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There are numerous resources in Nature that can be utilised, and frogs' intricate living circumstances enables them to serve a purpose by secreting substances on their skin that shield them from the outside environment. Some of the complex problems of today may have been aided by their evolution. As many bacteria have evolved resistance to current antibiotics in use, antimicrobial peptides from frog skin secretions are among the most promising novel antimicrobial agents due to their wide-ranging antimicrobial activities and different mechanisms of action. This project aims to identify two novel antimicrobial peptides and to evaluate their biological activities and to find peptides with drug potential through modification. And their structure-activity relationships were studied. In Chapter 3, the full-length cDNA sequences of brevinin-1HL and palustrin-2LTb, two novel antimicrobial peptides, were cloned from skin secretion of Hylarana latouchii through "shotgun" cloning. In Chapter 4, brevinin-1HL identified in the skin secretion of Hylarana latouchii was evaluated for its biological activity. Five analogues were designed through D-type amino acid substitution and changing the amino acids in the hydrophobic face. It was found that the secondary structure, especially the helicity between these peptides, plays a significant role in their actions. And the designed analogue, brevinin-1HL-D-9L, was considered as a potential drug candidate with potent antibacterial activity with low haemolysis at its effective values. In Chapter 5, a novel peptide, palustrin-2LTb, identified in the skin secretion of Hylarana latouchii was shown to have good antibacterial activity against selected pathogens. To reduce the manufacturing cost and decrease toxicity, trypsin-cleavage mimicking modification strategy was ultilised to produce predicted fragments. Seven such truncated derivatives of palustrin-2LTb were synthesised and tested for their biological activities. Functional screening data indicated that truncated fragment 3 not only maintained and optimised antimicrobial efficacy of the parent peptide, but also showed great in vivo therapeutic potential in an MRSA-infected insect larvae model. Due to its strong antibacterial activity, minimal haemolytic activity, and ability to kill MRSA in an in vivo model, fragment 3 showed outstanding effects and had obvious potential to become a new antimicrobial agent.

The focus of this thesis is the study of biological populations subject to external changing environments exploring a number of theoretical, numerical, and experimental approaches. We study different classes of environments, considering cases whose evolution over time is deterministic or stochastic. The first class of environments we consider vary deterministically. Here we focus on the study of microbial resistance in bacterial populations subject to therapies of one or two antibiotics. The environment specifies the drug concentration administered to the population, so it is determined by the type of dosing schedules. We investigate how subpopulations with higher mutation rates drive the emergence of multi-drug resistance. We approach this problem analysing experimental data (obtained by Dr. Danna Gifford), comparing their observations against stochastic simulations of a multi-type branching model we design. In separate work related to the previous one, we explore the delaying effect of competition on the emergence of single and double resistance through theoretical predictions of a similar stochastic model. We calculate the probability of having at least one resistant cell for dosing schedules with constant and time-dependent drug concentrations using a theoretical approach based on branching processes. The second class of environments we consider vary stochastically. The first system we study is a Moran-type model, describing a population subject to a switching environment that determines the type of reproduction, namely sexual or asexual. The population can exhibit several number of `mating types' (analog to male/female sexes, but not restricted to two) that depends on the rate of reproduction, as well as the mutation rate (i.e., inclusion of new types). We investigate the stationary distribution of the number of mating types for different switching regimes. We show that for slow switching regimes the distribution can become bimodal, while for fast switching the system behaves as if there was one single effective environment. Our approach exploits properties of branching processes and integer partitions in number theory. Lastly, we study and design an algorithm based on the so-called tau-leaping algorithm, focusing on systems with fast fluctuating environments. Our algorithm treats the input rates for the tau-leaping as (clipped) Gaussian random variables with first and second moments constructed from the environmental process. Several biological examples are explored, such as genetic circuits, birth-death processes, and genetic switches. We consider cases with discrete and continuous environmental spaces. The algorithm can produce results for macroscopic observables in fluctuating regimes beyond the adiabatic limit (i.e., infinitely fast switching) that are in good agreement with measurements from other simulation methods, but with a significantly reduced computing time.

To combat the ever-growing clinical burden imposed by antibiotic-resistant pathogens, multiple-antibiotic treatments are increasingly being considered as promising treatment options. The impact of multiple-antibiotic treatments on the evolution of resistance is not well understood however, and debate is ongoing about the effectiveness of various multiple-antibiotic treatments. In this thesis, I investigate how aspects of multiple-antibiotic treatments impact the rate of evolution of antibiotic resistance in the opportunistic human pathogen Pseudomonas aeruginosa. In particular, I look at the impact of interactions between antibiotics in combination on the evolution of resistance, and how creating heterogeneity in the antibiotic environment by rotating the antibiotics used may change the rate of evolution of resistance. I characterise the interactions present in 120 combinations of antibiotics and find that the type of interaction can be predicted by the mechanism of action of the antibiotics involved. I investigate the effect of a subset of these combinations on the evolution of antibiotic resistance. My results refute the influential but poorly-evidenced hypothesis that synergistic combinations accelerate the evolution of resistance, even when synergistic combinations have the same inhibitory effect on sensitive bacteria as additive or antagonistic antibiotic combinations. I focus on a combination of the antibiotics ceftriaxone and sulfamethoxazole and test whether it is more effective in preventing the evolution of resistance than predicted by the inhibitory effect of the combination on sensitive bacteria. I do not find the combination to be more effective than predicted. Finally, I create heterogeneous antibiotic environments by rotating the antibiotic present at different rates. For the first time in a laboratory setting, I test how varying the rate of fluctuation in the antibiotics present in a heterogeneous antibiotic environment impacts the rate of evolution of resistance. Unexpectedly, I find the rate of evolution of resistance increases with increasing levels of antibiotic heterogeneity.

Bacterial pathogens are evolving resistance to all available antibiotics and are a leading cause of death worldwide. Thus, the discovery of new antibiotics that inhibit multi-drug resistant (MDR) pathogens is urgent. We hypothesize that environmental Pseudomonas strains that can inhibit the growth of MDR pathogens may be source of novel antibiotics that may have new or multiple mechanisms of action. We isolated 288 environmental Pseudomonas strains from the Portage River in northwest Ohio which represents a polluted water source. Pseudomonas strains were isolated and phylogenetically characterized using the gyrB housekeeping gene. To test our hypothesis, the environmental strains were competed against a collection of human and animal MDR pathogens including nine Burkholderia species, seven Staphylococcus aureus, and nine Pseudomonas aeruginosa strains, all isolated from lungs of cystic fibrosis patients, and five Aeromonas fish pathogens. Results from the 8,640 individual antagonistic assays showed that 115 of the environmental Pseudomonas strains demonstrated the ability to inhibit at least one pathogen. This suggests that these environmental strains may produce novel compounds which effectively inhibit these MDR pathogens. We performed transposon mutagenesis and screened for loss of inhibition mutants with strains J4D8, JA1H2, J1A5 and J3E11 to determine biosynthetic gene clusters involved in antibiotics production. Whole genome sequencing and arbitrary PCR coupled with antiSMASH were used to identify specific gene clusters involved in antagonistic activity. The compounds were predicted to include pyochelin, pyoverdine, MA026, lankacin C and cepacin A which are all derived from non-ribosomal peptides. Further analysis of these compounds is required to determine their novelty.

Antimicrobial resistant bacteria existed before the medical use of antimicrobials but increasing antimicrobial use has escalated the development and spread of resistant bacteria in human and animal populations. Resistant bacteria and resistance genes spread between individuals through direct and indirect pathways, such that antimicrobial use in one population can result in increased morbidity and mortality from resistant bacteria in another population. In particular, there is a risk of resistant foodborne bacteria due to antimicrobial use in livestock, but this risk is challenging to quantify and likely varies with bacterial species, antimicrobial, and host species. Data of varying quality have been collected on antimicrobial use, pharmacokinetics, pharmacodynamics, resistance genes, and resistance phenotypes over several decades. Novel and innovative methodologies are required to maximize the utility of these data and improve our understanding of antimicrobial resistance ecology. In this dissertation, I first gather all available information on the impact of tylosin on beef cattle enteric bacteria with a systematic review. These data are used to inform and validate a meta-population mathematical model of tylosin pharmacokinetics and pharmacodynamics. The model uses previously published data to estimate the change in macrolide-resistant enterococci within cattle and in the feedlot environment during and after tylosin administration. The mathematical model and systematic review reveal a limited impact of tylosin on macrolide-resistant enterococci and other potential foodborne pathogens. Further along the foodborne transmission chain, the National Antimicrobial Resistance Monitoring System collects data on resistant bacteria at slaughterhouses, on retail meats, and from human infections. Multidrug resistant bacteria are associated with more severe health outcomes yet are more challenging to analyze because the number of multidrug resistance phenotypes grows exponentially with the number of antimicrobials considered. I present the first application of association rule mining, a machine learning methodology, to analyze multidrug resistance in chicken-associated Escherichia coli. Network analytics and the association rules demonstrated that E. coli from chicken carcasses and chicken meat had similar multidrug resistance phenotypes, despite the high variability in the dataset. Finally, I refine and expand the association mining techniques and apply them to hospital surveillance data, demonstrating that association mining can identify and quantify clinically important multidrug resistance in small, surveillance datasets. These studies establish the importance of using complementary analytic techniques to maximize the value of existing and future antimicrobial resistance data. Machine learning promises to substantially change the approach to antimicrobial resistance data, and may become necessary as the amount of antimicrobial resistance data collected expands, but my work on association mining indicates that efforts to validate and increase the rigor of these methods are essential. The merging of systematic reviews with mathematical models, and the union of machine learning with traditional statistics and visualization, will improve our understanding of antimicrobial resistance ecology.

The research described here focuses on the phylogenetic characterization of water-derived pseudomonads and their antagonistic activity against multi-drug resistance (MDR) P. aeruginosa and Burkholderia species. Phylogenetic work was based on the gyrB housekeeping gene. Genetic techniques have been optimized and employed to identify genes associated with antimicrobial production via transposon (Tn) mutagenesis using a triparental mating system approach with Pseudomonas as the model organism. This study expands on theses previous studies in the lab to identify biosynthetic gene clusters (BGC) involved in production of novel antibiotics capable of inhibiting the growth of MDR pathogens. We utilize a previously optimized workflow to identify genes from environmental isolates involved in the inhibition of MDR P. aeruginosa and species within the Burkholderia cepacia complex (Bcc). We show that both MDR Bcc and P. aeruginosa pathogens were inhibit by environmental Pseudomonas strains. Out of 7,784 interactions, 210 of these were antagonistic. Superkillers (SK), defined as strains that inhibit ≥3 of MDR pathogens, were selected for optimization of Tn mutagenesis to identify gene cluster whose products inhibit these MDR pathogens. Only six out of the 24 SK’s were capable of this process. Out of these six, three were selected for large scale mutagenesis to identify loss of inhibition (LOI) mutants. Four LOI mutations were found for strain S5F11, one of which had an insertion within a BGC predicted to produce an NRPS complex. Seven LOI mutants were found for S3E7. Although none of these insertions were identified within a BGC, genes have been identified that are observed to be heavily involved in antibiotic production. This study suggests that environmental Pseudomonas strains have the capacity to inhibit the growth of CF-derived MDR pathogens. Using Tn mutagenesis, we have identified novel loci that are associated with antibiotic production.

The emergence of antimicrobial resistance bacteria has become a major threat to human society. The rapid spread of resistant pathogens and the associated loss of efficacy of available drugs needs to be met with the development of antibiotics and alternative treatments. Pseudomonas aeruginosa is an opportunistic human pathogen evolving resistance to many currently used antibiotics. Chronic lung infections with the bacterium P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Escalating this problem is that pharmaceutical companies have dropped drug development due to low profitability, thus making the efforts of drug discovery of prime importance. To address this global health threat research institutes have now stepped forward to aid in discovery of novel compounds. P. aeruginosa dominates the lungs during chronic infections in CF patients, yet it’s abundance in non-human habitats such as water and soil is less compared to other diverse groups of pseudomonads. A trait that could contribute to such decreased abundance is bacterial competition from other Pseudomonas populations that dominate water and soil habitats. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. Here, we sought to determine if clinical isolates of P. aeruginosa are susceptible to competition by environmental pseudomonads which may provide a source of inhibitory factors. We have used a population based study in association with transposon mutagenesis, PCR techniques, whole genome sequencing and bioinformatic analysis to identify environmental Pseudomonas biosynthetic gene clusters (BGCs) and characterize antagonistic compounds that are effective against CF-derived P. aeruginosa. A total of five BGCs have been identified in this study from environmental Pseudomonas strains S4B6, S3F9 (soil-derived) and LE6C6 (water-derived) encoding diverse compounds such as bacteriocins, NRPSs, phenazines, and siderophores involved in antagonistic activity. Extending this analysis, we have also identified environmental Pseudomonas that inhibit not only CF-derived P. aeruginosa but are effective against other pathogens including ESKAPE pathogens and carbapenem resistant P. aeruginosa. Overall, this research serves as a platform for the identification of novel antibiotics from these environmental isolates.

The soil bacterium Streptomyces peucetius produces two widely used anticancer antibiotics doxorubicin and daunorubicin. Present within the biosynthesis gene cluster in S. peucetius is the drrAB operon which codes for a dedicated ATP-binding cassette type transporter for the export of these two closely related antibiotics. DrrAB system was believed to be the single-drug transporter due to its dedicated nature; however, our study demonstrated under both in vivo and in vitro conditions that DrrAB system can transport not only doxorubicin but also Hoechst 33342 and ethidium bromide. Moreover, many other well-studied multi-drug resistance proteins substrates (including verapamil, vinblastine and rifampicin) inhibit DrrAB-mediated doxorubicin transport, indicating they are also substrates of DrrAB pump. Kinetic studies show competitive inhibition of doxorubicin transport by Hoechst 33342 and rifampicin and non-competitive inhibition by verapamil, suggesting the possibility of more than one drug binding site in the DrrAB system. This is the first in-depth study of a drug resistance system from a producer organism, and it shows that a dedicated efflux system like DrrAB contains specificity for multiple drugs. Our study also provides the first direct evidence for the dual role of the metalloprotease FtsH in the biogenesis of membrane proteins. We found that FtsH is not only responsible for proteolysis of unassembled DrrB protein but it also plays a much broader role in biogenesis of the DrrAB complex. DrrA and DrrB proteins expressed together in a temperature sensitive ftsH mutant strain were found to be non-functional due to their incorrect assembly. Simultaneous expression of wild-type FtsH in trans resulted in normal doxorubicin efflux. Strikingly, doxorubicin efflux could be restored in mutant cells irrespective of whether FtsH was expressed simultaneously with DrrAB or expressed after these proteins had already accumulated in an inactive conformation, thus providing crucial evidence for the ability of FtsH to refold the misassembled proteins. Complementation experiments also showed that the catalytic ATP binding domain of FtsH contains a chaperone-like activity, however both the catalytic and the proteolytic domains of FtsH are required to be present and work coordinately to participate in biogenesis of DrrAB complex in the membrane.

Abstract: Arsenic is a potent environmental contaminant and human carcinogen, occurring naturally in the earth’s crust and entering the food chain through leeching into the water supply. Upon entering the body, inorganic arsenic is methylated to mono- and di-methylated forms, the trivalent versions of which have been shown to have increased toxicity over their inorganic counterparts. The majority of arsenic is methylated in the liver and is eliminated from the body in urine. Multidrug resistance protein 2 (MRP2) can transport the seleno-bis(S-glutathionyl) arsinium ion, [(GS)2AsSe]-, and coupled with the apical expression of MRP2 in the liver, provides a mechanistic explanation for the cooperative detoxification observed between arsenic and selenium. Multidrug resistance protein 1 (MRP1) was capable of transporting monomethylarsenic diglutathione (MMA(GS)2) and due to the broad tissue distribution of MRP1, is likely to be important for protecting cells from arsenic. Multidrug resistance protein 4 (MRP4) can transport MMA(GS)2 and dimethylarsinic acid (DMAV) and due to its basolateral localization in the hepatocyte and apical localization in the renal proximal tubule, is likely crucial in the disposition of arsenic from the liver into the blood stream and then into urine. The results of this thesis demonstrate the role and the importance of MRP1, MRP2, and MRP4 in the protection against arsenic and its metabolites.

Multi-drug resistance (MDR) has become a serious clinical problem for both cancer and infectious disease treatment. One of the leading causes of MDR is the drug efflux mediated by the ATP-binding cassette (ABC) super-family of proteins which are found among diverse groups of organisms. In our laboratory, we are interested in studying an ABC transporter DrrAB from Streptomyces peucetius. It confers resistance to two anticancer antibiotics doxorubicin and daunorubicin by its drug efflux function. Insights into the function of the DrrAB complex are expected to facilitate a better understanding of mechanisms of MDR. The normal function of the DrrAB complex depends on the cis expression of both subunits, DrrA and DrrB. DrrA forms the nucleotide binding domain (NBD) and provides energy for drug translocation, while the DrrB protein functions as the transmembrane domain (TMD) and forms the substrate translocation pathway. Studies on the detailed mechanisms of communication between DrrA and DrrB are critical for understanding the coupling of energy usage and substrate translocation. The present studies revealed the existence of two novel and functionally important modules in the C-terminal domain of DrrA that might be essential for conformational interplay between DrrA and DrrB during the catalytic cycle. One module present at the extreme C terminus of DrrA consists of two separate motifs, DEF and CREEM. CREEM motif together with its upstream region up to residue S319, interacts with the N-terminal cytoplasmic tail region of DrrB and forms an DrrA-DrrB interface, while the DEF motif regulates this interaction. The second novel module GATE is present 104 amino acids upstream of DEF. Based on our biochemical and structural analyses, we propose that GATE functions as a transducer of conformational changes (resulting from ATP binding) from DrrA to DrrB. Our studies provide important insights into the interplay between the NBD and TMD of the DrrAB drug transporter and their roles in communicating long range conformational changes during the catalytic cycle. These findings further deepen our understanding of the mechanisms of drug resistance.

Tubulysins are antimitotic natural products with potent anticancer activity against multidrug-resistant (MDR) cancer cells, acting by inhibition of tubulin polymerization. The marked difference in antiproliferative activity between tubulysins V and U exemplifies the importance of an acetate positioned alpha to the thiazole ring. However, this acetate has been shown to be labile under both acidic and basic conditions, so the effectiveness of this modification may be hindered due to this instability. Hence, the work presented here focuses on the synthesis of analogs that mimic and stabilize the acetate at this position.Heteroatom exchange at the α-thiazole position of tubuvaline was hypothesized to increase molecular stability while maintaining observed activity by bioisosteric replacement of the tubuvaline oxygen with a nitrogen. The nitrogen-containing analogs of tubulysin V and U, N tubulysin V and U, were the most important targets to test the singular modification of heteroatom exchange on bioactivity and to survey molecular stability. The synthetically derived N tubuvaline amino acid residue was generated following a rigorously controlled Mitsunobu reaction, but difficult final stage deprotections to N tubulysin V suggests a lowered stability compared to tubulysin V. N-acylation of a penultimate tetrapeptide intermediate led to generation of N tubulysin U and other acylated N tubulysin analogs to establish a more robust SAR at the α-thiazole position. N Tubulysin U was found to be more stable than tubulysin U under strongly basic conditions, and upcoming biochemical evaluation will determine the effect that these modifications have on antiproliferative activity.Investigations into the SAR at the tubuvaline α-thiazole position also included oxygen-based analogs, where two methods for acylation of tubulysin V were exploited to generate O-acylated analogs with various alkyl groups. Biochemical evaluation of antiproliferative activity, along with the use of two electrophilic analogs to act as affinity labels, will survey important interactions within the tubulysin-tubulin binding site.

Abstract: One of the major causes of failure in cancer chemotherapy is multidrug resistance (MDR), where cancer cells become resistant to different types of anticancer drugs. Over-expression of membrane efflux pumps like P-glycoprotein (P-gp), which recognizes different chemotherapeutic agents and transports them out of the cell play a major role in MDR. One of the major reasons for shortcomings of P-gp inhibitors in clinic is their non-selective distribution to nontarget organs, which leads to reduced elimination of P-gp substrates (e.g. anticancer drugs) and intolerable toxicities by anticancer drugs. The objective of this research is to develop a nanocarrier that permits a change in the pharmacokinetics of P-gp inhibitors, limiting their non-specific distribution. Polymeric micelles have shown promise in changing the pharmacokinetics of hydrophobic drugs in a favorable manner. Presented herein are the results of our investigation of self-associating poly(ethylene oxide)-block-poly(ε-caprolactone)(PEO-b-PCL) and PEO-b-poly(α-benzyl-ε-caprolactone) (PEO-b-PBCL) block copolymers as biodegradable polymeric nanocarriers for the solubilization and delivery a model P-gp inhibitor valspodar). It is hypothesized that encapsulation of valsopdar in polymeric nanocarriers can enhance its therapeutic efficacy by providing an inert alternative to Cremophor EL for solubilizing valspodar, favorably changing its pharmacokinetics and reducing its pharmcokinetic interaction with anticancer drugs (P-gp substrates) upon co-administration. PEO-b-PCL and PEO-b-PBCL were assembled to form carriers of 60-100 nm diameters, and were shown to be able to efficiently encapsulate valspodar: achieving a clinically relevant aqueous solubility of 2.8 mg/mL. Following intravenous administration of valspodar to healthy rats, there was nearly a 100% increase in plasma area under the curve (AUC) of valspodar when administered in the polymeric nanocarrier formulations as compared to when Cremophor EL formulation was used. Co-administration of doxorubicin, a model P-gp substrate anticancer agent, with valspodar in the standard Cremophor EL/ethanol formulation resulted in more than 50% reduction in doxorubicin clearance, which was accompanied by over a 100% increase in doxorubicin AUC. In contrast, no change was detected in doxorubicin clearance or AUC, when valspodar was administered in PEO-b-PCL polymeric nanocarrier formulation. Overall, our results suggest that PEO-b-PCL micelles hold great promise for solubilization of valspodar and the safe co-administration with doxorubicin.

Heterogeneous oxygen distribution in tumor microenvironment can contribute to therapy failure. Anoxia and severe hypoxia in microregions of solid tumors results in decreased activity of cells or up-regulation of hypoxia linked cell survival mechanisms. These adaptations are in favor of survival and proliferation of hypoxic cells in adverse conditions making them resistant to therapy. Knowledge of oxygen distribution inside tumor biopsies and models along with therapeutic efficacy of drug molecules would be of great value to improve clinical outcomes by personalizing therapy and and in-vitro screening of potential new drug molecules. Current methodologies for oxygen measurement in 3D tissues, based on both optical and electrochemical principles, are restricted to single time point measurements assuming steady state oxygen distributions. Both time- and depth-resolved continuous mapping of oxygen inside tumor tissue models has not been achieved. Development and validation of continuous oxygen concentration measurement approach for both 3D multicellular tumor spheroid models and single cells is reported here. A device with an array of gold microdisc electrodes is designed and fabricated using MEMS fabrication techniques. Oxygen concentration profiles inside model spheroids are measured using this device. Dependence of the rate of cellular oxygen consumption on extracellular oxygen concentration is demonstrated from the recorded data by discrete numerical analysis. A continuous model of oxygen concentration considering linearly decreasing consumption rates is developed which improves upon previous models that assume constant rate of consumption. Oxygen concentration of single cells and small cluster of cells is measured using a microring electrode platform. Oxygen consumption of single cell is quantified from the concentration data. Single cell processes like multi-drug resistance, metabolism, respiration etc. can be investigated using the microring electrode platform and cumulative tissue effects, also called multi-cellular resistance, in tumor biopsies and models can be investigated using the multielectrode array approach. Information obtained from both approaches combined together provides a comprehensive picture of cancer chemotherapy failure mechanisms. Finally, an interesting design of spatially averaging electrodes is validated for heterogeneous microliter size hemispherical drops. This design can be used for obtaining average concentrations of substances inside our model spheroids.

Abstract: Gene delivery for therapeutic purposes is quickly emerging as the best potential treatment option for inherited genetic diseases and cancer. Viral gene carriers have been the choice for this purpose due to their high efficiency, but harmful immunogenic and oncogenic host reactions have limited their in vivo use. Cationic polymers provide a safe alternative to viral carriers as they can be engineered to reduce immunogenic and toxic responses and serve therapeutic purposes in the body. Due to their strong positive charge, they are able to compact the negatively charged nucleotides to small nano-sized particles appropriate for cellular uptake. Additionally, they efficiently encapsulate the highly sensitive nucleotides, and protect them against degradation by the nucleases present at the physiological milieu. In this thesis work, I have used a novel approach for gene delivery by combining the critical properties of a cationic polymer (i.e., nucleotide condensing ability) with that of a fatty acid (i.e., lipid membrane compatibility). The resulting lipid modified polymer increased delivery of our gene of interest into target cells and resulted in increased siRNA delivery for cancer gene therapy.

Malignant melanoma is the most dangerous form of skin cancer and accounts for about 75% of skin cancer deaths. Once diagnosed at the metastatic stage, it has a very poor prognosis with a median survival rate of 6 months and a 5-year survival rate of less than 5%. In addition, melanoma has become an important public health hazard owing to its rising incidence, which has been well documented over the past 50 years. Currently there is no effective way to treat melanoma. It is highly resistant to existing chemotherapy, radiotherapy, and immunotherapy. Over the past 30 years, only two drugs have been approved by the Food and Drug Administration (FDA) for metastatic melanoma: dacarbazine (DTIC) and interleukin-2 (IL-2). But even with these two drugs, fewer than 15% of patients have a favorable response and fewer than 5% of patients reach complete remission. On the other hand, the toxicity associated with DTIC and IL-2 is often significant, resulting in serious or life threatening side effects in many patients. In recent years, great efforts have been made in fighting metastatic melanoma. But neither combinations of DTIC with other chemotherapy drugs (e.g., cisplatin, vinblastine, and carmustine) nor adding interferon-α2b to DTIC have shown a survival advantage over DTIC treatment alone. Extensive clinical trials with a lot of antibodies and vaccines to treat metastatic melanoma have also failed to demonstrate satisfactory efficacy. Therefore, developing more effective drugs for melanoma is urgently needed. We started our efforts in finding new drugs for melanoma by screening a large compound library. The in vitro cytotoxicity data on several melanoma cell lines led us to the discovery of three active structure scaffolds: serine amides, serine amino alcohols, and arylthiazolidine-4-carboxylic acid amides (ATCAAs). Because ATCAAs showed better selectivity between cancer cells and normal fibroblast cells, the chemists in our group focused on this scaffold and performed extensive structure modifications for structure-activity-relationship (SAR) studies. The SAR results were then used to guide further synthesis in an effort to maximize activity and selectivity. Two active compounds identified during the process were sent to the U.S. National Cancer Institute for anticancer screening using 60 human tumor cell lines. Results showed that these two compounds have extensive cytotoxic activity against all nine types of cancer cells with IC50values ranging from 120 nM (leukemia, CCRF-CEM cell line) to 11 μM (colon cancer, HCC-15 cell line). One compound showed particularly good activity against melanoma cells (IC50=130 nM~1 mM against all eight melanoma cell lines). I then evaluated ATCAAs inhibitory effect on melanoma colony formation and in vivo melanoma tumor growth. The in vivo data were very encouraging. One tested compound significantly inhibited melanoma tumor growth at a dose of 10 mg/kg and showed higher efficacy than did DTIC at a dose of 60 mg/kg. These findings built up a strong basis for the development of novel chemotherapeutic drugs for advanced melanoma. Furthermore, the chemists in our group also synthesized some new imidazole and imidazoline analogs by focusing on the SAR studies of the central five-member ring. Although the current compounds displayed lower potency when compared with our lead thiazolidine analogs, they may have the distinct advantage of being more stable in vivo with the reduced necessity of chiral separations. Some of these new compounds have activity similar to Sorafenib, an FDA-approved drug that has been tested clinically in melanoma patients. To further expand our understanding of SARs and to potentially identify new platforms for active compounds, Dr. Li and Dr. Seibel explored a compound library from the University of Cincinnati’s Drug Discovery Center. This library contains 342,910 small molecules. Based on the structure of our lead molecule, two ligand-based virtual screening approaches were used: 1) similarity search based on atom connectivity by using Scitegic Pipeline Pilot software and 2) similarity search based on molecular shape by using Schrodinger software. Results showed that these two approaches are highly complementary and lead to different active molecular structures. These structures are quite suitable for further structural modification and provide new platforms for our anticancer drug discovery efforts. Subsequently, further lead structure optimization led to the discovery of substituted methoxylbenzoyl-aryl-thiazole (SMART) compounds. To improve solubility and to circumvent the metabolic instability brought by the thiazole ring, our team designed and synthesized a new series of analogs: 2-aryl-4-benzoyl-imidazoles (ABIs). These two classes of compounds showed great in vitro cytotoxicity against melanoma, and the IC50 of the most active compound was below 10 nM. They also showed equal potency against multi-drug resistant melanoma cells and the sensitive parent cells, indicating that these compounds can effectively overcome multi-drug resistance, which is a major cause of cancer chemotherapy failure. In vivo testing on C57BL/6 mice bearing B16-F1 melanoma allograft and on double homozygous SCID (severe combined immunodeficiency) hairless outbred (SHO) mice or athymic nude mice bearing A375 human melanoma xenograft showed these two classes of compounds significantly inhibited melanoma tumor growth. Some compounds even showed substantially better activity than did DTIC, the gold standard anti-melanoma drug. Meanwhile, preliminary toxicity studies suggested that mice can tolerate tested compounds well at effective dose levels. No sign of acute toxicity was observed from the experiments. More importantly, I identified the cellular target for ABI and SMART compounds through a series of biotechniques and molecular modeling studies. Strong experimental evidence has shown that these compounds bind to tubulin at the colchicine binding site in the α/β-tubulin heterodimers to disrupt functional microtubule formation. In the meantime, I also tested the pharmacokinetic properties of some active compounds in mice together with Mr. Chien-ming Li. With their good in vivo anti-melanoma activity and their ability to overcome multi-drug resistance, these new classes of compounds have great potential for melanoma therapy.