15 research outputs found

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

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    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

    Oncogene and Cancer

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    This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

    Interrogation of Dynamic Proteins to Expand the Druggable Proteome

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    The human proteome is vastly complex, and our understanding of it is constantly evolving. There are roughly 20,000 protein-coding genes in the human genome, yet only about 10% of the resultant proteins are deemed “druggable” targets. And, only half of those have disease relevance. Thus, the druggable proteome is surprisingly narrow, consisting largely of structured proteins with defined binding pockets. With so many disease signatures residing in the “undruggable” portion of the proteome, there is much work to be done to expand the druggable landscape. An area rich with disease relevance is dynamic protein-protein interactions (PPIs), which underpin many regulatory cellular functions both in healthy and diseased states. However, devoid of typical binding pockets that enable traditional drug discovery approaches (i.e. substrate mimicry), dynamic PPIs occur over large, flat surface areas, which is why they have remained “undrugged.” A disproportionate number of dynamic proteins can be found in transcriptional regulation. As such, it provides an interesting avenue for chemical probe development and therapeutic intervention. For instance, a hallmark of cancerous cells are rampant growth and proliferation, with many proteins being overexpressed. While many research efforts have focused on targeting the overexpressed proteins themselves, halting the overexpression at the transcriptional level could stop the disease progression at its initiation. This dissertation works towards expanding the druggable proteome by establishing principles of molecular recognition that guide native PPIs. By primarily using molecular dynamics simulations, with complementary biophysical experimentation, I dissect coactivators and establish rules of activator recognition and engagement. In doing so, I demonstrate the utility of disorder in transcriptional regulation. In particular, I identify ways in which allostery manifests in dynamic coactivator proteins. Further, I explore how inhibition / enhancement of particular PPIs can be achieved using small molecules that attenuate fluctuations and disrupt binding allosterically.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169881/1/apeiffer_1.pd

    Innovations in Targeting Dynamic Proteins With Small Molecule Modulators

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    For decades, small molecule modulators of protein function have been vital for advancing both understanding of basic biology and therapeutic discovery; however, there are classes of proteins for which small molecule discovery has lagged. The majority of the proteins studied in this work are transcriptional coactivators, proteins that have great potential as therapeutic targets, yet are often classified as “undruggable” because of their atypical structure and mechanism of action. Coactivators are highly malleable and their interactions typically occur over broad surfaces areas with only moderate affinity, making them difficult to target. Recent advances in protein-protein interaction (PPI) inhibitor discovery, exploiting allostery and dynamic substructures within target proteins, has led to some success, but selectivity remains a challenge. Selectivity is pivotal for candidate probe molecules due to the extensive interaction network of these dynamic hubs. The following dissertation addresses the challenges of selectivity and presents small molecules that target coactivator interactions. A large portion of this work is dedicated to targeting the activator interaction domain (AcID) of the coactivator Med25. This domain contains a unique protein fold and is not required for basal transcription, but interacts with activators to regulate genes implicated in disease. Previous work from the Mapp lab led to identification of promising AcID PPI inhibitors. Here, this work was continued with the compound norstictic acid (NA). NA demonstrated high selectivity for AcID interactions and, using biochemical techniques in combination with molecular dynamics simulations, the mechanism of inhibition was elucidated: covalent modification of lysine residues within a dynamic loop leads to both orthosteric and allosteric inhibition of activator binding. NA proved useful for studying Med25 in a cellular context, engaging with full length protein from cell extracts and inhibiting the interaction between endogenous Med25 and transcriptional coactivators. Ultimately NA was used to probe the interaction of Med25 and ETV5, a transcription factor linked to metastasis, in a metastatic breast cancer cell line. Dosing with NA was able to decrease expression of the Med25•ETV5 regulated gene MMP-2, similarly to what was observed with KO of Med25. Inspired by the iterative screening approach used to discover NA, a new small molecule screening method for PPIs was developed, the first method for coactivator targets that directly incorporates selectivity at the primary level. A fluorescence polarization assay that simultaneously monitors multiple activator-coactivator interactions is presented. This method enables assessment of both selectivity and potency of candidate inhibitors in a single screen. A duplex assay containing AcID and CBP KIX has been optimized and a pilot screen has been conducted. The pilot screen was able to categorize compounds as Med25 AcID-selective, CBP KIX-selective, or dual inhibitors. Representative compounds from each subset were evaluated in secondary screening, leading to identification of novel inhibitors of both proteins. Ultimately, this approach is applicable to other coactivator-activator complexes. The final focus of this dissertation is development of a biochemical screen for the human serine protease TMPRSS2, which plays a key role in SARS-CoV-2 viral infection. TMPRSS2 is a difficult protein target because expression and purification is challenging, thus complicating inhibitor screening and in vitro studies. An expression and purification procedure to isolate active TMPRSS2 protease domain from E. Coli was developed. Biochemical methods were used to characterize the mechanism of known inhibitors, and, through integration with computational methodologies, novel TMPRSS2 targeting chemical scaffolds were identified.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169794/1/garlickj_1.pd

    Tuberculosis

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    Data are rapidly accumulating from all over the world regarding the efficacy of standardized treatment regimens for drug-sensitive, drug-resistant TB and latent TB infection. While we are facing the menace of multi drug-resistant TB [MDR-TB], extensively drug-resistant tuberculosis [XDR­ TB] has emerged threatening to undermine global efforts at TB control. Hence we have included chapters to cover all aspects of the diagnosis and management of MDR TB. This book will cover all these developments in great detail. With the widespread availability of internet globally various standard web resources available on TB have also been included so that the readers may get the comprehensive and updated guidelines from these resources. The changing clinical presentation of TB, advances in laboratory, imaging diagnostic modalities, therapeutic measures and emergence of MDR TB all suggest a pressing need to have a updated book on TB. Furthermore, while all physicians encounter the TB disease in their clinical practice, there have been a lot of controversies and misconceptions over various issues for the diagnosis and management of TB. Paucity of a well referenced, updated, standard book of TB has prompted us to undertake this venture sharing the clinical experience of global experts of TB. Our book contains chapters on epidemiology, immune-pathology, diagnosis, treatment and latest advances for TB, highlighting the global perspective of tuberculosis. World-wide resurgence of MDR TB indicates that the battle against this foe of mankind will continue in the coming years. TB still remains to be a research priority of paramount importance from medical, social and financial aspects and we have attempted to highlight all the aspects for the treatment of TB. We believe that this book will serve as a practical guide for the diagnosis and management of TB for practicing physicians (especially pulmonologists and internists) and all those who are involved in the management of TB

    Targeting LEDGF/p75 to Sensitize Chemoresistant Prostate Cancer Cells to Taxanes

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    Prostate cancer (PCa) is the second most diagnosed cancer in males. This disease disproportionately affects African American men, with a higher incidence and mortality compared to other ethnic/racial groups. An aging male population and the complexity of addressing the health disparities associated with this disease puts PCa into the spotlight due to its serious public health implications and the imminent fiscal challenge over the next decades. Chronic prostate inflammation resulting in activation of stress and prosurvival pathways contribute to disease progression and the development of chemoresistance. Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stressresponse protein that promotes cellular survival against environmental stressors, including oxidative stress, radiation, and cytotoxic drugs. It is overexpressed in PCa and other cancers and has been associated with features of tumor aggressiveness, including resistance to cell death and chemotherapy. This research work shows that the endogenous levels of LEDGF/p75 are upregulated in metastatic castration resistant prostate cancer (mCRPC) cells selected for resistance to the taxane drug docetaxel (DTX). These cells also showed resistance to the taxanes cabazitaxel (CBZ) and paclitaxel (PTX), but not to the classical inducer of apoptosis TRAIL. Silencing LEDGF/p75 effectively sensitized taxane-resistant PC3 and DU145 cells to DTX and CBZ, as evidenced by a significant decrease in their clonogenic potential. While TRAIL induced apoptotic blebbing, caspase-3 processing, and apoptotic LEDGF/p75 cleavage, which leads to its inactivation, in both taxane- resistant and -sensitive PC3 and DU145 cells, treatment with DTX and CBZ failed to robustly induce these signature apoptotic events. Also, pretreatment with caspase inhibitor zVAD partially rescued the cells from TRAIL-induced cell death. These observations suggested that taxanes induce both caspase-dependent and -independent cell death in mCRPC cells, and that maintaining the structural integrity of LEDGF/p75 is critical for its role in promoting drug-resistance. We also report the initial screening and selection of candidate small molecule inhibitors (SMIs) to target this protein and sensitize taxane-resistant cells to chemotherapy

    Doctor of Philosophy

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    dissertationThe dysregulation of proteinâ€"protein interaction (PPI) networks has been implicated in many diseases. Designing therapeutic small-molecule inhibitors of these interactions is a challenging field for medicinal chemistry. This work advances the techniques for discovering more potent PPI inhibitors through integration of computational and biochemical techniques. High-throughput screening using fluorescence polarization and AlphaScreen assays identified an acyl hydrazone-containing inhibitor of the β-catenin/Tcf4 PPI, a key mediator of the canonical Wnt signaling pathway. By removing the undesirable acyl hydrazone moiety, a new compound, 4-(5H-[1,2,5]oxadiazolo[3',4':5,6]pyrazino[2,3-b]indol-5-yl)butanoic acid, was developed to selectively inhibit the β-catenin/Tcf4 interaction. The ethyl ester of this compound was tested in zebrafish embryos and shown to inhibit Wnt signaling in vivo at 2 and 10 μM concentrations. Differences between the PPI interface and the active site of traditional targets add to the difficulty of discovering PPI inhibitors. Herein, the relationship between inhibitor potency and ligand burialâ€"defined as the fraction of the solvent accessible surface areas of the bound over unbound ligand, θlâ€"in the PPI surface was evaluated. A positive correlation between θl and inhibitor potency was discovered. However, this correlation was secondary to the strong nonbonding interactions. A study of five PPI targets with corresponding inhibitor-bound crystal structures also revealed that empirical scoring functions were slightly better at identifying known inhibitors out of the putatively inactive test set, and the Lamarckian genetic algorithm was more successful at pose prediction. Due to the nature of the PPI surface, directly targeting the binding site may be difficult. A novel combination of computational methods explored the druggability, selectivity, and potential allosteric regulation of PPIs. Solvent mapping confirmed that Tcf4, E-cadherin, APC and axin use the same binding site on β-catenin in different ways. Evolutionary trace analysis indicated that the region surrounding W504 of β-catenin might be a potentially allosteric site. Site-directed mutagenesis testing results for a W504I β-catenin mutant resulted in three-fold increased binding of Tcf4 to β-catenin over the wild-type. This new site is promising for the discovery of future allosteric inhibitors of the β-catenin/Tcf4 PPI. The combined results from these studies reveals ways to better design PPI inhibitors
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