This dissertation presents a collection of biological simulations and predictions in collaboration with experiment to support and elucidate the trends observed in various protein–ligand systems. Within the model systems, there is a strong focus on the support for the development of peptidomimetic inhibitors for post-translational reader proteins (CBX proteins). The systems studied throughout this document each present their own unique challenges but fall under the general theme of protein flexibility and the difficulties of sampling such systems. As part of this work, methodological advances were made to address the challenges of structural prediction on flexible proteins and ultimately form the method Selective Ligand-Induced Conformational Ensemble (SLICE). The development, validation, and future directions of the SLICE method are also discussed. Ultimately, the collaborative efforts presented in this dissertation bring forward a greater understanding of the drug design challenges on the CBX proteins as well a new methodology in the field of structure-based drug design. / Graduate

Writer, eraser, and reader proteins are three classes of proteins/enzymes that add, remove, and recognize post-translational modifications (PTMs) on histone tails, respectively. The orchestrated action of these protein classes controls dynamic state of chromatin and influences gene expression. Dysregulation of these proteins are often associated with disease conditions. All three classes are targeted with small molecule inhibitors for various disease conditions. This is a promising area of research to develop therapeutics for various clinical conditions. I worked on a methyllysine reader protein Cbx7, which belong to polycomb group of proteins. Cbx7 is a chromodomain containing protein and it uses its chromodomain to recognize methyllysine partners such as H3K27me3. Aberrant expression of Cbx7 is observed in several cancers including prostate, breast, colon, thyroid, etc. Hence targeting Cbx7 with potent and selective inhibitors would be beneficial for therapeutic intervention for Cbx7 associated diseases. Here I report my work on design, synthesis, and evaluation of Cbx7 inhibitors. In my work, we identified several potent and selective inhibitors for Cbx7 and we published first-in-class antagonists for Cbx7. Few of these inhibitors were tested on cancer stem cell models. Further, I propose future work for targeting Cbx7 and other chromodomain containing proteins. / Graduate / 2018-09-04

<p>Polycomb repressive complex 1 (PRC1) is critical for mediating gene expression during development. Five chromobox (CBX) homolog proteins, CBX2,4,6,7,8, 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 similarity in sequence and structure among the CBX ChDs provide a major obstacle in developing selective CBX ChD inhibitors. Here a selection of small, focused, DNA-encoded libraries (DELs) against multiple homologous ChDs was reported 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 Kd 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 significantly decreases 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.</p> <p>CBX2 is upregulated in a variety of cancers, particularly in advanced prostate cancers. Using CBX2 inhibitors to understand and target CBX2 in prostate cancer is highly desirable. Here, selections of focused DNA encoded libraries (DELs) were performed for the discovery of a selective CBX2 chromodomain probe, SW2_152F. SW2_152F binds to CBX2 ChD with a Kd of 80 nM and displays 24-1000-fold selectivity for CBX2 ChD over other CBX paralogs <em>in vitro</em>. SW2_152F is cell permeable, selectively inhibits CBX2 chromatin binding in cells, and blocks neuroendocrine differentiation of prostate cancer cell lines in response to androgen deprivation.</p> <p>Targeted covalent inhibitors (TCIs) are rationally designed inhibitors that bind to a target protein and specifically label a non-conserved amino acid on proteins by means of reactive moieties (warheads). TCIs typically function by two steps, in which inhibitors first non-covalently bind to the target protein and then covalent bond formation occurs between the inhibitor- warhead and a proximal nucleophile on protein. Covalent inhibitors or drugs have prolonged target engagement and enhanced pharmacokinetic potency in vivo, compared to non-covalent molecules. Strategies to develop effective warheads of TCIs have been reported for labeling different nucleophilic amino acid residues, of which cysteine and lysine are the most established for covalent labeling. Tyrosine is recently becoming an attractive nucleophile for TCIs as an alternative choice, yet currently developed warheads that label tyrosine do so with modest specificity over other side chains. Here, I report the development of novel Mannich electrophiles and use those electrophiles as covalent warheads on an inhibitor to specifically target tyrosine in protein labeling. To my knowledge, this is first demonstration of the use of Mannich electrophiles in covalent inhibitors. Specifically, I leveraged a previously developed CBX8 chromodomain inhibitor to specifically label a non-conserved tyrosine within CBX8 using cyclic imine derivatives as warheads. This ligand-directed, specific tyrosine conjugation on CBX8 but not on CBX2, significantly improves both the potency and selectivity of inhibition. Biochemical, proteomic, and cellular validation further showed the cyclic imine covalent inhibitors can increase both potency and selectivity to the target protein CBX8 in cells, serving as a robust chemical probe for target function evaluation and modulation. This new type of tyrosine labeling warhead is a useful addition to the toolbox of medicinal chemists for covalent inhibitor development.</p> <p>The following chapters are modified from following publications, with permissions from Sijie Wang, Emily C.Dykhuizen, and Casey J. Krusemark. </p> <p>Wang, S., Denton, K. E., Hobbs, K. F., Weaver, T., McFarlane, J. M., Connelly, K. E., Gignac, M.C., Milosevich, N., Hof, F., Paci, I., Musselman, C. A., Dykhuizen, E.C., Krusemark, C. J. Optimization of Ligands Using Focused DNA-Encoded Libraries To Develop a Selective, Cell-Permeable CBX8 Chromodomain Inhibitor. <em>ACS Chem Biol. </em>2020, 15, 112-131</p> <p>Wang, S., Alpsoy, A., Sood, S., Ordonez-Rubiano, S. C., Dhiman, A., Sun, Y., Krusemark, C. J., Dykhuizen, E. C. A Potent, Selective CBX2 Chromodomain Ligand and its Cellular Activity During Prostate Cancer Neuroendocrine Differentiation. <em>ChemBioChem.</em> 2021, 22, 2335-2344</p> <p>Wang, S., Ordonez-Rubiano, S. C., Dhiman, A., Jiao G., Strohmier B. P., Krusemark, C. J., Dykhuizen, E. C. Polycomb Group proteins in cancer: multifaceted functions and strategies for modulation Modulators. <em>NAR Cancer</em>. 2021, 3, zcab039</p>