Browsing by Subject "Medicinal Chemistry"
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Item Analysis of pyridyloxobutyl and pyridylhydroxybutyl DNA adducts in extra-hepatic tissues of rats treated with tobacco-specific nitrosamines.(2009-07) Zhang, SiyiThe tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) and N’-nitrosonornicotine (NNN) are potent carcinogens in tobacco products, and are believed to play a major role as causes of tobacco-related cancers. NNK and NNN require metabolic activation to exert their carcinogenic effects. Cytochrome P450-catalyzed α-hydroxylation of NNK and NNN generates a reactive intermediate, which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. NNK is reduced to its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reversible and stereoselective manner. NNAL similarly undergoes α- hydroxylation and produces pyridylhydroxybutyl (PHB)-DNA adducts. In this thesis, we used liquid chromatography-electrospray ionization-tandem mass spectrometry (LCESI- MS/MS) to analyze POB- and PHB-DNA adducts in extra-hepatic tissues of F344 rats treated chronically with NNK and enantiomers of NNAL and NNN. POB- and PHB-DNA adduct levels were remarkably similar in NNK- and (S)-NNAL-treated rats, while distinctively different from those in (R)-NNAL-treated rats. These data indicate extensive retention of (S)-NNAL in various tissues of NNK-treated rats, and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis. (S)-NNN treatment produced more POB-DNA adducts in the rat oral mucosa, whereas adduct formation from (R)-NNN treatment was more favored in the nasal olfactory and respiratory mucosa. These results suggest that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues, and that (S)-NNN might be an oral carcinogen in rats. In additional studies of this thesis, LC-ESI-MS/MS methods were developed for the quantitative analysis of 1, N2-propanodeoxyguanosine adducts derived from acrolein and crotonaldehyde (Acr-dGuo and Cro-dGuo). Acrolein and crotonaldehyde are widely-spread environmental pollutants, are present in cigarette smoke, and are formed endogenously through lipid peroxidation. Our methods for the analysis of Acr-dGuo and Cro-dGuo adducts are sensitive, accurate, and precise. These adducts were detected for the first time in the human lung. However, no differences were observed in adduct levels between self-reported smokers and non-smokers. The potential importance of these adducts in the human lung requires further study.Item Applications of Fluorine Magnetic Resonance for Small-Molecule Screening, Ligand Development, and Oxygen Sensing(2017-08) Gee, CliffordProtein-protein interactions (PPIs) play a vital role in biological processes but are difficult to target therapeutically. However, targeting PPIs is an important challenge because their dysregulation is linked to many various disease states including cancers and neurological disorders. While high throughput screening (HTS) has long been the standard method for drug discovery, fragment-based screening (FBS) has emerged as a promising alternative strategy due to its greater coverage of chemical space with smaller library sizes. Successful cases like Vemurafenib and Venetoclax, continue to bolster FBS efforts. Though many techniques, including X-ray crystallography, surface plasmon resonance, and thermal shift assays, have all been used as screening tools, the central hypothesis of this dissertation is that 19F NMR is a powerful and time efficient FBS tool that is complementary to existing tools and is useful for characterizing proteins and small molecule ligands. Protein-Observed Fluorine NMR Spectroscopy (PrOF NMR) due to its high speed, lack of background signals, environmental sensitivity, is an ideal method to use for both ligand discovery and characterization of ligand-protein interactions. Herein, we describe the application of PrOF NMR to two proteins in particular, the KIX domain of CBP/p300 which is part of a larger transcriptional activation complex, and the first bromodomain of BrdT, an epigenetic “reader” protein that has been validated as a target for male contraception. We demonstrate the use of PrOF NMR as a primary screening tool for KIX, identifying key pharmacophores for KIX binding. We also demonstrate the use of PrOF NMR for characterizing ligand-protein interactions, uncovering a new binding site in KIX, distinct from its two native transcription factor binding sites. Validation of hits from other screening campaigns can also be followed via PrOF NMR, and the quantitative information obtained can be used to guide the structure-activity relationship (SAR) process for further ligand development. Beyond ligand discovery in proteins, fluorine magnetic resonance can also be applied as an imaging and oximetry tool. Given the sensitivity of fluorine and its applications in both biophysical and biomedical contexts, fluorine magnetic resonance serves as a new tool for small-molecule screening, ligand development, and oxygen sensing.Item Chemical aspects of acylfulvene bioactivation to a cytotoxic reactive intermediate.(2009-12) Pietsch, Kathryn ElizabethUnderstanding molecular mechanisms of cytotoxicity is vital to the development of more effective chemotherapies. Acylfulvenes (AFs) are a class of semisynthetic analogues of the natural product illudin S. Minor structural changes between the parent compound and AFs have resulted in a more favorable selectivity profile in preclinical chemotherapy assays. AF cytotoxicity involves alkylation of biological targets, including DNA and cellular proteins. While AFs are capable of direct alkylation, reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Alkenal/one oxidoreductase (AOR) is a cytosolic enzyme implicated in activating AF in cells that are sensitive to the drug. This study aims to elucidate chemical aspects of acylfulvene activation mechanisms. We compared enzymatic versus chemical activation pathways for AF involving NADPH-dependent AOR or sodium borohydride, respectively. These two processes result in isomeric reactive intermediates. Despite structural differences, these isomers appear to have similar biological activity and give rise to similar patterns of DNA modification. Cell-based studies, utilizing human embryonic kidney cells transiently transfected with an AOR-overexpressing vector, were conducted to test the hypothesis that a chemically activated AF does not require further bioactivation to be cytotoxic. The reactivity of this activated compound was further assessed by measuring its half-life in the presence of acid. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful tool for anticipate that the chemically activated form of AF will serve as a useful tool for evaluating protein and nucleic acid interactions, and to gain a further understanding of their contributions to cytotoxicity, independent of bioactivation.Item Clinical pharmacokinetics study of phenytoin in epilepsy patients & expression of oxysterol 7 Alpha - hydroxylase (hCYP7B1) in E.coli(2010-09) Aliwarga, TheresaThe determination of pharmacokinetic parameters is crucial both for clinical studies and early in the drug discovery and development. This thesis describes a clinical pharmacokinetics study of one anticonvulsant drug, i.e. phenytoin (PHT) and the early screening study of potential chemopreventive agent for prostate cancer. PHT is extensively bound to plasma proteins, is excreted from the body as oxidative metabolites in the urine, and exhibits a non-linear pharmacokinetics profile. In this study, stable-labeled PHT was given either intravenously or intramuscularly. The simultaneous administration of oral and IV PHT enables a direct determination of the pharmacokinetic parameters of clearance, volume, half-life, and absolute oral bioavailability. Urine samples from epilepsy patients who were on maintenance therapy of PHT were collected from 0-12 hours and 12-24 hours after a single daily dose to measure the two principal PHT urinary metabolites, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (DHD). An isocratic HPLC-NI-APCI-MS method was used to quantify metabolites in urine. A weak, but significant, Spearman Rank Correlation was observed between the total urinary metabolites recovered and the oral bioavailability (p-value = 0.00924, r2= 0.166). The percent of dose recovered in urine ranged from 35.4% ± 15.7% in young adult patients (age 21-49 years old) and 32.9% ± 15.0% in patients of age 65-93 years indicating highly variable absorption. In contrast, absolute bioavailability was 0.864 ± 0.194 and 0.925 ± 0.252 for the two groups, as determined by the stable-isotope technique. Unaccounted biliary-fecal excretion of p-HPPH glucuronide, subjects’ noncompliance, and incomplete urine collection are possible explanations. Consequently, bioavailability is best determined by stable-isotope method. Chapter 2 of this thesis illustrates the early screening study of chemopreventive agent for prostate cancer. The rationale of this study was attempting to inhibit the metabolism of 5-androstane-3,17β-diol (3-Adiol). A metabolite of dihydrotestosterone, 3-Adiol, inhibits LNCaP prostate cell proliferation in the presence of Estrogen Receptor . CYP7B1 is the enzyme responsible for catalyzing the 6 and 7 hydroxylations of 3-Adiol in prostate. In this study, expression and purification of human CYP7B1 in E.coli was attempted. Despite spectroscopic evidence of P450 expression, the enzyme failed to turn over its endogenous substrate, DHEA to 7-hydroxy DHEA.Item Cyclic enaminones: synthons for piperidine containing natural products and natural product analogs.(2012-12) Gay, Bryant CharlesCyclic Enaminones: Synthons for Piperidine Containing Natural Products and Natural Product Analogs. Cyclic enaminones are important synthetic intermediates for the preparation of piperidine containing natural products and target molecules such as drugs, which require a piperidine moiety for bioactivity. They are valuable synthons because of their unique reactivity and chemical stability. In this thesis, I developed novel chemistry from which to derivatize enaminones, and additionally I utilized cyclic enaminones as synthons for the preparation of natural product derivatives. I discovered that α,β-unsaturated aldehydes add to the α-carbon of enaminones in the presence of organocatalysts and thereby, introduce aliphatic α-branched substituents; a reaction that was previously very difficult to accomplish. The chiral reaction products were obtained in good- to excellent yields and with high enantiopurity. The absolute stereochemistry of the reaction products was also determined. I prepared analogues of the cytotoxic phenanthropiperidines tylophorine and boehmeriasin A, and I synthesized dihydrolyfoline, a member of a group of natural biphenylquinolizidine lactone alkaloids that posses a wide variety of bioactivities. The natural occurring cytotoxic phenanthropiperidines suffer from poor physiochemical properties and adverse side effects. Thus, the target molecules were designed to mitigate the unwanted side effects by improving their physiochemical properties. I devised short, concise routes toward the synthesis of a library of simplified tylophorine analogs, as well as 15-hydroxyboehmeriasin A. The tylophorine analog library was screened for antiproliferative activity against several cancer cell lines and thus, insight was gained into the structure-activity relationships of this class of compounds including the indication that an intact indolizidine moiety is critical for high potency of tylophorine analogues. 15-Hydroxyboehmeriasin A was prepared and evaluated for cytotoxicity against the A549 human lung carcinoma cell line. An observed GI50 of 81 nM demonstrates that the addition of the 15-hydroxyl group was not detrimental to cytotoxicity and that this position should be explored for further modifications in efforts to improve the physicochemical properties. In addition I prepared (±)-dihydrolyfoline in a concise manner from a bicyclic enaminone precursor using a biomimetic oxidative biaryl coupling approach as the key reaction step.Item Defining the catalytic and kinetic mechanism and natural function of the highly conserved acyl-amp hydrolase, HINT1(2011-12) Bardaweel, SanaaHistidine triad nucleotide binding proteins (Hints) are members of the histidine triad (HIT) protein superfamily of nucleotidyl transferases and hydrolyases. It has been recently demonstrated that Hints are efficient phosphoramidases and therefore activators of potent antiviral and anticancer pronucleotides. In spite of their high evolutionary conservation among all kingdoms of life, and the several regulatory functions in which Hints have been implicated, a clear connection between their observed function and their catalytic efficiency has not been elucidated. To gain a comprehensive understanding of the essential role of these ubiquitous enzymes, our laboratory has devoted a considerable effort toward the delineation of the principles governing Hints catalysis and cellular function. Such understanding will provide an unprecedented ability to assess the role of these highly conserved, but functionally unknown enzymes. Since Hints are found in both prokaryotes and eukaryotes, we have attempted to understand their function, mechanism, and structural determinants in prokaryotes, under the assumption that their role may be at least partially conserved among members of the tree of life. Recently, we have demonstrated by E. coli gene disruption studies that the bacterial Hint enzyme is necessary for growth under high salt conditions, and when alanine is a carbon and nitrogen source. Through a combination of phenotypic screening and complementation experiments with wild-type and ecHinT knock-out E. coli strains, we have shown that catalytically-active ecHinT is required for growth on D-alanine. In addition, using Hint-inhibitors and active-site mutants, we have demonstrated that expression of catalytically-active ecHinT is essential for the activity of the enzyme D-amino acid dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-amino acids metabolic pathway. These results are considered as the first report in literature that shows a successful connection between a discovered Hint-related phenotype and the catalytic activity of Hint. Previously, we have demonstrated that lysyl-AMP generated by LysRS is a substrate for both human and E. coli Hints. In addition, we have shown that the ability of Hint to hydrolyze lysyl-AMP depends on its enzymatic activity. Here, we demonstrate that the molecular determinants governing this regulation appear to reside in the C-terminus region of Hint. Interestingly, the ecHinT-DadA interaction appears also to be governed by both ecHinT-activity and the C-terminus loop. We have also expanded our scope to look at possible toxicity of D-alanine in E. coli strains lacking dadA or hinT. Our results demonstrate that E. coli mutants lacking dadA or hinT are highly susceptible to D-alanine toxicity and that the catalytic activity of Hint is an essential requirement to protect E. coli from the observed toxicity of D-alanine. Based on careful analysis of the combined results from the ecHinT-LysRS and ecHinT-DadA potential interactions, and comprehensive understanding of the D-alanine metabolic pathway in bacteria, we proposed a possible regulatory mechanism of Hint, LysRS and DadA on global protein translational processes to prevent D-amino acids toxicity in E. coli.Item The design and synthesis of constrained AVPI peptidomimetics(2008-12) Kending, CoryRecent studies have demonstrated that breast cancer cell lines are sensitive to the XIAP (X-linked inhibitor of apoptotic proteins) antagonist Smac (second mitochondrial activator of caspases). Specifically the terminal tetrapeptide, AVPI, inhibits XIAP and thus sensitizes the breast cancer cells to various apoptotic therapeutics. These peptides are poor drug candidates due to their size, hydrophilicity and potential for peptidase cleavage. To address these issues we systematically synthesized peptide mimics that are constrained at various torsion angles along the peptide backbone. The compounds were tested in vivo and in vitro utilizing the laboratories of the Mayo Medical School. One compound, HCl*NH2-(R)-αMe-γ-Lactam-L-Val-L-Pro-L-Ile-OMe (3.1), increased the apoptotic signals above the assay background. Retro-modeling analysis utilizing the Schödinger flexible docking program suite did not provide an easy explanation for why this might be the case. The in silico models suggest that compound 3.1 interacts with only the primary pocket of the AVPI binding site and does not interact, like the native peptide, with both pockets in the active site. The R stereochemistry of the bicyclic compounds is predicted by our models to have a binding efficacy above that of the native peptide presumably due to the interaction with both pockets of the binding site as well as d-orbital interactions of the thiazole ring system with the neighboring tryptophan. The S stereochemistry was also initially proposed as a negative control for the biological assays, however, the instability of the molecule provided several daunting challenges to an already challenging and low yielding synthesis and the negative controls were abandoned when it was clear that the synthesis was futile. Our research shows limited evidence that our AVPI peptidomimetics are a potential scaffold to base other drug-like molecules upon. Future biological data from the bicyclic compounds will hopefully prove our hypothesis and molecular modeling studies correct.Item DNA-protein cross-linking by bifunctional DNA alkylating agents.(2010-03) Michaelson-Richie, Erin DeniseMany common DNA alkylating agents, such as environmental toxins and chemotherapeutic drugs, are bis-electrophiles capable of covalently cross-linking cellular biomolecules. While DNA-DNA cross-linking by such compounds is well-characterized, the identities and the biological effects of the corresponding DNA-protein cross-links (DPCs) are poorly understood. Furthermore, because bis-electrophiles produce DNA-DNA cross-links and DNA monoadducts in addition to DPCs, it is difficult to establish the biological outcomes specifically resulting from DPC lesions. The purpose of the present work was to characterize DNA-protein cross-linking by two bis-electrophiles, 1,2,3,4-diepoxybutane (DEB) and bis(2-chloroethyl)methylamine (mechlorethamine), and to evaluate the ability of DPCs to induce cytotoxic and mutagenic effects. Mass spectrometry-based proteomics and immunological detection methods identified 41 proteins participating in DPC formation in the presence of DEB in nuclear protein extracts prepared from human cervical carcinoma (HeLa) cells, and 38 proteins which formed DPCs to the chromosomal DNA of human fibrosarcoma (HT1080) cells treated with mechlorethamine. Relative to their cellular abundance, a disproportionately high number of the proteins involved in DPC formation were nuclear proteins with known nucleic acid-binding capabilities which participate in cellular processes such as transcriptional regulation and DNA repair. HPLC-ESI+-MS/MS analysis of total proteolytic digests of DPCs revealed the chemical structures of the cross-links produced by DEB and mechlorethamine to be 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-N7G-BD) and N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA), respectively. In order to analyze the biological consequences of DPC lesions, we selectively induced DPCs in mammalian cell cultures by electroporating them in the presence of epoxide-containing protein reagents. Significant levels of cell death and mutations were observed, suggesting that DPC lesions contribute to the biological effects of bis-electrophiles.Item DNA-protein crossing-linking by BIS-electrophiles(2008-10) Loeber, Rachel LeaA key carcinogenic metabolite of the important industrial chemical 1,3-butadiene (BD), DEB is a bifunctional alkylating agent capable of reacting with both DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)- guanine monoadducts, which can then react with a second nucleophilic site to form crosslinked adducts. A recent report revealed a strong correlation between expression of the human DNA repair protein AGT in bacteria and the cytotoxic and mutagenic activity of DEB (J. G. Valadez et al., Chem. Res. Toxicol. 17 (2004) 972-982). As AGT expression appeared to enhance the toxic effects of this bis-electrophile, the authors proposed that DEB induces AGT-DNA cross-links. The purpose of our study was to structurally characterize DEB-induced AGT-DNA conjugates and to identify amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was first detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of both wild-type AGT or a C145A AGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol- dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+- MS/MS sequencing of tryptic peptides obtained from dG monoepoxide-treated AGT indicated that the two cross linking sites were located at the alkyl acceptor site, Cys145 39 and a neighboring active site residue, Cys150. The same two active site cysteines became cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+-MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of AGT (C = Cys145 ). Replacement of the catalytic cysteine residue with alanine (C145A AGT) abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified AGT analyzed in parallel with an authentic standard. Based upon these results, the formation of AGT-DNA cross-links is a likely mechanism to explain the enhanced cytotoxicity of DEB in cells expressing this important repair protein.Item Exploration on atypical kinetics of UGT1A1 and UGT1A4(2010-08) Zhou, JinAtypical (non-Michaelis-Menten) kinetics confound straightforward in vitro-in vivo extrapolations on clearance and inhibition potentials of new chemical entities. However, unlike cytochrome P450s, studies on atypical kinetics of uridine 5'-diphospho-glucuronosyltransferases (UGTs) are much less prevalent. With the use of model substrates, the atypical kinetics of two important glucuronidation enzymes, UGT1A1 and UGT1A4, were explored. In Chapter 2 (Part I), two positional isomers dihydrotestosterone (DHT) and trans-androsterone (t-AND) were used as probe substrates and their glucuronidation kinetics with recombinant UGT1A4 were evaluated alone and in the presence of a UGT1A4 substrate (tamoxifen (TAM) or lamotrigine (LTG)). Interestingly, co-incubation with TAM, a high affinity UGT1A4 substrate, resulted in a concentration-dependent activation/ inhibition effect on DHT and t-AND glucuronidation. The glucuronidation kinetics of TAM and the interactions of DHT or t-AND on TAM glucuronudation were then evaluated. TAM displayed substrate inhibition kinetics, however, the substrate inhibition kinetic profile of TAM became more hyperbolic, as DHT or t-AND concentration was increased. Kinetic analysis with two-site kinetic models demonstrated that these atypical interactions can be explained by the existence of multiple aglycone substrate binding sites in UGT1A4. In this chapter, the glucuronidation kinetics of DHT, t-AND and TAM with two UGT1A4 variants (UGT1A4 P24T and UGT1A4 L48V) (Part II) and the interactions of ethinylestradiol and estradiol-3-sulfate on UGT1A4-catalyzed LTG glucuronidation (Part III) were also described. In Chapter 3, a robust bilirubin glucuronidation assay with recombinant UGT1A1 was established (Part I). With this assay, the correlation between UGT1A1-catalyzed bilirubin glucuronidation and estradiol-3-glucuronidation was studied in the presence model UGT1A1 substrates or inhibitors. Through this evaluation, we found estradiol-3-glucuronidation is a reasonable surrogate in vitro predictor for interactions with bilirubin even though they displayed different kinetic profiles. However, atypical interactions by some effectors were observed with estradiol-3-glucuronidation but not with bilirubin glucuronidation. The atypical interactions of daidzein and SN-38 on ethinylestradiol-3-glucuronidation were also analyzed with multi-site kinetic models. In conclusion, through kinetic studies with prototype substrates, we found evidence to support the existence of multiple aglycone substrate binding sites in UGT1A1 and UGT1A4. Thus, multiple probe substrates may be needed to evaluate drug-drug interactions involving UGT1A1/UGT1A4-catalyzed metabolism.Item Formation, Persistence, and Repair of 1,2,3,4-Diepoxybutane-Induced bifunctional DNA adducts in tissues of rodents exposed to 1,3-Butadiene by Inhalation.(2009-09) Goggin, Melissa Mary1, 3-butadiene (BD) is an industrial chemical and environmental pollutant. It is also classified as a probable human carcinogen based on animal tumorigenesis studies and human epidemiologic evidence. BD is metabolized to three reactive epoxides, of which 1,2,3,4-diepoxybutane (DEB) is the most mutagenic, likely due to its bifunctional electrophilic structure that enables it to form DNA-DNA cross-links, 1,4-bis-(guan-7- yl)-2,3-butanediol (bis-N7G-BD) and 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7GN1A- BD), and 1,N6-(2-hydroxy-3-hydroxymethyl-1,3-propanodiyl)-dA (1,N6-HMHPdA) exocyclic DNA adducts. The purpose of this research is to identify mechanismbased biomarkers of exposure to BD. Our laboratory has identified DNA-DNA crosslinks and exocyclic deoxyadenosine adducts formed from DEB that could potentially be used as biomarkers of exposure. Highly specific and sensitive isotope dilution HPLCESI+- MS/MS methods have been developed to analyze DEB-induced DNA-DNA crosslinks and exocyclic DEB-dA adducts in DNA extracted from laboratory animals exposed to BD by inhalation. bis-N7G-BD was the most abundant adduct, N7G-N1ABD was ~10 times less abundant than bis-N7G-BD, and 1,N6-HMHP-dA was the least abundant (~4 fold lower than N7G-N1A-BD). The quantitative isotope dilution mass spectrometry methods developed in this work were employed to investigate the dose dependent formation, persistence, and repair of the BD-induced DNA adducts in laboratory rodents. Species, gender, and tissue differences in adduct levels were observed. Mouse DNA contained a greater number of DEB-specific adducts than rat DNA at identical exposures. Adduct levels were also higher in female as compared to male rodents, and in liver DNA (compared to lung, brain, kidney, and thymus). Although bis-N7G-BD was most abundant DEB-DNA adduct immediately following exposure, it did not persist in mouse or rat liver while the other adducts did. We did not observe repair of the bifunctional lesions by BER or NER, however, in vitro studies suggest that 1,N6-HMHP-dA is repaired by AlkB. The research presented in this thesis is consistent with animal inhalation studies where mice were more susceptible to tumor formation. The data also suggests that these differences are due to species differences in the extent of BD metabolism to DEB. Two of the adducts, N7G-N1A-BD and 1,N6- HMHP-dA were persistent in DNA and may be responsible for the mutagenicity of BD.Item Library Synthesis Of Piperidinone Sulfonamides, Transition Metal-Free C-H Trifluoromethylation Of Cyclic Enaminones, And Elucidating The Binding Site Of Epothilones On Beta-Tubulin With Epothilone Photoaffinity Probes(2014-07) Ranade, AdwaitChapter 1 focuses on synthesizing a library of piperidinone sulfonamides. The piperidinones serve as valuables intermediates for the synthesis of nitrogen-containing bioactive molecules, various alkaloids, and drug candidates. Amongst the myriads of highly derivatized N-heterocyclic compounds, molecules possessing the piperidinone sulfonamide moiety in their structures show interesting biological activities. A library of 18 piperidinone sulfonamides was prepared under a Pilot Scale Library grant and submitted to NIH for testing in various biological assays. Two compounds from the library were identified as active hits. One of the compounds showed prion protein 5' UTR inhibition while the other showed inhibition of human platelet-activating factor acetylhydrolase 1b, catalytic subunit 2. Chapter 2 focuses on direct C-H trifluoromethylation of cyclic enaminones. Cyclic enaminones are of interest in natural product synthesis and are regarded as valuable synthons due to unique structural and chemical properties. They serve as versatile precursors for synthesizing piperidine-containing alkaloids and drug molecules. In this project, transition metal free, direct C-H trifluoromethylation of cyclic enaminones was developed with trimethyl(trifluoromethyl)silane (TMSCF3). This method proceeds under mild conditions at room temperature and possibly involves a radical mechanism. The C-H functionalization was successful with both electron-rich and electron-deficient cyclic enaminones. This methodology circumvents substrate prefunctionalization and transition metal catalysis, and allows a convenient and direct access to a variety of medicinally significant 3-trifluoromethylpiperidine derivatives. This chemistry also presents a rare example of a direct trifluoromethylation of an internal olefinic C-H bond. Chapter 3 focuses on efforts toward elucidating the binding site of epothilones on ß-tubulin. Epothilones are potent cytotoxic tubulin-binding polyketide-derived macrolides. Even though the binding sites for epothilones and paclitaxel on ß-tubulin overlap, epothilones show efficacy against paclitaxel-resistant cancer cell lines. This implies a significantly different binding mode for epothilones. To date, two epothilone binding models have been proposed based on NMR and electron-crystallography data. In order to differentiate the proposed binding modes, four epothilone A photoaffinity analogues were designed. Three of those analogues were successfully synthesized and showed excellent cytotoxicity as well as the required tubulin assembly. It was hypothesized that the protein region labeled by these photoprobes is dependent on the epothilone conformation at the binding site. For one of the analogues, the probe-labeled peptide fragment `TARGSQQY' (residues 274 to 281) in the ß-tubulin isoform TBB3 was identified by MS analysis. Our experimental results corroborated the consensus of both the models that Thr 274 and Arg 276 are necessary for binding of epothilones to ß-tubulin. However, based on the photoaffinity labeling studies results and molecular modeling studies, an orientation of the epothilone in the binding site is proposed that is significantly different from those previously proposed.Item Macrocyclization Through Ene-Yne Cross-Coupling/Alkyne Reduction Tandem Reaction And Its Application In Natural Product Synthesis(2015-09) Li, WeiChapter 1 — Macrocyclization Through Copper-Catalyzed Castro–Stephens Coupling/Alkyne Reduction Tandem Reaction Macrocycles, incorporating conjugated polyene subunits within the ring, are structural features found in a number of natural products that exhibit diverse and potent biological activities. Existing methods for the construction of such structures are limited and in many cases inefficient. We discovered an unprecedented copper-mediated reductive ene–yne macrocyclization reaction during our pursuit of the total synthesis of oximidine II. The reaction selectively generates an endocyclic Z-double bond through an intramolecular coupling of a vinyl iodide and a terminal alkyne fragment followed by in situ alkyne reduction. We developed this transformation as a general method for the preparation of polyunsaturated macrocycles. The reaction conditions were optimized and the scope of the reaction was extensively explored. It was found that the alkyne reduction step is driven by the release of the ring strain. Thus, the reaction is particularly efficient for suitably strained 11- to 13-membered E,Z-1,3-diene macrocycles. A complementary stepwise procedure was employed for the synthesis of larger rings. Finally, a plausible reaction mechanism was proposed based on experimental findings. HASH(0x7f87dd8493f8) Chapter 2 — Formal Total Synthesis of Lactimidomycin Lactimidomycin is a macrocyclic natural product that possesses potent in vitro and in vivo anti-tumor activities. We accomplished a facile, 9-step synthesis of an advanced intermediate for the total synthesis of lactimidomycin. The crucial 12-membered polyene lactone core structure was constructed employing our newly developed Castro–Stephens coupling/alkyne reduction tandem reaction. The stereocenters were established via asymmetric a vinylogous aldol reaction and a Marshall’s propargylation reaction. Chapter 3 — Synthesis and Biological Evaluation of Oximidine II Analogues Oximidine II belongs to a family of benzolactone enamide natural products that exert their cytotoxic effects through inhibition of V-ATPases. Unlike other members of this family, the structure-activity relationship (SAR) of oximidines has not been extensively investigated. Guided by computational analysis and previous studies in our group, we designed and synthesized two oximidine II analogues with simplified scaffold. The simplified benzolactone core was accessed through a ring-closing metathesis (RCM) reaction and the enamide side chain was installed via a copper-mediated C–N coupling reaction. The analogues were evaluated for their biological activity. The results revealed that these molecules were weakly cytotoxic to a number of cancer cell lines.Item Studies on Human arylamine N-acetyltransferases.(2009-01) Liu, LiArylamine N-acetyltransferases (NATs, EC 2.3.1.5) are important phase II drug metabolism enzymes that are expressed in most human tissues. Humans express two NAT isozymes, NAT1 and NAT2, which share 81% sequence identity. NATs catalyze the AcCoA-dependent N-acetylation of arylamines to arylamides, which is a critical detoxification process and competes with cytochrome P450-catalyzed oxidation of arylamines to N-arylhydroxylamines. In this thesis, recombinant human NAT2 was successfully overexpressed and purified to homogeneity; the substrate specificities and molecular interactions of environmental arylamines with human NAT1 and NAT2 were characterized; the in vitro and intracellular inactivation of human NAT1 and NAT2 by the nitrosoarene and N-arylhydroxamic acid metabolites of toxic and carcinogenic arylamines was investigated. To investigate the substrate specificities and inhibition of human NATs, we developed a method to produce homogeneous human "wild type" NAT2 in milligram quantities. Human NAT2 was overexpressed as a L54F dihydrofolate reductase (DHFR) fusion protein linked with a TEV protease-cleavage linker. Chromatography with a methotrexate affinity column, followed by a DEAE column, afforded partial purification of the fusion protein. The fusion protein was digested with TEV protease, and human NAT2 was purified to homogeneity with a second DEAE column. A total of 2.8 mg of human rNAT2 from 2 L of cell culture was purified to homogeneity with this methodology. The kinetic specificity constants (kcat/Km) for N-acetylation of arylamine environmental contaminants were characterized for human NAT1 and NAT2. The dramatic effects of small alkyl substituents on the relative abilities of NAT1 and NAT2 to acetylate substituted anilines was reflected by the 1000-fold difference in the NAT1/NAT2 ratio of the specificity constants for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents. A NMR-based model was used to interpret the interactions of binding site residues with the alkylanilines and to provide insight into how NATs achieve certain substrate specificities. Arylamines and their N-hydroxylation products, N-arylhydroxylamines, can undergo oxidation to form nitrosoarenes in vivo. We investigated the inactivation of human NAT1 by the nitrosoarene metabolites of four environmental arylamines in vitro and in human cells. 4-Nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, were found to be potent inactivators of human NAT1. 4-NO-BP is an affinity label for NAT1 (kinact/KI = 59,200 M-1s -1), whereas 2-NO-F inactivates NAT1 through an apparent bimolecular process (k2 = 34,500 M-1s -1). Glutathione (GSH) afforded only partial protection of NAT1 from inactivation by the two nitrosoarenes in vitro. Nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, were much weaker inhibitors of NAT1. Treatment of HeLa cells with 4-NO-BP (10 uM) for 15 minutes and 60 minutes caused 39% and 58% losses of NAT1 activity, respectively, without causing a decrease in either glyceraldehyde phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F was an even more effective inhibitor of HeLa cell NAT1 than 4-NO-BP. Tandem mass spectrometric analysis indicated that 4-NO-BP treatment of HeLa cells in which 3FLAG-NAT1 had been overexpressed resulted in a formation of (4-biphenyl)sulfinamide with the active site Cys68 of NAT1. This is consistent with the results obtained with recombinant NATs in vitro. It was also demonstrated that the nitrosoarene metabolites of arylamines that are efficiently N-acetylated by NAT2 are potent inactivators of NAT2 in vitro and in human cells. The second order rate constants for inactivation of NAT2 by 4-NO-BP and 2-NO-F were 80,400 M-1s -1 and 50,500 M-1s -1, respectively; the values for NO-B and 2-NO-T were 14 M-1s -1 and 16 M-1s -1. Treatment of HeLa cells with 4-NO-BP (5 uM) for 1 h caused a 23% reduction in NAT2 activity, and exposure to 2-NO-F (2.5 uM) for 1 h caused a 22% loss of NAT2 activity, without inhibiting GAPDH and GR activities. Therefore, HeLa intracellular NAT2 is less susceptible to the effects of the lower concentrations of the two nitrosoarenes than is NAT1. It is concluded that NAT1 and NAT2 are intracellular targets of the nitrosoarene metabolites of 4-aminobiphenyl and 2-aminofluorene. Low concentrations of nitrosoarenes may cause a loss of NAT1 and NAT2 activities and impair a key detoxification pathway. N-Arylhydroxamic acids, which are also potentially reactive metabolites of arylamines, have been shown to inactivate hamster NATs, in vitro and in vivo, and human recombinant NAT1. It was previously established that inactivation of NATs by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP) involves an initial NAT-mediated deacetylation to form N-OH-4-aminobiphenyl, which undergoes oxidation to the electrophilic 4-nitrosobiphenyl (4-NO-BP), followed by reaction with the nucleophilic active site Cys68 to form a sulfinamide. We hypothesized that the relative stabilities of the acetyl-enzyme intermediates influence the susceptibilities of NATs to inactivation by N-arylhydroxamic acids. The second order rate constant for inactivation of human NAT2 by N-OH-AAF was 459 M-1s-1, which was 8-fold greater than that for NAT1. Mass spectrometric analysis of both NATs after treatment with N-OH-AAF revealed that the principal adducts were sulfinamide conjugates of Cys68. Kinetic analysis revealed that the hydrolysis rate of acetyl-NAT2 was 4.7-fold greater than that of acetyl-NAT1. Thus, the more rapid inactivation of NAT2 was facilitated by the rapid hydrolysis of the Cys68 thioacetyl ester to free the Cys68 thiol group for reaction with 2-NO-F. The hypothesis was further supported by the results from inactivation of human NATs by N-OH-4-AABP. Treatment of HeLa cells with N-OH-4-AABP (50 uM) for 6 hours had no effect on intracellular NAT2 activity, but caused a 24% decrease in NAT1 activity. Incubation with N-OH-4-AABP (100 uM) for 6 h reduced NAT2 and NAT1 activities by 19% and 56%, respectively. Treatment of HeLa cells with 50 uM N-OH-AAF for 6 hours reduced NAT2 and NAT1 activities by 11% and 33%, respectively. Therefore, approximately 10-fold greater concentrations and longer incubation times were required for the N-arylhydroxamic acids to produce effects on intracellular NATs than were required for 2-NO-F and 4-NO-BP. HeLa NAT1 is more susceptible to the effects of the N-arylhydroxamic acids than NAT2.Item Synthetic approaches toward azabicyclic and azaspirobicyclic scaffolds relevant to peptide conformation control.(2009-12) Bhagwanth, SwapnaA ring closing metathesis (RCM) strategy was explored toward synthesis of a conformationally constrained Type VIa #1;-turn mimic of the tripeptide dopaminergic receptor modulator, Pro-Leu-Gly-NH2 (PLG). It was anticipated that this strategy would result in a generalized route to synthesize the indolizidinone scaffold-based Type VIa #1;- turn mimic of any Xaa-Pro dipeptide. Although this strategy was unyielding in the desired result, the study highlighted the recalcitrant reactivity of sterically hindered amino acid residues. The interaction between the N-terminal tetrapeptide, Ala-Val-Pro-Ile (AVPI) of the pro-apoptotic protein, second mitochondrial activator of caspases (Smac), and the binding site on the X-linked inhibitor of apoptosis protein (XIAP) has been previously validated for its chemotherapeutic potential. We, designed and synthesized, conformationally-constrained diastereoisomeric AVPI analogues, containing the 5.5- fused bicyclic thiazolidine scaffold. Although the synthesis of this scaffold presented several challenges; the base- and acid-lability of the thermodynamically unstable diastereomer as well as difficulties in the vital amidation step notwithstanding; synthesis of one diastereomer was successfully completed. A diastereomeric pair of 5.6.5-azaspirobicyclic lactams, synthesized to mimic the biological activity of PLG and possessing opposite stereochemistry at the C-8a’ position, was recently found to have opposing dopamine D2 receptor modulatory activities. Our goal was to identify the structural elements in this scaffold that were responsible for this opposite activity. We therefore, utilized molecular modeling on existing PLG modulators containing this scaffold and designed more 5.6.5-azaspirobicyclic lactams containing: (1) different stereochemistry at the C-3’ position and (2) various substitutions at the C-2’ position. Following the established paradigm toward synthesis of the target molecules only led to inconvenient epimerization/racemization issues. However, with a seemingly simple change in the order of events provided the desired target molecules. In addition, our initial hypothesis was verified by pharmacological testing of the synthesized molecules. Eventually it is anticipated that use of both positive and negative modulators will serve to understand the overall structural and biochemical mechanism of PLGmediated D2 receptor modulation.Item Toward therapeutic nanoassemblies: the design and modeling of protein-protein interactions.(2009-11) White, Brian RichardUnraveling the nanoscale processes of biological pathways via the testing, replication, and visualization of the underlying mechanisms remains a persistent challenge in the study of these critical life-governing systems. Recent advances in the field of chemically induced dimerization have unlocked multiple tools for the exploration of these facets of biology, including the development of switchable signaling systems, assertion of control over protein localization in the cell, and regulation of gene expression. An additional revelation through protein complexation by chemical induction is the construction of multivalent protein-based nanostructures, capable of bearing multiple targeting agents. However, stochastic assembly of these proteins has proven unsatisfactory in generating homogeneous populations. Herein, we have taken the initial steps toward developing a protein-based biomolecular language for nanostructural assembly. Through gel filtration analysis, we have characterized the ability of interfacial point mutations to modulate the stability of a bis-methotrexate (bis-MTX) induced E. coli dihydrofolate reductase (DHFR) dimer over a dynamic range of 1.5 kcal/mol. Furthermore, we have employed single-molecule fluorescence assays to demonstrate the stabilization of a heterodimeric DHFR dimer, yielding 4-fold selectivity for the heterodimer over either corresponding homodimer. In addition to our experimental characterization of the chemically induced DHFR dimer, we have also taken steps toward the construction of a tripartite computational model of dimerization in an effort to predict the effects of further mutations. We have tested a number of molecular mechanics force fields against quantum mechanical benchmarks and discovered that the MMFF94, OPLS2005, and AMBER force fields yield the most accurate electrostatic and configurational treatment of the complex bis-MTX dimerizer. While initial attempts at calculating the binding free energy of the macromolecular complex have been unsuccessful, we have gleaned important insights into the complexities of modeling this three-body system. The advances described within the following work delineate important aspects of protein interface remodeling in a chemically induced system and provide an avenue toward the further development of both a computational model of protein interactions and the future directed assembly of protein based materials and therapeutic nanostructures.