<p dir="ltr">Humans encode approximately 100 deubiquitinating enzymes (DUBs) which are categorized into seven distinct subfamilies. Each family and representative has a unique expression, function and binding topology to ubiquitin. In addition to human DUBs, parasites, bacteria, and viruses contain DUBs with unique structures and functions. One subfamily of DUBs, the ubiquitin C-terminal hydrolases (UCH), has four structurally similar human members and two known members within the <i>Plasmodium falciparum</i> genome. Human UCHL1 and UCHL3 are genetically validated targets in oncology and <i>Plasmodium falciparum</i><i> </i>UCHL3 (PfUCHL3) is a prospective target for antimalarial drug development. Though these three UCH enzymes have potential as therapeutic targets, there is a significant lack of quality small molecule chemical probes to understand the underlying biology and function of the enzymes, pharmacologically validate the targets, and serve as leads for drug development in oncology and malaria.</p><p dir="ltr">The UCH enzymes are cysteine proteases, which our lab has leveraged to identify novel covalent small molecule inhibitors of each enzyme. The workflow for each hit identification and optimization campaign is similar. Covalent fragment screening of electrophilic small molecule libraries against the respective recombinant enzyme was performed to identify chemical space around each enzyme. Subsequent medicinal chemistry hit-to-lead optimization was undertaken to improve upon the moderately potent hit molecules to provide improved small molecule inhibitors for each enzyme. Inhibitor identification and optimization for UCHL1 is described in Chapter 2, revealing a novel scaffold and a cocrystal structure reveals a unique binding pose for UCHL1 inhibitors. These molecules were also characterized in breast cancer cells to validate UCHL1 as a therapeutic target in breast cancer. First-in-class covalent inhibitors of UCHL3 are described in Chapter 3. Medicinal chemistry optimization along with a cocrystal structure of the initial hit has revealed the molecular interactions of this novel inhibitory scaffold. PfUCHL3 inhibitor identification is described in Chapter 4. Characterization of these molecules against Plasmodium falciparum is described along with a comparison to a recently identified reversible PfUCHL3 inhibitor. Finally, conclusions and future directions toward the development of potent, drug-like inhibitors of each UCH enzyme is presented in Chapter 5.</p>

The development and advancement of prostate cancer is supported by a plethora of genetic and proteomic abnormalities, including events of post-translational modifications. The protein arginine methyltransferase 5 (PRMT5) enzyme regulates epigenetic events of histone modifications and protein post-translational modifications within protein signaling pathways. PRMT5 functions by catalyzing the symmetric dimethylation of terminal arginine residues on target protein substrates. Under abnormal conditions of overexpression and upregulation, PRMT5 methyltransferase activity constitutively drives the growth and proliferation of dysregulated cells. Overexpression or upregulation of PRMT5 correlates with disease progression as observed among numerous cancer types, including breast, colorectal, leukemia, lung, melanoma and prostate cancers. We demonstrated previously that PRMT5 knockdowns attenuated both growth and proliferation of lung and prostatic tumors, in vitro and in vivo. Plants naturally produce chemical toxins as mechanisms of defense against microbial and other biological threats. Human exploitation, consumption and application of agents isolated from plants for therapeutic intervention dates back throughout the millennia. In this study, we extracted, purified and evaluated natural, small, chemical compounds from plant products that antagonize PRMT5 activity in prostate cancer cells. We found that crude and purified extracts of Dendrobium aurantiacum var. denneanum (D. denneanum) plants attenuated prostate tumor growth and proliferation by selective inhibition of PRMT5 methyltransferase activity. These findings establish the first set of natural PRMT5-specific inhibitors reported.

Indiana University-Purdue University Indianapolis (IUPUI) / The human genome encodes 19 members of the aldehyde dehydrogenase (ALDH) superfamily, critical enzymes involved in the metabolism of aldehyde substrates. A major function of the ALDH1A subfamily is the oxidation of retinaldehyde to retinoic acid, a key regulator of numerous cell growth and differentiation pathways. ALDH1A1 has been identified as a biomarker for both normal stem cells and cancer stem cells. Small molecule probes are needed to better understand the role of this enzyme in both normal and disease states. However, there are no commercially available, small molecules that selectively inhibit ALDH1A1. Our goal is to identify and characterize small molecule inhibitors of ALDH1A1 as chemical tools and as potential therapeutics. To better understand the basis for selective inhibition of ALDH1A1, we characterized N,N-diethylaminobenzaldehyde (DEAB), which is a commonly used inhibitor of ALDH1A1 and purported to be selective. DEAB serves as the negative control for the Aldefluor assay widely utilized to identify stem cells. Rather than being a selective inhibitor for ALDH1A1, we found that DEAB is a slow substrate for multiple ALDH isoenzymes, and depending on the rate of turnover, DEAB behaves as either a traditional substrate or as an inhibitor. Due to its very slow turnover, DEAB is a potent inhibitor of ALDH1A1 with respect to propionaldehyde oxidation, but it is not a good candidate for the development of selective ALDH1A1 inhibitors because of its promiscuity. Next, to discover novel selective inhibitors, we used an in vitro, high-throughput screen of 64,000 compounds to identify 256 hits that either activate or inhibit ALDH1A1 activity. We have characterized two structural classes of compounds, CM026 and CM037, using enzyme kinetics and X-ray crystallographic structural data. Both classes contained potent and selective inhibitors for ALDH1A1. Structural studies of ALDH1A1 with CM026 showed that CM026 binds at the active site, and its selectivity is achieved by a single residue substitution. Importantly, CM037 selectively inhibits proliferation of ALDH+ ovarian cancer cells. The discovery of these two selective classes of ALDH1A1 inhibitors may be useful in delineating the role of ALDH1A1 in biological processes and may seed the development of new chemotherapeutic agents.

The canonical Wnt signaling pathway is one of the most important pathways involved in cell proliferation and differentiation. It is highly conserved in evolution and participates not only in embryonic development but also in adult tissue homeostasis. In the intestine, Wnt signaling is closely connected to maintenance of intestinal stem cells and renewal of the epithelia. Conversely, aberrant activation of the Wnt signaling pathway underlies different types of human diseases. Its constitutive activation results in neoplasia and specifically in development of colorectal cancer, which is the third most common malignancy in western world. The aim of this thesis was to uncover various aspects of the regulatory mechanisms of the Wnt/β-catenin signaling cascade. Furthermore, I headed to find novel Wnt pathway modulators and confirm their function in vivo. The results are presented in four publications. The first study examines murine Wnt proteins processing and the sequential order of Wnt post-translational modifications which are required for the secretion and signaling activity of the ligands. Next publication focuses on the gene Troy, which we identified as negative regulator of Wnt signaling. TROY was discovered as a Wnt target gene during DNA microarray profiling of human colorectal cancer cells....

Hepatitis C virus (HCV) represents a global health threat. HCV is a blood-borne positive-strand RNA virus belonging to the Flaviviridae family that infects ~160 million people worldwide. About 70% of infected individuals fail to clear the virus and subsequently develop chronic hepatitis, frequently leading to liver cirrhosis and in some cases hepatocellular carcinoma. Therapeutic options for HCV infection are still limited and a protective vaccine is not yet available. Currently available therapies include administration of pegylated alpha interferon in combination with ribavirin. The recently approved protease inhibitors Boceprevir and Telaprevir are also included in the treatment regimen. However, limitations to the treatment with direct-acting antivirals (DAAs) are associated with severe side effects and low sustained virological response (SVR) rates that vary depending on the virus and host genotype. The replication step of the viral life cycle is mostly targeted by majority of DAAs. Recent findings have suggested that a combination of entry inhibitors together with DAAs exhibit a synergistic effect in the treatment of HCV. Therefore, identification of efficient HCV entry inhibitors is of high priority In vitro studies have shown that HCV attachment and subsequent entry into the host cells is mediated by E1 and E2 viral envelope proteins. HCV entry requires interaction with a number of receptors which include CD81, scavenger receptor B1 (SR-B1) and the tight junction proteins, claudin 1 (CLDN1) and occludin (OCLN). Since the E2 glycoprotein is reported to interact directly with cellular receptors, it is an attractive target for neutralisation. The present study focuses on the establishment and characterisation of entry inhibitors as antivirals for HCV. The thesis is presented in three chapters: Chapter 1- ‘Introduction’, provides a brief overview on HCV genotypes, genome organisation, life cycle including details on the entry process and therapies used for the treatment of HCV. Chapter 2 describes the generation of monoclonal antibodies (mAbs) against HCV envelope proteins as potent anti-viral agents for the prevention of HCV infection. Data on the identification and characterization of the neutralizing epitopes of HCV envelope proteins have been presented. Chapter 3 includes isolation of entry inhibitors of HCV from natural sources and identification and characterization of the active components exhibiting antiviral property. A number of studies have reported the role of neutralizing antibodies in the course of HCV infection and emerging data suggest protective effect of antibodies against HCV infection. Most of the ongoing studies are based on HCV genotype 1a which is prevalent globally. However in India, the prevalent genotype is 3a. Therefore, we established a panel of mAbs against HCV-LPs comprising of core-E1-E2 derived from genotype 3a as described in chapter 2. HCV-LP based system has been used in this study since it mimics the biophysical conformation, morphology and antigenic properties of the native virion and represents a model system for studies on viral binding and entry. MAbs were characterised and analyzed for their ability to prevent viral binding and entry into host cells. Three mAbs namely E3D8, H6D3 and A10F2 were identified to recognize the E2 viral glycoprotein which significantly inhibited HCV-LP binding to Huh7 cells in vitro. The neutralizing epitopes corresponding to the mAbs were identified using overlapping truncated fragments and synthetic peptides of the E2 protein. Our experiments suggest that the epitopes recognised by the inhibitory mAbs are unique and different from those reported till now. The synergistic effect of a combination of mAbs on virus neutralization has shown promising results for treatment of viral infections. Since in the present study the epitopes recognised by the mAbs are non-overlapping, we went ahead to determine whether a combination of these mAbs would enhance the ability to block HCV-LP binding. Indeed, flow cytometry and fluorescence microscopy studies revealed that a combination of the antibodies efficiently blocked the binding of HCV-LP to human hepatoma cells. More importantly and of relevance is the observation that the mAbs in combination inhibited viral infection (JFH1 strain) and replication in permissive human hepatocytes as determined by real time RT-PCR. Phytochemicals present in plants have been considered as conducive for prevention of several viral infections and are found to be promising antiviral agents. Natural products which are biologically active disclose drug-like properties since they are small molecules and can be easily metabolised and absorbed by the body. In our study as described in chapter 3, we evaluated extracts from Indian medicinal plants and fruits which are known to have hepato-protective effect, for natural potent attachment and entry inhibitors for HCV. Flow cytometric analysis suggested that the root extract of the herb Boerhavia diffusa and fruit extract of Prunus domestica exhibited high antiviral activity by inhibiting the binding of Hepatitis C virus like particles (HCV-LPs) to the human hepatoma cells. We went on to isolate, identify and confirm the active principles to be Boeravinone H, a dehydrorotenoid, (from Boerhavia diffusa) and Rutin, a flavonoid, (from Prunus domestica) by LC-ESI-MS, NMR, UV and IR spectral analysis. Our study revealed that the compounds block the attachment as well as entry step probably by targeting the viral particle. We also assessed the efficiency of these small molecules (Boeravinone H and Rutin) to inhibit HCV negative strand synthesis post entry by real time RT-PCR. Results suggest significant inhibition of viral entry and infection in the HCV cell culture (ex vivo). To our knowledge it is the first report on Boeravinone H and Rutin as entry inhibitor for HCV. In conclusion, our findings support the potential of employing a cocktail of neutralizing mAbs and antiviral agents from natural source in the management of HCV infection.

<p>Coronaviruses (CoVs) are single-stranded, positive-sense RNA viruses in the Coronaviridae family. Within this family are four different genera, Alpha-, Beta-, Gamma-, and Deltacoronaviruses with human-infecting CoVs spanning the Alpha- and Beta-CoV genera. Most notably, Severe Acute Respiratory Syndrome Coronavirus-1 (SARS-CoV-1) and SARS-CoV-2 are Betacoronaviruses that spread worldwide in their outbreaks from 2002-2003 (SARS-CoV-1) and 2019-2020 (SARS-CoV-2). Human-infecting Alphacoronaviruses, NL63-CoV and 229E-CoV, have caused milder infections involving respiratory disease, gastroenteritis, and in more severe cases, death. Despite milder disease, Alphacoronaviruses are the cause of 15-30% of severe upper and lower respiratory tract infections each year. There have been recent efforts in the development of potent, small-molecule inhibitors to treat SARS-CoV-2 infection but there is an ongoing need to develop new and effective anti-coronavirus therapeutics to treat other human-infecting CoVs circulating society. Coronaviruses encode two essential proteases, the papain-like protease (PLP) and the 3C-like protease. PLPs are cysteine proteases located in non-structural protein 3 (nsp3). PLPs processes the viral polyprotein, releasing the first three nonstructural proteins encoded in the virus, and also are involved in evading the innate immune response through deubiquitinating (DUB) and deISGylating activity. </p> <p><br></p> <p>This study compares the substrate specificity and catalytic function of multiple human-infecting PLPs from both Alpha- and Beta-CoVs including NL63-CoV PLP2, 229E-CoV PLP2, Canine-CoV PLP2, FIPV-CoV PLP2, PEDV-CoV PLP2, SARS-CoV-1 PLpro, and SARS-CoV-2 PLpro. Interestingly, Alphacoronavirus PLP2s have a >400-fold greater catalytic efficiency for ubiquitin compared to Betacoronaviruses PLpro. This work also identifies a non-covalent scaffold of inhibitors that has pan-CoV inhibition; however, the IC50 values are >30-fold higher for NL63-CoV PLP2 than for SARS-CoV-1 PLpro. The X-ray structures of NL63 PLP2 and 229E PLP2 were determined to 2.1 Å and 1.8 Å, respectively, and provide structural information about the substrate and inhibitor binding region that could be the result in the differences in Alpha- and Betacoronavirus PLP function. Since PLP does not function as a single-domain in vivo, it is critical to understand the function of PLP when tethered to other domains of nsp3. This study also investigates nine different constructs of SARS-CoV-2 nsp3 with increasing domains, ranging from the single PLpro domain to Ubl1-Ydomain ΔTM1-TM2. Interestingly, the longer constructs of SARS-CoV-2 nsp3 show less catalytic efficiency for Ub-AMC and greater affinity for ISG15-AMC, with 8-fold lower Km values compared to PLpro alone. Lastly, each SARS-CoV-2 nsp3 construct was inhibited by a known PLpro inhibitor, GRL-0617, with reported IC50 values ranging from 0.91 μM to 1.9 μM. These data show that GRL-0617 still remains a lead compound to be optimized for cellular potency. </p> <p><br></p> <p>Overall, this dissertation advances the understanding of the kinetic and structural differences between Alphacoronavirus PLP2 and Betacoronavirus PLpro enzymes in the efforts of developing a pan-CoV inhibitor. Additionally, these data provide initial kinetic and biophysical characterization of PLpro within the larger context of nsp3 to elucidate the function of PLpro in its most native context during coronaviral infection.</p>

<p dir="ltr">The BRG1/BRM-associated factor (BAF) complexes, also called SWI/SNF, are multi-subunit chromatin remodelers that regulate chromatin compaction in an ATP-dependent manner. In the past decade, BAF complexes have been under the spotlight in cancer research, especially after proteomic analyses revealed the genes encoding the subunits are amongst the most frequently mutated genes in cancer. The present dissertation focuses on prostate cancer (PCa), a disease in which the role of the BAF subunits is increasingly being explored but is yet to be defined as a potential therapeutic target. According to the GLOBOCAN report, PCa is the second most frequent cancer in males worldwide. Since most of the variants of PCa rely on the androgen receptor (AR) axis, surgical or chemical castration and androgen deprivation therapy (ADT) are the main treatment strategies for PCa patients. Even though these therapeutic approaches prolong survival, reduce tumor burden, and relieve symptoms, PCa patients eventually relapse and develop castration resistant PCa (CRPC). At present, the mechanisms underlying ADT resistance are not fully understood, current efforts focus on finding new targets for PCa treatment.</p><p dir="ltr">In the projects included in this dissertation we explored the function of the PBAF complex, a BAF subtype, in a variety of models of PCa and its potential as a therapeutic target by inhibiting or depleting its different subunits. To do so we (i) developed the first inhibitors for BRD7 (a subunit unique to PBAF) and (ii) established cell-based assays in multiple PCa cell lines to study BRD7 and other PBAF unique subunits.</p><p dir="ltr">Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromodomain-containing protein 7 (BRD7) has been implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a series of ligands designed to occupy this binding region and identified two inhibitors with increased selectivity towards BRD7, 1-78 and 2-77, which bind with submicromolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands occupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.</p><p dir="ltr">There are three BAF complexes that have been biochemically characterized up to date: canonical BAF (cBAF), polybromo-associated BAF (PBAF) and GLTSCR1/like-containing BAF (GBAF or ncBAF). All BAF complexes are characterized by containing an ATPase and accessory subunits that may be shared between them or unique to each subtype. PBAF, the BAF subtype of interest of this dissertation, contains four unique subunits: BRD7, PBRM1, ARID2 and BAF45A. We showed that knocking down BRD7 and ARID2 leads to reduction of cell viability in PCa cells with ligand-dependent and independent AR signaling, while knocking down PBRM1 leads to reduction in viability of cells with only ligand-dependent AR signaling. We also performed a chromatin immunoprecipitation assay with BAF45A and observed that it does not colocalize with AR binding sites, indicating that the mechanism by which PBAF regulates AR signaling is indirect. This observation was further supported by the fact that knocking down BRD7 prevents expression of genes related to adaptive processes, but not AR target genes, in response to androgen treatment. Further mechanistic studies will aid in understanding the function of PBAF in PCa. However, overall, our results indicate that PBAF is a promising therapeutic target in PCa models expressing AR, including CRPC systems.</p>

Dans l'introduction, diverses voies de trafic intracellulaire et endocytose seront discutées. Je familiarise le lecteur avec des protéines inactivant les ribosomes, en mettant l'accent sur la structure, l'endocytose, et le trafic intracellulaire de la toxine bactérienne Shiga toxin (STX). STx et la ricine suivent la voie rétrograde pour exercer leur effet toxique sur les cellules. Ils sont respectivement, une menace maladie infectieuse pour la santé humaine et des outils potentiels pour le bioterrorisme pour lequel aucun antidote n’existe actuellement. D'un criblage à haut débit, Retro-1 et Retro-2 avaient déjà été identifiés comme de puissants inhibiteurs de la voie rétrograde à l'interface des endosomes précoces-TGN, et Retro-2 a été démontré pour protéger les souris contre la ricine. Parmi les facteurs de trafic analysés, seule la protéine SNARE syntaxine-5 a été ré- localisée dans les cellules traitées avec Rétro - 2. / In the introduction, various endocytic and intracellular trafficking pathways will be discussed. I acquaint the reader with ribosome-inactivating proteins, with emphasis on the structure, endocytosis, and intracellular trafficking of the bacterial toxin Shiga toxin (STx). STx and ricin follow the retrograde route to exert their toxic effect on cells. They are respectively, an infectious disease threat to human health and potential tools for bioterrorism for which no antidote currently exists. From a high throughput screening, Retro-1 and Retro-2 had previously been identified as potent inhibitors of the retrograde route at the early endosomes-TGN interface, and Retro-2 was demonstrated to protect mice against ricin. Of the trafficking factors analyzed, only the SNARE protein syntaxin-5 was re-localized in Retro-2 treated cells. Yet, whether syntaxin-5 is the direct target of Retro-2 and whether its re-localization was directly responsible for retrograde transport inhibition remained to be established.