound protein structure. To specifically target the structural adaptations of the complex at the binding location, conformational jumps are selected in SLICE from among highly ranked structures generated by the docking software, AutoDock Vina.Binding trajectories are followed using molecular dynamics in the Amber code, until the next configurational jump that starts the next SLICE iteration. The method is verified against a test system consisting of Chromobox protein homolog 8 (CBX8) and its ligand, H3K9Me 3 , for which the final (bound) configuration is known. Application of the SLICE simulation method to the test system yielded a predicted structure in agreement with crystal structure coordinates. Several concurrent direct molecular dynamics simulations