Cumulative evidence suggests that redox dysregulation in cancer cells represents a chemical vulnerability that can be targeted by pharmacological modulation of cellular oxidative stress. According to this emerging mechanism, pharmacological prooxidants may induce deviations from redox homeostasis causing cytotoxicity confined to malignant cells already at a high set point of constitutive oxidative stress leading to functional impairment, cell cycle arrest, and cell death. In contrast, the same prooxidant deviation from redox homeostasis is tolerated by nonmalignant cells that operate at a lower redox set point. This work focuses on experimental redox drug discovery targeting metastatic melanoma cells by pursuing the following specific aims: I. To identify drug-like lead compounds containing redox-directed pharmacophores for prooxidant intervention targeting melanoma in relevant models of the human disease. II. To investigate the molecular mechanism of action underlying antimelanoma activity of our lead compounds comprising Michael acceptors [cinnamaldehyde (CA) and 2,6-dichlorophenolindophenol (DCPIP)] and endoperoxides [dihydroartemesinin (DHA)]. III. To explore the therapeutic potential of drug-like electrophiles for non-melanoma indications including skin photoprotection and genotype-directed cancer chemotherapy. First, we have explored the possibility that prooxidant dietary constituents containing an electrophilic Michael acceptor pharmacophore may display chemotherapeutic activity. Focusing on the cinnamon-derived Michael acceptor CA we have demonstrated significant anti-melanoma activity of this dietary prooxidant observed in vitro and in vivo. Second, we have demonstrated that the synthetic quinoneimine and redox dye DCPIP targets human melanoma cells in vitro and in vivo. DCPIP-apoptogenicity observed in the human melanoma cell lines A375 and G361 was inversely correlated with NAD(P)H:quinone oxidoreductase (NQO1) expression levels. Efficacy against tumors with low NQO1 enzymatic activity including those displaying the human homozygous NQO1*2 missense genotype suggests feasibility of DCPIP-based genotype-directed redox intervention. Third, we demonstrated that the endoperoxide-based antimalarial DHA may serve as an experimental redox chemotherapeutic that selectively induces iron-dependent melanoma cell apoptosis without compromising viability of primary human melanocytes. Given the causative role of redox dysregulation in melanoma and the shortage of efficacious agents currently available, it seems that the emerging therapeutic potential of redox-directed chemotherapeutics for melanoma intervention deserves further evaluation.

Objectives: Evaluate the association of cancer on net worth, consumer debt, mortgage debt, home equity and changes in retirement trends. Methods: Data from the Health and Retirement Study from 1998-2010 was used. Persons had to have a diagnosis of cancer. The index date was the corresponding HRS wave of the year of the first diagnosis of cancer. The pre-index date was 2 years and a 2-year and 4-year post index was observed. Primary outcomes of interest were zero/negative net worth and net worth. Multiple logistic regression was used to test for the association between demographic, economic, human capital, and cancer-related variables on outcomes. Generalized linear models were conducted to assess the association of cancer on net worth, consumer debt, mortgage debt, and home equity. Multinomial logistic regression was performed to assess the association of cancer on retirement. Results: A total of 6,055,110 individuals (weighted) qualified. The majority of patients in this sample were male (53.8%), non-Hispanic (95.5%), and white (90.3%). Marital status (p<0.05), alcohol consumption (p=0.046), hypertension (p = 0.034), private insurance (p=0.001), cancer status (p<0.001), and cancer treatment (p=0.022) were significant predictors of zero/negative net worth 4-years after cancer diagnosis. Patients receiving treatment for their cancer were 71% more likely to have consumer debt 4-years post diagnosis (p=0.006). Patients who reported their cancer improving 4-years post diagnosis were significantly less likely (p=0.008) to have consumer debt (OR=0.59; 95%CI: 0.41-0.87). Cancer treatment and cancer status were significant predictors of mortgage debt (p<0.001 and 0.024, respectively). For individuals whose cancer either improved (OR=1.46; 95%CI: 1.04-2.06) or worsened (OR=4.09; 95%CI: 1.38-12.15), both groups were significantly more likely (p=0.030 and 0.011, respectively) to have home equity 4-years post diagnosis. Cancer status was a significant predictor of individuals transitioning from working to retired (p=0.022).Conclusion: This nationally representative investigation of 6.1 million patients over 50 years of age with cancer found that approximately 65% of cancer patients reported zero/negative net worth of cancer and almost 45% of cancer patients reported consumer debt four-years post diagnosis. Cancer-related characteristics explain a significant amount of the change in net worth four-years post diagnosis of cancer.

Background: Commonly used response scales in patient-reported outcome (PRO) measures include the visual analogue scale, 11-point numeric rating scale, 5-point numeric rating scale, 5-point verbal rating scale, and 5-point verbal-numeric rating scale. Although prior studies have explored the interpretation of response scale labels and compared scores resulting from the response scale, many questions remain. Purpose: To identify sets of verbal descriptors interpreted with the least variation and to explore whether the response scales provide interval level data and whether the scales are interchangeable. Methods: Subject recruitment and screening was through an online drug-drug interaction service (MediGuard.org). Via an online survey platform, subjects used a scale (0 = lowest possible and 10=highest possible) to assign interpretation scores to verbal descriptors. Repeated measures analysis of variance informed the test interval data between scores. Subjects also completed repeated administrations of four symptom-specific item stems with different response scales. Ordinal regression informed the analysis of scores assigned to verbal descriptors, comparison of probabilities of responding in given categories across scales, and prediction of response category on one scale conditional on observed response on another scale. Cut-points informed tests for interval level data. Results: The sample (n=350) comprised 223 females and 127 males with a mean (SD) age of 56.9 (12.1) years. Number of health conditions per subject ranged from 1 to 12 (median = 5). Age, sex, level of education, and number of health conditions were associated with the interpretation of verbal descriptors. Scores assigned to "poor," "fair," "good," "very good," "excellent," "somewhat," "sometimes," and "quite a bit," had the largest variation. The probability of responding in the same categories on the different response scales was significantly different across scales before and after collapsing categories. No scale yielded interval level data. The 11-NRS data tended more towards interval level than the data from other scales. Conclusions: Using different response scales with verbal descriptors in non-randomized studies may introduce bias. Differential item functioning and subgroup analyses should be investigated in the development and use of these response scales. The scales are not interchangeable. Compared with other scales the 11-NRS produced data approaching interval level. Collapsing categories entails significant probabilities of misclassification.

Administering drug therapy through the intravenous route ensures rapid, and complete, bioavailability, which can be critical in an emergency situation. However, bypassing all of its protective barriers leaves the body vulnerable to harm if the parenteral formulation becomes unstable when mixed with the blood. An example of this formulation instability is the precipitation of poorly water-soluble drugs after mixing with the blood's aqueous environment. This happens when parenteral formulations rely too heavily upon the solution pH, and excipients, to increase the drug solubility. This precipitation in the blood can damage venous cell membranes producing symptoms ranging from mild skin irritation to death. To screen potential drug formulations for problems such as injection site drug precipitation, pharmaceutical companies have traditionally used costly and time consuming animal studies. To reduce the amount of pre-clinical animal studies necessary to find an optimal IV formulation, an in vitro device to detect injection site drug precipitation is introduced. In addition to the device, software that simulates the dilution of a parenteral drug formulation with blood upon administration has been developed and is introduced. Both the device and software were tested on commercially available formulations plus one formulation currently in clinical trials. The results and capabilities of the new device were compared to those obtained using an earlier in vitro device. Finally, a robust model for early screening of injection site precipitation is developed using both the in vitro device and software.

The incidence of neurodegenerative disorders like Parkinson’s disease (PD) and Alzheimer’s disease (AD) are increasing as the population ages. Slowing the rate of neurological decline can have a huge impact on health care costs and quality of life for both the patients and those caring for them. Pituitary adenylate cyclase activating peptide (PACAP) is a Secretin family peptide that activates the PAC1, VPAC1 and VPAC2 receptors and is associated with neuroprotection and neuronal differentiation. PACAP administration protects neurons against toxic, hypoxic, traumatic or inflammatory insults. The receptors of the Secretin family are unique due to the large extracellular domain (ECD) necessary to bind the endogenous ligand prior to receptor activation. The Secretin family ligands are all peptides, this family of receptors being responsible for regulating and maintaining homeostasis within the organism. PACAP is a pleiotropic peptide acting both centrally and peripherally. Exogenously administered peptide is rapidly metabolized. For neuroprotective effects, PACAP must cross the blood brain barrier (BBB). Enhancing the transport across the BBB has been accomplished through peptide glycosylation. Here we design and synthesize a series of glycosylated PACAP agonists and antagonists to evaluate them for receptor activity and ability to cross the BBB. A homology model was constructed of the full length PAC1R based on the transmembrane portion of both the mu opioid receptor and the corticotropin releasing factor-1 receptor combined with the NMR derived solution structure of the PAC1R ECD bound with the receptor antagonist, PACAP6-38. Using this model to guide us, the decision was made to place the glycosylated residue at the C-terminus of the peptide. A series of PACAP based glycopeptide agonists and antagonists were prepared using solid phase peptide synthesis (SPPS). Synthesis of PACAP analogs is complicated by the inclusion of two sites of aspartimide formation, the D3-G4 and D8-S9 sequences. Initial SPPS trials resulted in very little desired peptide formation. Reagent adjustments and using an amino-group protection strategy improved peptide yield. Methionine sulfoxide formation occurs in PACAP analogs. Substitution of methionine with leucine avoids this oxidation issue. An initial screen of PACAP and two glycosylated analogs using PC12 cells for PAC1R activation indicated that all three promoted neurite-like process outgrowths indicating PAC1R activation. The diluent treated cells did not exhibit this morphological change. Quantification of cells for assessing antiproliferative effects was not performed. More PC12 experiments should be performed to assess antiproliferative action and to screen additional glycosylated PACAP analogs for PAC1R activation. One of the glycosylated PACAP analogs was detected in CSF after i.p. administration in a mouse. Microdialysis samples obtained in vivo were analyzed by a newly developed LC/MS² technique and found to contain the administered glycosylated PACAP still intact, demonstrating that the glycopeptide crosses the BBB. Additional experiments using other glycosylated PACAP analogs are planned.

The use of solid dispersions to formulate poorly water soluble drugs is a growing field in the pharmaceutical sciences. Hot-melt extrusion is a common method for producing dispersions. However, bench-top extruders require large amounts of powder to run and are inappropriate for early formulation screens. Plastic and glass syringes are readily available in most laboratories. 250 mg of drug-polymer blend is placed in a plastic syringe body that has the tip covered with a bent needle. The syringe is heated for 5 minutes and the extrudate is pushed through the syringe. Extrudates are characterized by differential scanning calorimetry and powder x-ray diffraction. Acetaminophen, griseofulvin, indomethacin, salicylamide, and sulfamethoxazole extruded with polyvinylpyrrolidone K12 match or exceed the performance of solvent evaporated controls. Glass syringes can be used when polymers have processing ranges above the melting point of the plastic syringes. Syringe extrusion is effectively demonstrated as a rapid process for early formulation screening.

<p>The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens. </p><p>In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the &#61538;a-&#61538;b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections. </p><p>In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.</p> / Dissertation

The metastasis suppressor gene NM23-H1 has been shown to possess three enzymatic activities including nucleoside diphosphate kinase, histidine-dependent protein kinase and 3’-5’ exonuclease activity. While these properties have been demonstrated in vitro using recombinant proteins, the contribution of these activities to suppression of metastatic dissemination is unknown. Site-directed mutagenesis studies were used to identify amino acid residues which are required for proper function of each enzymatic activity associated with H1, providing a platform for studying the importance of each function on an individual basis. To assess the relevance of these activities to melanoma progression, a panel of mutants harboring selective lesions disrupting the enzymatic activities of H1 were overexpressed using stable transfection in two melanoma cell lines, WM793 (isolated from a vertical growth phase human melanoma), and the metastatic derivative cell line 1205LU. In vitro correlates of metastasis measuring motility and invasion were used in an attempt to identify the mechanism mediating H1-dependent motility suppression of cancer cells. Surprisingly, all mutants studied retained full motility suppression in this setting, suggesting that the enzymatic functions associated with H1 are not required for inhibiting cell migration. Instead, gene expression analyses conducted on the panel of stable transfectants indicate that differences in steady-state mRNA levels of genes involved in mitogen-activated protein kinase (MAPK) signaling showed significant correlations with H1 expression and motility suppression. RNAi studies have confirmed that H1-dependent modulation of the expression of two genes in particular, BRAP and IQGAP2, contribute to the observed phenotype, suggesting a novel mechanism used by NM23 to control cellular migration in human melanoma.

Natural products occupy a central role as the majority of currently used antibiotic and anticancer agents. Among these are type-II polyketide synthase (PKS)-derived molecules, or polyketides, which are produced by many representatives of the genus Streptomyces. Some type-II polyketides, such as the tetracyclines and the anthracycline doxorubicin, are currently employed as therapeutics. However, several polyketide molecules exhibit promising biological activity, but due to toxic side effects or solubility concerns, remain undeveloped as drugs. Gilvocarcin V (GV) (topoisomerase II inhibitor) has a novel mechanism of action: [2+2] cycloaddition to thymine residues by the 8-vinyl side chain and cross-linking of histone H. Mithramycin blocks transcription of proto-oncogenes c-myc and c-src by forming an Mg2+-coordinated homodimer in the GC-rich minor groove of DNA. The purpose of this research was to investigate the biosynthesis of several type II polyketide compounds (e.g. chrysomycin, elloramycin, and mithramycin) with the goal of improving the bioactivities of these drugs through combinatorial biosynthesis. Alteration of the glycosylation pattern of these molecules is one promising way to improve or alter the bioactivities of these molecules. To this end, an understanding of the glycosyltransferases and post-polyketide tailoring enzymatic steps involved in these biosynthetic pathways must be established. Four specific aims were established to meet these goals. In specific aim 1, the biosynthetic locus of chrysomycin A was successfully cloned and elucidated, which afforded novel biosynthetic tools. Chrysomycin monooxygenases were found to catalyze identical roles to their gilvocarcin counterparts. Cloning of deoxysugar constructs (plasmids) which could direct biosynthesis of ketosugars, NDP-D-virenose, and NDP-D-fucofuranose in foreign pathways was undertaken in specific aim 2. Finally, these “sugar” plasmids were introduced into producer organisms of elloramycin and mithramycin pathways in specific aims 3 and 4 to interrogate the endogenous glycosyltransferases in order to alter their glycosylation patterns. These experiments resulted in the successful generation of a newly glycosylated tetracenomycin, as well as premithramycin, and mithramycin analogues. In specific aim 4, a new mithramycin analogue with an altered sugar pattern rationally designed and improved structural features was generated and structurally elucidated.

Resveratrol has been reported as a potential anticancer agent but cannot be used as an antitumor drug due to its chemical and metabolic instability. We have designed and synthesized 184 novel compounds related to resveratrol in an attempt to produce more potent and drug-like molecules. We have identified a tetrazole analog of resveratrol, ST-145(a) as a lead anticancer agent from the resveratrol analog series of compounds with a GI50 value of less than 10nM against almost all the human cancer cell lines in the National Cancer Institute’s screening panel. In a separate study, we tested the hypothesis that the limited bioavailability of resveratrol, can be improved by synthesizing analogs which would be glucuronidated at a lower rate than resveratrol itself. We demonstrated that ST-05 and ST-12(a) exhibit lower glucuronidation profiles when compared to resveratrol and that these synthesized stilbenoids likely represent useful scaffolds for the design of efficacious resveratrol analogs. We have also initiated a new discovery program to identify selective CB1 and CB2 receptor ligands from a library of novel stilbene scaffolds structurally related to the resveratrol molecule. From the screened resveratrol analogs, two compounds were identified as selective CB2 and CB1 ligands. Compound ST-179 had 47-fold selectivity for CB2 (Ki = 284 nM) compared to CB1, while compound ST-160 was 2-fold selective for CB1 (Ki = 400 nM) compared to the CB2 receptor. These structural analogs have the potential for development as novel cannabinoid therapeutics for treatment of obesity and/or drug dependency. Combretastatin A4 (CA-4) is one of the most potent antiangiogenic and antimitotic agents of natural origin. However, CA-4 suffers from chemical instability due to cis-trans isomerism in solution. To circumvent this problem, we have developed a facile procedure for the synthesis of novel 4,5-diaryl-2H-1,2,3-triazoles as CA-4 analogs to constrain the molecule to its cis-configuration. Twenty three triazoles were prepared as CA-4 analogs and submitted for anticancer screening. Among these CA-4 analogs, ST-467 and ST-145(b) can be considered as lead anticancer agents from this series, and further investigation against various cancer cell types in vivo with this class of compound may provide novel therapeutic avenues for treatment.