PURPOSE: The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes. METHODS: Hypoxia inducible factor-1 alpha (HIF-1alpha) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1alpha transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay. RESULTS: SB202190 decreased HIF-1alpha protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated. CONCLUSIONS: SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1alpha activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.

Cancer is one of the major causes of morbidity in the world. Although the overall survival of cancer has been significantly improved by chemotherapy in the last three decades, the success of cancer chemotherapy is still severely limited by the lack of selectivity of anti-cancer drugs to malignant cells leading to dose-limiting toxicity and the resistance of cancer cells to the conventional anti-cancer drugs. Gene-directed enzyme prodrug therapy (GDEPT) was designed to direct the anti-cancer drugs to specifically target the cancer cells by using cancer specific promoter to drive the expression of enzyme which can convert prodrug into anti-cancer drug specifically in cancer cells. However, this strategy is hindered by the lack of strong cancer specific promoters to specifically express drug-converting enzymes in cancer cells. In consequence, there is not enough anti-cancer drug activated inside the cancer cells. The first part of this study was to employ NF-κB binding sites as a novel enhancer system to improve the promoter activity of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) for GDEPT. In this system, the basal CEA promoter sequences were placed downstream of the 4 or 8 NF-κB DNA binding sites linked in tandem (κB4 or κB8). The system was designed to serve two particular purposes: to exploit the high levels of intratumoural NF-kB expression and keep the relative tumour specificity of the CEA and hTERT promoters. The results demonstrated that κB enhancer systems increased the transcriptional activity of CEA and hTERT promoter without compromising its cancer specificity. The fidelity of the κB4-CEA enhancer-promoter system was therefore improved by the increased transcriptional contrast between the cancer and normal cells. Moreover, in comparison with CEA promoter alone, κB-CEA enhancer-promoter system expressed human thymidine phosphorylase (TP) protein at significantly higher levels which were comparable to those expressed by CMV promoter. The κBCEA- TP system transfected cells demonstrated significantly higher sensitivity to 5'-Deoxy-5-Fluorouridine (5'-DFUR), a prodrug of 5-fluorouracil (5-FU). The second part of this study was involved in using NF-κB inhibitor as a chemosensitizer to sentizise the anti-cancer drug-induced chemoresistance cells to anti-cancer drugs. The results derived from this study manifested that the anti-alcoholism drug, Disulfiram (DS), and anti-inflammatory drug, triptolide (PG490), markedly enhanced the cytotoxicity of several conventional anti-cancer drugs in colon, lung and breast cancer cell lines. PG490 induced caspase-dependent cell death accompanied by a significant decrease in Bcl-2 levels. PG490 induced the expression of p53 and down-regulated p21 expression. This study indicated that some clinically used non-cancerchemotherapeutic drugs may be developed as chemosensitizers for cancer chemotherapy

Bcl-2 family members are important regulators of apoptosis, and their tampered expression is often involved in oncogenesis. Of particular importance are the levels of Bcl-2 family members in forming lymphomas. We studied two groups of murine thymic T cell lymphomas derived from either Bcl-2 or Bax overexpression in order to predict their sensitivity and resistance to treatments. While the growth rate and histological characteristics were similar for both lymphoma groups, Bax-derived lymphomas failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2-derived lymphomas often halted proliferation following radiation delivery and rarely had p53 mutations. Bax-derived lymphomas were uniformly sensitive to treatment with 2-deoxy-D-glucose (2DG) while all Bcl-2-derived lymphomas were resistant. This led us to hypothesize that the Bcl-2 family is involved in 2DG-induced cell death. Focusing on the mechanism of 2DG toxicity in Bax-derived lymphomas, our studies demonstrate the following: cell death involved the activation of proapoptotic Bax, was effectively blocked by anti-apoptotic Bcl-2, and was mediated, at least in part, by the BH3-only family member Bim. Based on these results, we explored whether a BH3 mimetic (ABT-737) could sensitize lymphomas to 2DG killing. Indeed, a combination of ABT-737 with 2DG enhanced killing in Bax-derived lymphomas and resensitized Bcl-2-overexpressing lymphomas to 2DG. Since both 2DG and BH3 mimetics are currently in clinical trials, understanding their killing mechanisms and optimal combinations are of potential clinical significance. The work in this dissertation demonstrates a novel role of Bcl-2 family member proteins in regulating 2DG toxicity and may predict response to 2DG treatment. The information found presents a new strategy of combining 2DG with BH3 mimetics to improve existing lymphoma therapies.

Isoprenoids are the largest family of biologically active compounds, synthesized by five carbon subunits namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). For long time the mevalonate-dependent (MVA) pathway has been considered as the sole source of IPP and DMAPP, until recently a new non-mevalonte dependent (NMVA) pathway was discovered. This new pathway utilizes entirely different set of enzymes for isoprenoids synthesis and don't have any homologues in humans. NMVA pathway is the only source of isoprenoids for certain eubacteria, parasite and plants. Absence of the NMVA pathway in higher organisms has opened a new platform for the development of novel antibiotics and antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme in NMVA pathway has been reported as the rate limiting enzyme in the synthesis of IPP and DMAPP and has been the center of interest for inhibitor development. Reaction mechanism of thiamine pyrophosphate (TPP) and Mg2+ dependent DXS enzyme has been studied in this report. Using steady state kinetics analysis, product inhibition and dead end inhibitor, the mechanism of substrate (pyruvate and D-glyceraldehyde-3-phosphate) addition was studied. Due to different domain organization in DXS as compared to theother TPP dependent enzyme, the mechanism of addition was found to be random sequential rather than ping-pong mechanism. Based on bioinformatics tool and in vitro studies it has been established that NMVA exists in all the plasmodium species, thus making the enzymes involved in NMVA as an alluring target for new antimalarial drugs. All the plasmodium species and other member of the phylum apicomplexa harbor apicoplast an organelle which is homologous to the chloroplast of plants and algae. All the enzymes from NMVA pathway translocate to apicoplast from nucleus through a secretory pathway using signaling and transit peptide. In this study DXS from P. vivax has been cloned and expressed in E. coli using genomic DNA and codon optimized synthetic DNA as a source. Expression of full length DXS with signal and transit peptide as well as mature protein without these peptide using serial deletion has been studied. Kinetic parameters of P.vivax DXS have been calculated and found to be comparable to the DXS from other species.

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyses the aldol condensation of the five-carbon sugar phosphate, arabinose 5-phosphate (A5P), and phosphoenol pyruvate (PEP) to give the eight-carbon phosphorylated sugar, KDO8P. It is the second committed step in the synthesis of KDO, a necessary component of the cell wall of Gram-negative bacteria. This thesis describes the design, synthesis and evaluation of a number of inhibitors of KDO8P synthase that utilise the functionality of one or both substrates. The KDO8P synthase family can be divided based on the requirement of a divalent metal ion. Chapter 2 describes the growth, purification and characterisation of an example from both the metal-independent KDO8P synthases (Neisseria meningitidis, Nme) and metal-dependent KDO8P synthases (Acidithiobacillus ferrooxidans, Afe) in order to utilise these enzymes for the inhibition studies described in this thesis. In Chapter 3, a number of small molecule PEP analogues were selected as mimics of KDO8P synthase reaction intermediates and tested as inhibitors of KDO8P synthase from N. meningitidis and A. ferrooxidans. Glyphosate, (E)-vinyl phosphonate and the fluorinated analogue of (E)-vinyl phosphonate were selected as mimics of the high-energy oxocarbenium intermediate through which the KDO8P synthase reaction is thought to occur. The two enantiomers of phospholactate were selected in order to investigate the chirality of the tetrahedral intermediate and determine the importance of this chirality for inhibition of KDO8P synthase. All five inhibitors were found to be moderate to poor inhibitors of both the KDO8P synthase from N. meningitidis and A. ferrooxidans. Chapter 4 describes the design and synthesis of inhibitors that incorporated structural features of the second substrate, A5P, in order to improve inhibition from that observed for the PEP analogues investigated in Chapter 3. A bisphosphate inhibitor was designed that incorporated a terminal phosphate moiety, representative of the phosphate of A5P. A large increase in inhibition was found, compared to the phospholactates from which it was derived. A structure-activity-relationship study was undertaken on this compound by design of compounds that lacked one of the two phosphate moieties of the bisphosphate inhibitor, in order to determine their relative importance. The inhibition results indicate that the primary terminal phosphate, thought to bind in the A5P phosphate binding site, is more important for inhibition of KDO8P synthase than the secondary phosphate. In Chapter 5 these investigations into the inhibition of KDO8P synthase are discussed in detail, and interpreted using the aid of computational studies. In addition several approaches are described for the completion and advancement of the studies presented here in this thesis.

(a) The Robins reagent [2-acetamido-6-O-(diphenylcarbamoyl)purine] was utilized for glycosylation under Lewis acid conditions. Regioselectivity of glycosylation depends on the glycosyl donor and its 2-O- or 2-N-protecting group. Regioselective N9 glycosylation of 2-acetamido-6-O-(diphenylcarbamoyl)purine with problematic glucosamine has been accomplished by protecting the amino function as a phthalimido group with consequent stabilization of the oxocarbenium cation, and lowering the activation energy by introduction of trichloroacetimidate at the anomeric carbon. (b) 6-Heteroaryl functions [6-(1,2,4-triazol-4-yl) and 6-(imidazol-1-yl)] were introduced into purine derivatives for regioselective N9 alkylation. The regiospecificity of alkylation mainly results from steric effects due to the coplanar conformation of the two linked heterocyclic rings governed by conjugation. Several of the obtained acyclic derivatives showed antiviral and antitumor activities. (c) Glycosylation of purine derivatives with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride using the sodium salt method usually gave a mixture of both anomers. Lipophilic groups were introduced into the imidazole ring of 6-(imidazol-1-yl)purine derivatives to increase the solubility of the sodium salts in moderately polar solvents. Differential solvation effects in binary solvent mixtures were utilized to improve the stereoselectivity of glycosylation. The stereoselectivity varied with the sizes of lipophilic groups and the polarity of solvents. With the propyl group, and in CH3CN/toluene (1:1) and/or CH3CN/CH2Cl2 (1:1), regiospecfic and highly stereoselective glycosylation of purines with 2-deoxy-3,5-di-O-(p-toluoyl)-a-D-erythro-pentofuranosyl chloride was achieved. (d) Using the above method, a low cost and efficient synthesis of 2-chloro-2'-deoxyadenosine (2-CdA, cladribine) was accomplished with an overall yield of 48% from inexpensive guanosine and 57% from 2,6-dichloropurine. 2-Chloro-6-(2-propylimidazol-1-yl)purine was prepared either from guanosine in a yield of 61% in 5 steps or from 2,6-dichloropurine in a yield of 72% in one step. Coupling of this 2-chloro-6-heteroarylpurine with 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentofuranosyl chloride in binary solvent mixtures, followed by activation of imidazolyl as a better leaving group via benzylation at N3 and then ammonolysis gave cladribine in good yield (79%) for 3 steps. Analogs of purine derivatives with lipophilic groups (butyl, pentyl and 2-phenylpropyl) worked almost as well.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first reaction in the shikimate pathway, leading to the biosynthesis of aromatic compounds including the aromatic amino acids. The catalytic activity of DAH7PS is regulated through feedback inhibition and is the major control point for the pathway. DAH7PSs are divided into two families, type I and type II, based on molecular weight and amino acid sequence. Type I DAH7PSs can be further divided based on sequence similarity. All DAH7PS enzymes with their crystal structures solved share a basic (β/α)₈-barrel fold in which the key catalytic components are housed. Furthermore, all structurally characterised DAH7PSs, except Pyrococcus furiosus DAH7PS (PfuDAH7PS) and Aeropyrum pernix DAH7PS, have recruited extra structural motifs that are implicated in allosteric regulation. However, there are significant differences in the additional structural elements. This thesis investigates the hypothesis that the diverse regulation strategies for controlling DAH7PS activity have evolved by domain recruitment, whereby regulatory domains have been added to the catalytic barrel. Chapter 2 describes the functional characterisation of the type Iβ Thermotoga maritima DAH7PS (TmaDAH7PS), and the exploration of its response to inhibitors. The catalytic activity of TmaDAH7PS was found to be substantially inhibited by tyrosine (Tyr) and to a lesser extent, phenylalanine (Phe). The putative regulatory domain previously identified as a ferredoxin-like domain was recognised as an aspartate kinase-chorismate-mutase-tyrA (prephenate dehydrogenase) or ACT domain. Chapter 3 describes the characterisation of TmaDAH7PS with the N-terminal domain removed. The truncated enzyme was found to be more catalytically active than wild-type TmaDAH7PS and insensitive to inhibition by the aromatic amino acids, Tyr, Phe and tryptophan. Apart from the truncation of the ACT domain, the crystal structure of truncTmaDAH7PS showed no major changes to the monomer structure when compared to wild-type TmaDAH7PS. However, truncTmaDAH7PS crystallises as a dimer, unlike wild-type TmaDAH7PS. In Chapter 4, the solution of the crystal structure of TmaDAH7PS with Tyr bound is presented. Tyr binding was shown to induce a significant conformational change, and Tyr is observed to bind at the interface between the ACT domains from two diagonally located monomers of the tetramer. The major reorganisation of the regulatory domain with respect to the barrel observed in the crystal structure, was confirmed by small angle X-ray scattering. The closed conformation adopted by the protein on Tyr binding physically gates the neighbouring barrel and blocks substrate entry into the active site. Chapter 5 explores the interactions between TmaDAH7PS and the allosteric inhibitor, Tyr. The residues His29 and Ser31, which form hydrogen bonds with the hydroxyl moiety of the Tyr ligand, were examined for their impact on the sensitivity and selectivity of the enzyme for the inhibitors Tyr and Phe. The hydroxyl side chain of Ser31 was found to be important for both the preferential inhibition by Tyr over Phe and the inhibitory mechanism. His29 (the hydrogen-bonding partner of Ser31) appears to play a secondary role in determining ligand selectivity and the relative positioning of these two residues is crucial to the inhibition of the enzyme. Chapter 6 evaluates the transferability of allosteric control of catalytic activity. The ACT domain of TmaDAH7PS was fused onto the barrel of the unregulated PfuDAH7PS. This chimeric enzyme was found to be catalytically active, inhibited by Tyr (although less sensitive) and preliminary crystallographic results show inhibition occurs via the same conformational change observed for wild-type TmaDAH7PS.

Mechanical ventilation in itself can harm the lung and cause ventilator-induced lung injury (VILI), which can induce or aggravate acute respiratory distress syndrome (ARDS). Much debate remains over pivotal concepts regarding the pathophysiology of VILI, especially about the precise contribution, kinetics, and primary role of potential VILI mechanisms. Consequently, it remains largely unknown how best to design a well-timed and full-bodied mechanical ventilation strategy. Little is known also about small airways dysfunction in ARDS. Dynamic positron emission tomography (PET) with [18F]fluoro-2-deoxy-D-glucose (18F-FDG) can be used to image cellular metabolism, which during lung inflammation mainly reflects neutrophil activity, allowing the study of regional lung inflammation in vivo. We studied the regional evolution of inflammation using dynamic PET/CT imaging of 18F-FDG in VILI and during different lung-protective mechanical ventilation strategies. By dynamic CT we investigated also the location and magnitude of peripheral airway closure and alveolar collapse under high and low distending pressures and high and low inspiratory oxygen fraction. Piglets were submitted to an experimental model of early ARDS combining repeated lung lavages and injurious mechanical ventilation. The animals were subsequently studied during sustained VILI, or submitted to distinct approaches of lung-protective mechanical ventilation: the one recommended by the ARDS Network (ARDSNet), or to one defined as open lung approach (OLA). The normally and poorly aerated regions - corresponding to intermediate gravitational zones - were the primary targets of the inflammatory process accompanying early VILI, which may be attributed to the small volume of the aerated lung that receives most of ventilation. The ARDSNet strategy did not attenuate global pulmonary inflammation during 27h and led to a concentration of inflammatory activity in the upper and poorly aerated lung regions. The OLA, in comparison with the ARDSNet approach, resulted in sustained and better gas exchange and lung mechanics. Moreover, the OLA strategy resulted in less global and regional inflammation. Dynamic CT data suggested that a significant amount of airway closure and related reabsorption atelectasis occurs in acute lung injury. Whether potential distal bronchioles injury (“bronchiolotrauma”) is a critical and decisive element in ventilator-associated lung injury is a matter for future studies.

Interferons (IFNs) are an essential part of innate immunity and contribute to adaptive immune responses. Here, we employed a loss-of-function analysis with human A549 respiratory epithelial cells with a knockout (KO) of the type I IFN receptor (IFNAR KO), either solely or together with the receptor of type III IFN (IFNAR/IFNLR1 KO). The course of rubella virus (RuV) infection on the IFNAR KO A549 cells was comparable to the control A549. However, on the IFNAR/IFNLR1 KO A549 cells, both genome replication and the synthesis of viral proteins were significantly enhanced. The generation of IFN β during RuV infection was influenced by type III IFN signaling. In contrast to IFNAR KO A549, extracellular IFN β was not detected on IFNAR/IFNLR1 KO A549. The bioenergetic profile of RuV-infected IFNAR/IFNLR1 KO A549 cells generated by extracellular flux analysis revealed a significant increase in glycolysis, whereas mitochondrial respiration was comparable between all three cell types. Moreover, the application of the glucose analogue 2-deoxy-D-glucose (2-DG) significantly increased viral protein synthesis in control A549 cells, while no effect was noted on IFNAR/IFNLR KO A549. In conclusion, we identified a positive signaling circuit of type III IFN signaling on the generation of IFN β during RuV infection and an IFN signaling-dependent contribution of glycolysis to RuV infection. This study on epithelial A549 cells emphasizes the interaction between glycolysis and antiviral IFN signaling and notably, the antiviral activity of type III IFNs against RuV infection, especially in the absence of both type I and III IFN signaling, the RuV replication cycle was enhanced.