Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors. Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity. Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively. Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione. The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22412号 / 工博第4673号 / 新制||工||1729(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.

Le domaine de l'épigénétique couvre l'ensemble des phénomènes héritables et transmissibles qui interviennent dans l'expression du génome sans modifier la séquence nucléotidique. L'information épigénétique est régulée par les modifications de la chromatine impliquant les histones et l'ADN. La méthylation de l'ADN est un phénomène réversible jouant un rôle crucial dans l'expression des gènes puisque la méthylation des promoteurs de gènes empêche leur transcription. La modulation aberrante de cette marque épigénétique est associée à diverses pathologies telles que le cancer. Cette méthylation étant réversible, elle peut être ciblée afin de reprogrammer la cellule cancéreuse. Les méthyltransferases d'ADN (DNMT), étant les enzymes responsables de la méthylation, représentent la cible principale de notre stratégie de recherche. Leur inhibition par des petites molécules est au centre de nos recherches de thérapies anticancéreuses dont les bases sont représentées par deux catégories d'inhibiteurs de DNMT existant. Les premiers sont des analogues de cytosine qui est la cible de la méthylation. Ils sont connus pour s'intégrer dans l'ADN et former un complexe covalent irréversible avec l’enzyme (complexe suicide) mais ils souffrent d'un manque de stabilité et de certains effets indésirables dus à leur incorporation dans l’ADN. Les seconds sont les inhibiteurs non nucléosidiques qui sont divers et parfois connus pour cibler d’autres enzymes. Ils ont l’avantage de pouvoir être utilisés comme sondes pour comprendre plus précisément le mécanisme d'inhibition mais ils manquent de spécificité et de sélectivité. Au cours de cette thèse, une banque de molécules a été criblée à partir de la combinaison d'un test enzymatique et d'un test cellulaire visant à inhiber ces enzymes. Les synthèses de trois familles de molécules potentiellement inhibitrices de DNMT issus de ce criblage sont décrites en expliquant le chemin de drug design emprunté pour obtenir des informations mécanistiques d’inhibition de la méthylation d’ADN, notamment de réactivité avec la cible. Les découvertes ont été inspirées par des études de modélisation permettant de mettre en évidence une sélectivité de certains inhibiteurs. La synthèse chimique a également abouti à une nouvelle voie de synthèse d’accès aux diaminopyrimidines dont l’impact permet de faciliter les études chimiques de dérivés quinazolines comme inhibiteur non nucléosidiques utiles pour les thérapies anticancéreuses. / Epigenetic is defined as the study of heritable changes in the genes expression without altering the DNA sequence. Two main processes are implicated in this field, the histones modifications and the DNA methylation. By introducing an acetyl or a methyl group on the histone tails or by methylation of DNA, the chromatin state is modified and the gene expression is controlled. Aberrant epigenetic modifications are associated with several diseases, in particular with cancer. In cancer cells, the whole DNA is hypomethylated, thus promoting genome instability, while the promoter region is hypermethylated, inducing silencing of these genes. Overall, these observations indicate that DNA methylation is a central epigenetic process in cancerogenesis. Since DNA methylation is reversible, it is possible to target the methylation process in order to reactivate tumor suppressor genes. The DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, use the SAM co-factor at specific CpG sites to product 5-methylcytosine. Three main isoforms (DNMT1, DNMT3A and DNMT3B) are described to ensure efficient methylation process during replication. Two families of DNMT inhibitors already exist, the nucleosidiques analogues are cytidine derivatives and are toxic molecules because of their incorporation into DNA, and the non-nucleosidic analogues are less toxic but also less potent. Our strategy of drug design is based on docking study and high throughput screening (HTS) information. First, novel potent derivatives of reference inhibitors are designed from molecular modelling. Then, three different families of compounds from HTS are described with appropriate structure-activity relationship studies. Mechanistic information on DNA methylation process are described through the discovery of a reactive inhibitor of DNMT3A. The study on a family of hydrazone derivatives of gallic acid is depicted and shows its selectivity for DNMT3A, compared to DNMT1, based on docking study. An alternative chemical pathway to diaminopyrimidines is described and extended to the synthesis of quinazolone in order to synthesize new quinazoline derivatives as potent inhibitors of DNMT. Promising informations are described in this thesis to enrich epigenetic knowledge of tumor genesis and to provide new molecules for anticancer therapy.

<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>

<p dir="ltr">In this study, we presented the discovery of the first-in-class covalent inhibitor specific to Src homology 2 domain containing phosphatase 1 (SHP1), an overlooked cancer immunotherapy target. Through high-throughput screening, we identified a chloroacetamide fragment highly selective for SHP1. This fragment was subsequently refined to yield M029, a covalent inhibitor characterized by low-micromolar potency, heightened selectivity, enhanced stability, and improved bioavailability. Notably, M029 targets a cryptic, non-conserved cysteine residue on SHP1, thereby illuminating novel avenues for future drug development focused on SHP1. This presented study also marked the first characterization of SHP1 pharmacology inhibition <i>in vivo</i> using M029 as a tool compound. Consistent to previous genetic studies, SHP1 inhibition was observed to markedly bolster anti-tumor efficacy, primarily through the activation of CD8+ T cells and NK cells, coupled with a reduction in T cell exhaustion. While no synergistic effects were noted in conjunction with anti-PD-1 treatment, M029 as a standalone therapy showcased more favorable responses compared to anti-PD-1 therapy alone, underscoring its potential for clinical application.</p><p dir="ltr">Meanwhile, we also demonstrated the effects of targeting both protein tyrosine phosphatase 1B (PTP1B), and T cell protein tyrosine phosphatase (TC-PTP) using proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional small molecules comprising a targeted protein ligand, an E3 ligase ligand, and a linker. By recruiting an E3 ligase to the targeted proteins, PROTACs leverages the cell's ubiquitin-proteasome machinery to achieve selective target protein degradation. In contrast to traditional occupancy-based inhibitors, event-driven PROTACs show improved efficacy by promoting target protein degradation in a catalytic mode of action and greater selectivity through the obligatory formation of the target-PROTAC-E3 ternary complex, which is essential for efficient target degradation. Through optimizing the previously reported PROTAC DU-14, we acquired a cereblon (CRBN)-based PTP1B/TC-PTP dual targeting PROTAC X1 of higher bioavailability than DU-14. X1 showed enhanced efficacy than DU-14 in multiple cell lines and manifested anti-cancer efficacy <i>in vivo</i>. Additionally, employing X1 as a tool compound, we validated the anti-cancer potential of PTP1B/TC-PTP degradation in STAT3 dependent malignancies, such as non-Hodgkin’s lymphomas. Treatments with X1 or DU-14 effectively induced tumor cell apoptosis, whereas the dual inhibitor ABBV-CLS-484 failed to produce comparable outcomes.</p>

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