Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.

Covalent bond-forming reactions between small molecules and proteins are ubiquitous. These reactions play a central role in the diversification and functionalization of proteins, enabling normal cell growth and life. Scientists routinely employ electrophilic compounds to modify proteins by exploiting the intrinsic nucleophilicity found on amino acid side-chains. These modifications permit a wide variety of experiments and allow for new insights and a deeper understanding of the chemistry and biology of living systems. The three research projects described in this thesis employ electrophilic, protein-modifying agents to meet unique goals. The first study (Chapter 2) details the development of a novel class of compounds that enhance the efficacy of therapeutic oncolytic viruses specifically in cancer cells. A medicinal chemistry-based approach was used to understand, measure and improve physicochemical and pharmacological properties of these small molecules. Inspired by the unique scaffold identified in Chapter 2, the second study of this thesis (Chapter 3) explores the bioactivity of the structurally related armeniaspirole natural products. Chemical synthesis enabled the uncovering of structure-activity relationships and ultimately allowed for the design of an activity-based probe. The final study (Chapter 4) details investigations of the terminal thioesterase involved in the biosynthesis of valinomycin. Small molecules substrates for the enzyme were synthesized and used to reveal details of the enzymatic mechanisms.

The discovery of antibiotics and their subsequent clinical use has had a tremendous and beneficial impact on human health. The β-lactam antibiotics, which include penicillins, cephalosporins, carbapenems, and monobactams, constitute over half of the global antibiotic market. However, like all antibiotics, the β-lactams are susceptible to bacterial antibiotic resistance. One of the most disconcerting manifestations of bacterial resistance to β-lactam antibiotics is the evolution and dissemination of β-lactamases, enzymes able to chemically inactivate β-lactam antibiotics. These resistance determinants are the key contributing factor to extensively-drug resistant Gram-negative pathogens, for which we are already bereft of chemotherapeutic treatment options in some cases. The coadministration of a β-lactamase inhibitor (BLI) with a β-lactam antibiotic is a proven therapeutic strategy to counter β-lactamase expression. Unfortunately, the emergence of both serine β-lactamases (SBLs) that are resistant to BLIs and metallo-β-lactamases (MBLs), which are intrinsically resistant to BLIs due to a discrete mechanism of β-lactam hydrolysis, threaten the efficacy of combination therapy. Notwithstanding this bacterial adaptation, the discovery and development of novel BLIs is an attractive strategy to evade resistance, as evidenced by the recent clinical approval of the diazabicyclooctane (DBO) SBL inhibitor, avibactam. Herein, I describe efforts directed at understanding the mechanism of avibactam SBL inhibition. Furthermore, DBO derivatives are shown to display bifunctional properties in inhibiting both β-lactamases and the targets of β-lactam antibiotics, the penicillin-binding proteins. In addition to understanding the enzymology and chemical biology of DBOs, I describe two screening campaigns directed towards discovering inhibitors of MBLs, an unmet clinical need. Using target and cell-based screening of both synthetic and natural product chemical libraries, a fungal natural product inhibitor of clinically relevant MBLs was discovered and characterized. This study expands our understanding of the mechanisms by which DBOs can be used to combat extensively drug-resistant Gram-negative pathogens. It also describes the discovery of a new natural product MBL inhibitor using a workflow that should be amenable to other resistance determinants. It’s hoped that these studies can contribute meaningfully to countering antibiotic resistance observed in clinical settings. / Thesis / Doctor of Philosophy (PhD) / Beta-lactam antibiotics like penicillin are a mainstay for treatment of bacterial infections. Bacterial resistance to these antibiotics threatens their utility and therefore new strategies are required to counter this phenomenon. Herein I describe efforts aimed at understanding new drugs and candidate drugs that act by inhibiting the function of enzymes produced by bacteria that are able to degrade beta-lactam antibiotics. Through the discovery of new molecules and an understanding of their chemical mechanism of inhibition it is believed that bacterial resistance to beta-lactam antibiotics can be reversed.

Synthetic immunology aims to utilize synthetic chemistry and molecules to modulate natural immunological functions against cancer cells. Synthetic molecules developed for use in immunotherapy are antibody recruiting molecules (ARMs) that can utilize antibodies in human serum as a weapon against cancer cells. The development of ARMs has inspired the creation of a novel class of synthetic immunotherapeutics - covalent antibody recruiting molecules (cARMs). cARMs not only bind but covalently label specific antibodies to recruit them to the tumour cell surface. First generation cARM molecules consist of a human prostate specific membrane antigen (hPSMA) target binding domain, an antibody labeling acyl imidazole ester domain, and an anti-dinitrophenyl (DNP) antibody binding domain. The ALD domain instigates an irreversible covalent bond to label anti-DNP antibodies which enables the generation of a tumour therapeutic antibody directly in vivo which can enact immune cell function against tumour cells. cARMs have been proven to recruit anti-DNP antibodies to hPSMA expressing cells and mediate selective antibody-dependent cellular phagocytosis/cytotoxicity (ADCP/ADCC) against cancer cells in in vitro assays. Following the development of a mouse model, initial in vivo studies demonstrated significantly improved survival in tumour bearing mice that were treated with ARM and cARM. In vivo labeling studies displayed successful labeling of anti-DNP antibodies in circulation by cARMs and the presence of labeled antibodies for at least 72hrs in circulation. Conversely, ARM mediated antibody binding was considerably less at all examined time points. Resolving autoinhibition limitations of cARMs allowed for enhanced and improved labeling of anti-DNP antibodies both in vitro and in vivo. This enhancement in labeling could allow for improved ternary and immune active quaternary complex formation to mediate tumour cell death. In vivo tumour studies demonstrated increased survival in tumour bearing mice treated with non-autoinhibited cARM when compared to ARM. Furthermore, innate immune cell populations were shown to increase in peripheral blood of tumour bearing mice treated with ARM and cARM. While cARM treatment dosing and concentration are yet to be optimized, this thesis demonstrates the potential of cARMs as an immunotherapeutic. cARMs can be synthesized to target a variety of cancer cell specific antigens, selectively label antibodies, and has the potential to recruit multi-specific therapeutic antibodies against heterogeneous tumours. / Thesis / Master of Science (MSc)

Antimicrobial resistance is a significant threat to global health, and it is necessary to identify new drugs and drug targets for pathogenic bacteria, parasites, viruses, and fungi. Novel small molecules with antimicrobial activity may be discovered in the lab through chemical synthesis or from nature as secondary metabolites. This thesis describes our efforts to synthesize and identify antiparasitic and antiviral small molecules. The preparation of 3-diarylether quinolines with 5 μM activity against the parasite T. gondii, through a novel TFA-catalysed Povarov reaction using enol ethers as carbonyl surrogates is described. Libraries of quinazolinone and dihydroquinazolinone derivatives have been prepared through a multicomponent synthetic route. Structure activity relationship analysis allowed for differentiation of the antiparasitic pharmacophore from the antiviral pharmacophore, as well as the identification of compounds with single digit micromolar activity against both T. gondii and Herpes Simplex Virus 1. This work also details the design and synthesis of B-ring aza-analogs of bioactive Amaryllidaceae alkaloids in just 5 steps from chiral pool reagents. Aza-substitution of the B-ring eliminated antiviral activity, and this modification may also affect anticancer activity. Analysis of several natural product sources has also identified novel small molecules. Isolation of metabolites from Xylaria polymorpha identified three novel polyketide derivatives with unknown biological activity. The alkaloid candicine was found to be the primary polar metabolite from Ficus benjamina latex, as well as a potent inhibitor of murine cytomegalovirus. By identifying the mechanisms of action of these bioactive small molecules, we may identify targets for further drug development. / Thesis / Doctor of Philosophy (PhD) / There is a need to discover new antimicrobial drugs to combat drug-resistant infections. We are trying to find new molecules that can prevent the growth of parasites and viruses by developing and using novel chemical reactions, as well as by isolating new products from plants and fungi. This text describes a new way to make quinolines, a type of molecule found in many drugs. A molecule prepared by this method inhibited the parasite T. gondii at low concentrations. We have also identified quinazolinones, molecules that can be rapidly assembled by combining three components, which inhibit parasites and viruses. The thesis also includes a faster way to make derivatives of an antiviral molecule from daffodils, which can help determine which parts of the molecule are important for antiviral activity. We have also identified new molecules from the fungus Xylaria polymorpha and an antiviral compound from the Ficus benjamina tree.

Thesis advisor: Eranthie Weerapana / Protein activity is modulated by hundreds of post-translational modifications (PTMs). This thesis will describe the development and application of proteomic methods to study three chemically distinct PTMs. In the first project we describe the development of a proteomics platform to identify cysteine oxidation sites on interactors of the NADPH oxidase complex in response to EGF activation. The NADPH oxidases (Nox) are the source of H(2)O(2) which acts as a secondary messenger during EGFR activation. Known targets of Nox H(2)O(2) include phosphatases PTP1B and PTEN. Oxidation of the active site of PTP1B and PTEN temporarily inactivates their phosphatase activity which allows for EGF signal propagation. The platform involves combining TurboID with OxICAT to identify proteins which are oxidized by Nox2 in a spatially and temporally controlled manner. In the second project, our goal is to identify proteins which recognize two Met oxidation sites in actin known to play a role in regulating the transition between F and G actin. We utilized a peptide based photo-crosslinking approach to identify PFKL and HSP70s HSPA8 and HSPA1B as putative "readers" of oxidized or unoxidized methionine in actin respectively. Finally, protein citrullination is a enzyme catalyzed PTM where the guanidinium on arginine is converted into a urea by a family of enzymes called protein arginine deiminases. Aberrant citrullination is linked to many human diseases including rheumatoid arthritis. Therefore, proteomic methods to characterize citrullination can provide insights into disease pathophysiology. We describe the identification of novel protein targets of with a chemoselective biotin phenyl glyoxal probe, and the development of a label free proteomic method to identify sites of citrullination. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Post-translational modifications (PTMs), which biochemically modify proteins to generate diversity and control functionality, are important in the field of chemical biology research. However, typical bioconjugation methods involving nucleophilic addition of cysteine and lysine residues are limited by their lack of site-specificity. In this thesis, we have demonstrated the site-selective chemical modification of a variety of proteins through protein halogenation and subsequent pallado-biology strategies. The "Tag-and-Modify" approach, developed previously in our group and involving the creation of a protein tag for further manipulation, is a useful tool for site-selective chemical modification. Here, we envisioned extending its utility to a novel concept of [¹⁸F]-radiolabelling of proteins involving direct electrophilic fluorination, thus forming a C-F bond at a specific modification site and leading to the generation of protein species applicable for in vivo [¹⁸F]-PET imaging and diagnosis. We also explored several alternative methods for the installation of C-Br/C-I bonds onto a protein of interest. Our first attempts involved the biosynthetic incorporation of a synthetic bromotryptophan into the protein, however these processes were unsuccessful. As a workaround, we found IPy₂BF₄, an iodinating agent, to be very effective as a direct method for the site-specific installation of C-I bonds on tyrosine/histidine residues of proteins. We subsequently demonstrated novel and efficient palladium-catalysed cross-couplings of the resultant iodinated tyrosine/histidine moieties, leading to the creation of new C-C bonds. This approach was compatible with a wide range of functionally diverse boronic acids, using a water-soluble and phosphine-ligand-free Pd-complex catalyst under fully aqueous conditions. Our novel, successful method for C-C bond formation onto proteins is potentially applicable to the initial investigation of challenging in vivo Pd cross-coupling of thyroglobulin, based on our achievement of protein labelling under ex vivo conditions. In summary, we are able to create new chemical tools for the site-selective/site-specific chemical modification of proteins, enabling their use as biological probes.

The aim of this project is to develop general approaches to the control of protein structures and functions. The project mainly consists of three aspects: (1) stabilizing folded protein structures, (2) reversible photo-control of protein folding and function, (3) design of a photo-switchable dominant negative protein to photo-control DNA binding. (1) Most proteins adopt specific folded structures to perform biological functions. Stabilizing the active folded forms of peptides or proteins is thus important for maintaining or enhancing the functions of these molecules. We hypothesized that a protein’s folded structure could be stabilized by introduction of a rigid cross-linker with its length matching the distance of the two attachment points in the folded structure. To test this, we synthesized a thiol-reactive alkyne-based rigid cross-linker and tested its effect. When it was introduced at i and i+11 positions of a model α-helical peptide, a significant promotion in the folded structure as well as strong resistance against thermal melting was observed. The rigid cross-linker was also applied to the Fyn SH3 domain, a protein with tertiary structure and a similar stabilization effect was obtained. This work demonstrates that a rigid cross-linker can be generally used to stabilize folded peptide/protein structures. (2) Reversible photo-switch of protein folding/unfolding offers exciting prospects for external manipulation of protein function because of its fast response, high spatial resolution and compatibility with living systems. I developed a general approach to the design of photo-switchable proteins based on the introduction of photo-switchable intramolecular cross-linkers. An azobenzene based photo-switch was used because the energy available from photoisomerization is higher than the free energy of protein folding. I chose the FynSH3 domain as a model protein. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the L3C-L29C-T47AFynSH3 mutant was cross-linked with the trans cross-linker, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded state of the protein. Photo-control of FynSH3 binding to a proline-rich peptide was also demonstrated. This work shows that structure-based introduction of switchable cross-linkers is a feasible general approach for photo-control of global folding/unfolding of globular proteins, and thereby photo-control of their activity. (3) The third aspect of my PhD research is to apply the photo-switchable proteins to photo-control of Jun/Fos DNA binding activity. Fos and Jun are important transcription factors implicated in numerous cancers. They form a hetero-dimer that binds to specific DNA sequences. Dominant negative proteins are mutants of Fos or Jun that prevent native Jun/Fos DNA binding activity. I designed photo-switchable versions of these dominant negative proteins by covalently introducing photo-switchable cross-linkers. These proteins do not have any function in the dark due to their disrupted structures induced by the trans form cross-linkers. They only function as dominant negative proteins when the cross-linker is in the cis form after photo-irradiation. Several such proteins were synthesized and their effectiveness was tested. These photo-switchable dominant negative proteins are powerful tools for temporal control of Jun/Fos regulated gene expression.

The aim of this project is to develop general approaches to the control of protein structures and functions. The project mainly consists of three aspects: (1) stabilizing folded protein structures, (2) reversible photo-control of protein folding and function, (3) design of a photo-switchable dominant negative protein to photo-control DNA binding. (1) Most proteins adopt specific folded structures to perform biological functions. Stabilizing the active folded forms of peptides or proteins is thus important for maintaining or enhancing the functions of these molecules. We hypothesized that a protein’s folded structure could be stabilized by introduction of a rigid cross-linker with its length matching the distance of the two attachment points in the folded structure. To test this, we synthesized a thiol-reactive alkyne-based rigid cross-linker and tested its effect. When it was introduced at i and i+11 positions of a model α-helical peptide, a significant promotion in the folded structure as well as strong resistance against thermal melting was observed. The rigid cross-linker was also applied to the Fyn SH3 domain, a protein with tertiary structure and a similar stabilization effect was obtained. This work demonstrates that a rigid cross-linker can be generally used to stabilize folded peptide/protein structures. (2) Reversible photo-switch of protein folding/unfolding offers exciting prospects for external manipulation of protein function because of its fast response, high spatial resolution and compatibility with living systems. I developed a general approach to the design of photo-switchable proteins based on the introduction of photo-switchable intramolecular cross-linkers. An azobenzene based photo-switch was used because the energy available from photoisomerization is higher than the free energy of protein folding. I chose the FynSH3 domain as a model protein. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the L3C-L29C-T47AFynSH3 mutant was cross-linked with the trans cross-linker, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded state of the protein. Photo-control of FynSH3 binding to a proline-rich peptide was also demonstrated. This work shows that structure-based introduction of switchable cross-linkers is a feasible general approach for photo-control of global folding/unfolding of globular proteins, and thereby photo-control of their activity. (3) The third aspect of my PhD research is to apply the photo-switchable proteins to photo-control of Jun/Fos DNA binding activity. Fos and Jun are important transcription factors implicated in numerous cancers. They form a hetero-dimer that binds to specific DNA sequences. Dominant negative proteins are mutants of Fos or Jun that prevent native Jun/Fos DNA binding activity. I designed photo-switchable versions of these dominant negative proteins by covalently introducing photo-switchable cross-linkers. These proteins do not have any function in the dark due to their disrupted structures induced by the trans form cross-linkers. They only function as dominant negative proteins when the cross-linker is in the cis form after photo-irradiation. Several such proteins were synthesized and their effectiveness was tested. These photo-switchable dominant negative proteins are powerful tools for temporal control of Jun/Fos regulated gene expression.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references.