The Histone Methyltransferase DOT1L: Discovery of Small-Molecule Inhibitors and its Role in Wnt Signaling.

Covalent post translational modifications of histone proteins are an important mechanism of epigenetic gene regulation that modulate chromatin structure. Methylation of histone lysine residues is one of several chemical marks that establish the “histone code” essential for proper temporal and spatial expression of gene programs required for cell fate determination in development. Dysregulation of histone methylation contributes to the development of numerous human diseases, particularly cancer. The sole histone H3 lysine 79 (H3K79) methyltransferase, DOT1L, is required for leukemogenic transformation in a subset of leukemias bearing translocations of the MLL gene. Human leukemias carrying MLL gene rearrangements aberrantly recruit DOT1L to leukemogenic genes leading to increased H3K79 methylation and their transcriptional activation. There are also reports that DOT1L plays a role in Wnt signaling, a pathway frequently dysregulated in colon cancer. Small molecule inhibitors of DOT1L are highly sought for the development of therapeutics in leukemia and as chemical tools to probe the role of DOT1L in other human diseases. We applied several approaches for the identification of DOT1L inhibitors, virtual screening, de novo design, and biochemical screening. Here we present the biochemical, biophysical, and cellular characterization of different classes of DOT1L inhibitors. Several S-adenosylmethionine (SAM) analogues have been identified as DOT1L inhibitors by virtual screening and we developed a novel pathway for synthesis of additional 5’ modified adenosine analogues. Additionally, we identified UMD-7, which inhibits H3K79 methylation by a unique mechanism of histone binding and phenocopies genetic loss of DOT1L. Employing a chemical biology approach the requirement for H3K79 methylation in Wnt signaling was investigated by inhibiting DOT1L with EPZ004777, a selective and potent SAM competitive inhibitor. Our findings indicate that H3K79 methylation is not essential for maintenance or activation of Wnt pathway target gene expression in colon cancer cell lines. Furthermore, H3K79 methylation is not elevated in human colon carcinoma samples in comparison with normal colon tissue. Therefore, our findings indicate that inhibition of DOT1L histone methyltransferase activity is likely not a viable therapeutic strategy in colon cancer.PhDMolecular and Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/110377/1/gsgibbon_1.pd

Characterization of the Protein-Protein Interactions between Histone Methyltransferase DOT1L and MLL Fusion Proteins towards Developing Small Molecule Inhibitors.

The MLL fusion proteins activate target genes in part via recruitment of DOT1L (disruptor of telomeric silencing 1-like), a histone H3 lysine 79 (H3K79) methyltransferase. The resulting hypermethylation of H3K79 at the Hox and MEIS1 loci is a pivotal event for leukemogenesis in acute leukemia, suggesting that the protein-protein interactions (PPIs) between DOT1L and MLL oncogenic fusion proteins represent a potential therapeutic target. The PPIs between DOT1L and the MLL fusion proteins, AF9 and ENL, were characterized. It was determined that full length DOT1L protein binds to AF9 and ENL with Kd of 33 nM and 206 nM respectively. The AF9/ENL binding site in human DOT1L was mapped and 10 amino acids (DOT1L865-874), highly conserved in DOT1L from a variety of species, were identified as essential for binding to AF9/ENL. Alanine scanning mutagenesis studies demonstrated that four conserved hydrophobic residues are crucial for the binding. Functional colony forming unit (CFU) assay showed that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that the physical interaction between DOT1L and MLL-AF9 is required for transformation by MLL fusions. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy for MLL leukemias with potentially fewer adverse effects than enzymatic inhibition of DOT1L. High-throughput screening (HTS) approach was employed for the discovery of small-molecule inhibitors targeting the PPI between DOT1L and MLL-fusion proteins. Approximately 101,000 compounds were screened and 39 compounds demonstrated dose-dependent inhibition of AF9-DOT1L interaction. For the first time, four compounds with diverse chemical structure were identified and validated as inhibitors of the DOT1L - AF9 PPI with IC50 values ranging from 10 – 50 µM. This is an important initial proof-of-concept for the development of small molecules targeting C-terminal hydrophobic domain in AF9, indicating it is a “druggable” target. Such inhibitors can serve as chemical probes to understand the biological consequences of inhibiting DOT1L recruitment by MLL-fusion proteins, AF9 and ENL, and the impact on leukemia development, as well as on normal hematopoiesis. Ultimately, this work may result in the development of novel drugs for treatment of MLL acute leukemias.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99831/1/shenchen_1.pd

Interrogating DOT1L Recruitment by MLL-Fusion Proteins MLL-AF9 and MLL-ENL Towards the Development of Novel Targeted Therapy

The Mixed Lineage Leukemia (MLL) is a Trithorax (Trx) transcriptional regulator often implicated in leukemic oncogenesis. MLL1 rearrangement leukemia is a very aggressive form of leukemia that results from a translocation of chromosome 11q23. The translocation gives rise to a chimeric protein that consists of the N-terminus MLL and 1 of more than 80 fusion partners. The most common fusion partners are ENL, AF9, and AF4. ENL and AF9 are a part of the YEATS family of proteins, containing an N-terminal histone acetylation reading YEATS domain and a C-terminal ANC1 homology domain (AHD). AF9 and ENL’s AHD recruit either the Super Elongation Complex (SEC) or the histone 3 lysine 79 (H3K79) histone methyltransferase (HMT) disrupter of telomeric silencing 1-like (DOT1L) to activate gene transcription. Recruitment of DOT1L proved to be essential for the transforming activity of multiple MLL fusion proteins. Our lab mapped the 10 amino acid (865 – 874) binding site on DOT1L, which binds to the AHD domain of AF9/ENL. Applying alanine mutagenesis studies, a point mutation, I867A, was identified sufficient to disrupt the AF9-DOT1L interaction in vitro and demonstrated that DOT1L lacking the 10 amino acids (Δ10) was unable to support transformation by MLL-AF9. In this study, we used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis to further validate the disruption of the AF9-DOT1L and ENL-DOT1L protein-protein interactions (PPI) as a potential therapeutic approach. We demonstrate that disrupting the AF9-DOT1L PPI inhibits leukemic cell growth, downregulates HOXA9 and MEIS1 gene expression, leading to cell differentiation and inducing apoptosis. These observed effects were similar to enzymatic inhibition. Nevertheless, PPI deficient adult hematopoietic cells completely reconstituted the bone marrow of mice; whereas, cells lacking DOT1L or its catalytic activity were not. We successfully designed a DOT1L peptidomimetic with a KD of 10 nM against AF9 and 25 nM to ENL that is cellularly active and selective for MLL-AF9 transduced murine cells over a non-DOT1L dependent, E2A-HLF, cells. These results emphasize the critical role of the AF9-DOT1L PPI in leukemic cell growth, but not for adult hematopoiesis making it an attractive therapeutic approach for MLL-rearrangement leukemia. Due to the homology between the AHD domain of AF9 and ENL, we utilized the same genetic approach as with MLL-AF9 to determine if blocking DOT1L recruitment in MLL-ENL cells would yield the same effect on leukemogenesis. We showed that blocking DOT1L recruitment is not sufficient to fully inhibit leukemic cell growth. We postulate that this is due the retention of the YEATS domain in the MLL-ENL fusion that is lost in MLL-AF9. We characterized two interactions that could be contributing to leukemogenesis, Paf1-YEATS, and YEATS-H3. We demonstrate that Paf1 directly interacts with ENL YEATS with a binding affinity of 15 nM and confirm the YEATS-H3K27ac interaction with a binding affinity of 80 M. We developed a fragment-based screening method using DSF to identify compounds that bind to the YEATS domain. The development of these tools will allow us to probe both the YEATS-Paf1 and YEATS-H3 interactions to determine which interactions are critical for MLL-ENL driven leukemia. Overall, these studies show the characterization of several PPIs involved in MLL-AF9/ENL leukemia and the development of tools to further elucidate their roles in leukemic and non-leukemic contexts towards novel therapy.PhDMolecular & Cellular PathologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149845/1/smgrigs_1.pd

Novel epigenetic therapies in hematological malignancies: Current status and beyond

Over the last decade transcriptional dysregulation and altered epigenetic programs have emerged as a hallmark in the majority of hematological cancers. Several epigenetic regulators are recurrently mutated in many hematological malignancies. In addition, in those cases that lack epigenetic mutations, altered function of epigenetic regulators has been shown to play a central role in the pathobiology of many hematological neoplasms, through mechanisms that are becoming increasingly understood. This, in turn, has led to the development of small molecule inhibitors of dysregulated epigenetic pathways as novel targeted therapies for hematological malignancies. In this review, we will present the most recent advances in our understanding of the role played by dysregulated epigenetic programs in the development and maintenance of hematological neoplasms. We will describe novel therapeutics targeting altered epigenetic programs and outline their mode of action. We will then discuss their use in specific conditions, identify potential limitations and putative toxicities while also providing an update on their current clinical development. Finally, we will highlight the opportunities presented by epigenetically targeted therapies in hematological malignancies and introduce the challenges that need to be tackled by both the research and clinical communities to best translate these novel therapies into clinical practice and to improve patient outcomes.We would like to thank all the members of the Huntly laboratory and our funders including the European Research Council, MRC, Bloodwise, the Kay Kendall Leukaemia Fund, the Wellcome Trust, the Cambridge NIHR Biomedical Research Centre, and core support grants to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute

Design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and ubiquitination facilitator Keap1

This thesis details the design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and the ubiquitination facilitator Keap1. The thesis is in two parts as outlined below. Part 1: The first part of this thesis details efforts towards identification of novel small molecule inhibitors of DOT1L, a histone methyltransferase which has been implicated in the development and proliferation of mixed lineage leukaemias (MLL). This work aims to optimise the drug-like properties of published DOT1L inhibitors while retaining potency, through further exploration of the nucleobase template. Structure-activity relationships (SARs) identified polar substituents and small heterocycles as favourable replacements for the halogen in 5-ITC, a small molecule inhibitor of DOT1L. Alternative nucleobase templates also demonstrated comparable DOT1L inhibition. To demonstrate proof of concept, a polar nitrile substituent was translated into the inhibitor Br-SAH as a direct replacement of the bromide. Activity was retained and a crystal structure obtained which demonstrated the nitrile occupied the same hydrophobic pocket. This work also demonstrated the use of a nitrile as a non-traditional replacement for heavy halogen atoms. Part 2: The second part of this thesis details identification of novel inhibitors of the Keap1-Nrf2 protein-protein interactions (PPI) using an approach based on kinetic target-guided synthesis (kTGS). Keap1 is a dimeric cytoplasmic protein that mediates the ubiquitination of Nrf2, a transcription factor that acts as a regulator of cellular antioxidant responses. Disruption of the PPI between Keap1 and Nrf2 has been shown to have a therapeutic benefit in diseases associated with oxidative stress and inflammation as well as providing a potential route to chemopreventative agents for cancer. Biased kTGS was applied as proof of concept. A biased ligand was designed and screened against a focused library of azides. The 1,3-dipolar cycloaddition products formed in the presence of the Keap1 Kelch domain were evaluated and validated through chemical synthesis and screening. A novel triazole structure was identified with improved activity over the initial biased fragment thus demonstrating kTGS as a valid approach for identifying novel inhibitors of the Keap1-Nrf2 PPI interaction

Therapeutic targeting in pediatric acute myeloid leukemia with aberrant HOX/MEIS1 expression

Despite advances in the clinical management of childhood acute myeloid leukemia (AML) during the last decades, outcome remains fatal in approximately one third of patients. Primary chemoresistance, relapse and acute and long-term toxicities to conventional myelosuppressive therapies still constitute significant challenges and emphasize the unmet need for effective targeted therapies. Years of scientific efforts have translated into extensive insights on the heterogeneous spectrum of genetics and oncogenic signaling pathways of AML and identified a subset of patients characterized by upregulation of HOXA and HOXB homeobox genes and myeloid ecotropic virus insertion site 1 (MEIS1). Aberrant HOXA/MEIS1 expression is associated with genotypes such as rearrangements in Histone-lysine N-methyltransferase 2A (KMT2A-r), nucleoporin 98 (NUP98-r) and mutated nucleophosmin (NPM1c) that are found in approximately one third of children with AML. AML with upregulated HOXA/MEIS1 shares a number of molecular vulnerabilities amenable to recently developed molecules targeting the assembly of protein complexes or transcriptional regulators. The interaction between the nuclear scaffold protein menin and KMT2A has gained particular interest and constitutes a molecular dependency for maintenance of the HOXA/MEIS1 transcription program. Menin inhibitors disrupt the menin-KMT2A complex in preclinical models of KMT2A-r, NUP98-r and NPM1c acute leukemias and its occupancy at target genes leading to leukemic cell differentiation and apoptosis. Early-phase clinical trials are either ongoing or in development and preliminary data suggests tolerable toxicities and encouraging efficacy of menin inhibitors in adults with relapsed or refractory KMT2A-r and NPM1c AML. The Pediatric Acute Leukemia/European Pediatric Acute Leukemia (PedAL/EUPAL) project is focused to advance and coordinate informative clinical trials with new agents and constitute an ideal framework for testing of menin inhibitors in pediatric study populations. Menin inhibitors in combination with standard chemotherapy or other targeting agents may enhance anti-leukemic effects and constitute rational treatment strategies for select genotypes of childhood AML, and provide enhanced safety to avoid differentiation syndrome. In this review, we discuss the pathophysiological mechanisms in KMT2A-r, NUP98-r and NPM1c AML, emerging molecules targeting the HOXA/MEIS1 transcription program with menin inhibitors as the most prominent examples and future therapeutic implications of these agents in childhood AML.</p

The role of VRK1 in chromatin remodeling: regulation of histone post-translational modifications and epigenetic enzymes

[ES]La organización del ADN es esencial para el correcto empaquetamiento de la cromatina y necesaria para facilitar distintos procesos celulares que requieren una remodelación dinámica de la cromatina. La modulación de su estructura es esencial para la regulación de la expresión génica, ya que determina qué genes son accesibles y qué factores reguladores son reclutados al ADN. Por ello, las células eucariotas han desarrollado mecanismos que permiten la modulación y apertura de la cromatina cuando esta es inaccesible. Las alteraciones epigenéticas son un mecanismo para controlar la accesibilidad del ADN y regular los patrones de expresión génica y el desarrollo normal. El control epigenético puede producirse a través de la metilación del ADN, las modificaciones postraduccionales (PTM) de las histonas, la interpretación de estas modificaciones por las enzimas epigenéticas, el intercambio de variantes de las histonas y el ARN no codificante. Los errores en estos mecanismos pueden desregular el control de los procesos basados en la accesibilidad de la cromatina, lo que conduce a una expresión anormal de distintos genes. Se ha descubierto que condiciones patológicas como el cáncer, los trastornos metabólicos y las enfermedades inflamatorias y neurodegenerativas están relacionadas con errores epigenéticos. Las modificaciones postraduccionales (PTM) de las colas N-terminales de las histonas regulan el acceso al ADN. Es importante destacar que las PTM de las histonas son reversibles y su coordinación requiere la regulación estricta de múltiples enzimas epigenéticas, conocidas como writers (enzimas que añaden una modificación) y erasers (enzimas que eliminan una modificación). Cabe destacar la acetilación y la metilación entre las diversas PTMs, ya que ha sido ampliamente estudiada su relación con el cáncer y su respuesta a tratamientos. El equilibrio entre la de- y la acetilación está controlado por las deacetilasas (HDAC) y las acetiltransferasas (HAT), mientras que la de- y la metilación están reguladas por las demetilasas (KDM) y las metiltransferasas (KMT). Una actividad anómala de las enzimas epigenéticas y, por consiguiente, una alteración del patrón de PTM de histonas pueden alterar distintos procesos celulares como la proliferación, la reparación del ADN, la transcripción génica y la replicación del ADN, así como la expresión de genes supresores de tumores o asociados al cáncer. Por ello, es crucial desvelar los mecanismos moleculares que controlan estos cambios y las enzimas epigenéticas implicadas en dicha regulación. VRK1 (Vaccinia-related kinase 1) es una quinasa nuclear implicada en diferentes procesos celulares, como la modulación de factores de transcripción o proteínas implicadas en la respuesta al daño del ADN. La localización de VRK1 en el núcleo la convierte en una posible candidata para participar en la remodelación de la cromatina y, por tanto, en una potencial diana terapéutica. Sin embargo, el diseño de inhibidores específicos para quinasas, especialmente para VRK1 debido a su estructura, sigue siendo un reto importante. Por ello, esta tesis pretende ampliar el conocimiento del papel de la quinasa humana VRK1 en la remodelación de la cromatina, descifrando la regulación del patrón de PTMs de histonas y caracterizando nuevos posibles sustratos como las enzimas epigenéticas. Otro objetivo de este trabajo es caracterizar un nuevo inhibidor de VRK1, VRK-IN-1, para comprender su mecanismo de acción y proponer así la inhibición de VRK1 como posible tratamiento del cáncer. En este trabajo, se ha demostrado que la ausencia de VRK1 altera completamente el patrón de PTM de histonas, siendo capaz de imitar el efecto de algunos inhibidores de enzimas epigenéticas. La depleción de VRK1 causa una disminución de los niveles de H3K4me3, H3K9ac, H3K27ac, H3K79me2 y H4K16ac, y un aumento de H3K9me3 y H3K27me3 en células de adenocarcinoma de pulmón y osteosarcoma. Además, VRK1 puede formar un complejo proteico estable con HDAC1, SIRT1, SIRT2, SETDB1, LSD1, JMJD1A y JMJD2A, lo que sugiere que VRK1 podría estar controlando la actividad de estas enzimas epigenéticas. Además, se observó que la inhibición de la actividad quinasa de VRK1 mediante el tratamiento con VRK-IN-1 muestra resultados similares a la disminución de la expresión de VRK1, bloqueando la proliferación celular, alterando el patrón PTM de histonas y la respuesta génica tras daño en el ADN. En conjunto, estos hallazgos proponen a VRK1 como un regulador de la cromatina, capaz de interactuar con diferentes enzimas epigenéticas y mantener los PTMs de histonas asociados un estado relajado. Además, los datos presentados, no sólo proporcionan un nuevo recurso para investigar inhibidores más específicos de VRK1, sino que también abren una puerta a nuevas oportunidades para la inhibición de VRK1. [EN]DNA organization is essential for proper chromatin packaging and necessary to facilitate different processes that require dynamic chromatin remodeling. Modulation of chromatin structure is critical for the regulation of gene expression, since it determines which genes are accessible for transcription and the sequential recruitment of regulatory factors to the underlying DNA. Thus, to deal with inaccessible chromatin, eukaryotic cells have developed mechanisms that facilitate the opening of chromatin. Epigenetic alterations are defined as mechanisms that control DNA accessibility for the regulation of gene expression patterns and normal development. The epigenetic transcriptional control can occur through DNA methylation, histone post-translational modifications (PTMs), the reading of these modifications by epigenetic enzymes, histone-variants exchange, and noncoding RNA. Errors in the epigenetic regulation can alterthe control of chromatin-based processes, ultimately leading to abnormal gene expression. Pathological conditions such as cancers, metabolic disorders, and inflammatory and neurodegenerative diseases have been found to be related to epigenetic errors. Post-translational modifications (PTMs) of the N-terminal tail of histones regulate DNA access. Importantly, histone PTMs are reversible, and their coordination requires a tight regulation of multiple epigenetic enzymes, known as writers (enzymes that add a mark) and erasers (enzymes that remove a mark). Among the different PTMs, acetylation and methylation are especially important. They have been extensively investigated in a context of cancer and therapy responses. The balance between de- and acetylation is controlled by deacetylases (HDACs) and acetyltransferases (HATs), while de- and methylation are regulated by demethylases (KDMs) and methyltransferases (KMTs). An abnormal activity of the epigenetic enzymes and, subsequently, a disturbed histone PTM landscape can alter different cellular processes such as proliferation, DNA repair, gene transcription, and DNA replication, together with the expression of tumor-suppressor or cancer-associated genes. Thus, it is crucial to unveil the molecular mechanisms that modulate these changes and the histone-modifying enzymes involved in such regulation. VRK1 (Vaccinia-related kinase 1) is a nuclear kinase implicated in different cellular processes, such as modulation of transcription factors (TFs) or proteins implicated in the DNA damage response (DDR). The location of VRK1 as a nucleus-resident kinase makes it a suitable candidate to participate in chromatin remodeling and, thus, a potential therapeutic target. Therefore, this thesis aims to broaden the knowledge of the role of the human kinase VRK1 in chromatin remodeling, deciphering the regulation of histone PTMs patterns and characterizing new possible epigenetic enzymes substrates of VRK1. Another goal of this work is characterizing a novel VRK1 inhibitor, VRK-IN-1, to understand its mechanism of action and propose VRK1 inhibition as a potential cancer therapy. In this work, we demonstrate that the absence of VRK1 alters the histone PTM landscape, mimicking the effect of some epigenetic enzyme inhibitors. VRK1 depletion causes a decrease of H3K4me3, H3K9ac, H3K27ac, H3K79me2 and H4K16ac levels, and an increase of H3K9me3 and H3K27me3 levels in lung adenocarcinoma and osteosarcoma cellular models. Furthermore, VRK1 can form a stable protein complex with HDAC1, SIRT1, SIRT2, SETDB1, LSD1, JMJD1A and JMJD2A, suggesting that VRK1 controls the activity of these epigenetic enzymes. In addition, we observed that the inhibition of VRK1 kinase activity by using VRK-IN-1 resembles the VRK1 depletion, thus blocking cell proliferation, impairing histone PTM patterns and altering the DDR upon DNA damage induction. Altogether, these findings reveal VRK1 as an orchestrator of chromatin remodeling, capable of interacting with different epigenetic enzymes and maintaining histone PTMs associated with relaxed chromatin. Moreover, the present data provides a resource for investigating novel VRK1 inhibitors, as well as exploring new target therapies through the biochemical mechanisms here uncovered

Innovative approaches to cancer therapy

Cancer is a term used to describe complex diseases characterized by abnormal cell-proliferation with different factors to take into consideration, reason for which nowadays there are many types of treatments with limited success. Therefore, innovative anticancer agents are required. Recently, anticancer research has been focused on epigenetics. Among epigenetic actors, Chromodomain (ChD)-containing proteins have gained ever-increased interest. These proteins, whose altered expression or mutations in cancer lead to abnormal gene transcription, are involved in epigenetic regulation of transcription by recognizing methylated lysine residues on histones. According to this information, in the first part of this thesis, an innovative epigenetic approach was applied to design and synthetize small-molecules as potential ChDs binders for cancer treatment. In detail, we reported the identification of two hit compounds (2 and 3) and the further structure-activity relationship studies. A fluorescence polarization competition assay was employed to evaluate the obtained derivatives. Cancer research activities have also taken advantage of innovation in emerging technologies. As an example, technical advances have allowed to apply X-ray crystallography in a high-throughput way for compound screening. Therefore, in the second part of this work a fragment-based drug discovery approach based on high-throughput X-ray crystallography was applied, in collaboration with Paul Scherrer Institute, for identifying new binders of tubulin. In this context, compound 29 was identified as a binder of tubulin Colchicine-site, previously validated for cancer treatment. Structural similarity of this compound with Nocodazole, an antineoplastic agent co-crystallized with tubulin at the same Colchicine-site, and the capability of both molecules to bind the same tubulin pocket, encouraged us to develop two series of 29 analogs by combining synthetic efforts with computer-aided drug design protocols. For some synthetized derivatives, X-ray crystallography and Resazurin assay were employed to evaluate the binding ability at the tubulin Colchicine-site and the capacity to affect cell viability, respectively

Structure-Based Drug Discovery Against Human ENL YEATS Domain and SARS-CoV-2 Main Protease

Structure-based drug design is a drug discovery strategy where rational design of drug molecules take place based on the structural information of therapeutic targets. With the development of structural biology technologies such as protein crystallography and cryo-electron microscopy, which results in the availability of more and more proteins in a higher and higher resolution, structure-based drug design has become one of the most useful drug discovery strategy in both academia and pharmaceutical industry. This dissertation discusses applying structure-based drug design strategies in inhibitor development targeting ENL (eleven-nienteen leukemia) protein, which is an important protein in the mix lineage leukemia (MLL)-rearranged leukemia, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease, a vital viral enzyme for its replication. Chapter I is a brief introduction to the topics of this dissertation. Starting with a short introduction of the concept of structure-based drug design, it then mainly discusses the molecular mechanism of the pathogenesis and potential therapeutic targets of the following two diseases: MLL-rearranged leukemia and COVID-19. Chapter II describes the development of a series of selective ENL YEATS domain inhibitors. ENL is a histone acetylation reader essential for disease maintenance in acute leukemias, especially the MLL-rearranged leukemia. The function of ENL is dependent on the recognition of histone acetylation by its YEATS domain, suggesting that inhibition of the ENL YEATS domain is a potential strategy to treat MLL-rearranged leukemia. In our study, high-throughput screening of a small molecule library was carried out to identify inhibitors for the ENL YEATS domain. Structureactivity relationship studies of the hits and structure-based inhibitor design led to two compounds with IC50 values below 100 nM in inhibiting the ENL-acetyl-H3 interaction. Both compounds and their precursor displayed strong selectivity toward the ENL YEATS domain over all other human YEATS domains. One of these compounds also exhibited on-target inhibition of ENL in cultured leukemia cells and a synergistic effect with the BET bromodomain inhibitor JQ1 in killing leukemia cells. Together, we have developed selective inhibitors for the ENL YEATS domain, providing the basis for further medicinal chemistry-based optimization to advance both basic and translational research of ENL. Chapter III and IV describes the development of SARS-CoV-2 main protease inhibitors and the assessment of their selectivity against host proteases. The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2Mpro) to digest two of its translated long polypeptides to form mature viral proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV main protease (SC1Mpro), we designed and synthesized a series of peptidyl aldehyde inhibitors that reversibly covalently bind to the active cysteine of SC2Mpro. The most potent compound has an IC50 of 8.3 nM. Crystallographic analysis confirmed the covalent linkage between the aldehyde inhibitors and active cysteine and showed structural rearrangement of the apoenzyme to accommodate the inhibitors. Two inhibitors completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5–5 μM and A549/ACE2 cells at 0.16–0.31 μM. Even though a number of inhibitors have been developed for the SARS-CoV-2 main protease as potential COVID-19 medications, little is known about their selectivity. Using enzymatic assays, we characterized inhibition of TMPRSS2, furin, and cathepsin B/K/L by 11 previously developed Mpro inhibitors. Our data revealed that all these inhibitors are inert toward TMPRSS2 and furin. Diaryl esters also showed low inhibition of cathepsins. However, all aldehyde inhibitors displayed high potency in inhibiting three cathepsins. A cellular analysis indicated high potency of MPI5 and MPI8 in inhibiting lysosomal activity, which is probably attributed to their inhibition of cathepsins. Among all aldehyde inhibitors, MPI8 shows the best selectivity toward cathepsin L. With respect to cathepsin B and K. MPI8 is the most potent compound among all aldehyde inhibitors in inhibiting SARS-CoV-2 in Vero E6 cells. Cathepsin L has been demonstrated to play a critical role in the SARS-CoV-2 cell entry. By selectively inhibiting both SARS-CoV-2 MPro and the host cathepsin L, MPI8 potentiates dual inhibition effects to synergize its overall antiviral potency and efficacy. Due to its high selectivity toward cathepsin L that reduces potential toxicity toward host cells and high cellular and antiviral potency, we urge serious consideration of MPI8 for preclinical and clinical investigations for treating COVID-19