The focus of the research described in this section of the thesis is the synthesis of compounds expected to bind strongly to both the estrogen β and α receptors and act as estrogen agonists. Based on earlier results in our group and docking studies we prepared a series of A-CD analogs, compounds 1, in which the usual 13-methyl group was replaced by an ethyl group. Docking studies also indicated that substituents at C8 could lead to enhancement of binding to the estrogen receptor. With this in mind two such derivatives, compounds 2 were prepared. A major concern in the use of estradiol in hormone replacement therapy is its potential metabolism of dangerous ortho-quinones. The 1,2-naphthalenediol derivatives 3 avoid this possibility. They were predicted to be potent binders to the estrogen receptors with the naphthalene diol portion serving as rings A and B and the hydroxyl group taking the place of the 17-OH group of estradiol. The preparation of several derivatives of 2 is reported. The estrogen receptor binding [ERB] relative to estradiol as standard has been determined at the University of Illinois for a number of the compounds prepared in this thesis. Unfortunately, the results were not as encouraging as expected. Importantly, all of the 13-ethyl derivatives tested showed lower binding affinity compared to the 13-methyl analogs. Similarly, the derivatives with substituents at C8 do not show higher activity than those having only hydrogens at C8. Finally, the situation with the naphthalene derivatives is, at this stage, still not completely resolved. The binding for the compounds thus tested is quite low, but it must be admitted that the structures thus far synthesized have a much lower LogP than estradiol, a factor known to greatly decrease the binding constants to the estrogen receptors.

Infectious diarrhea results in 2 to 5 million deaths worldwide per year, and treatments that are safe, effective, and readily available are under investigation. The field of medicinal ethnobotany focuses on plants that are used by different cultural groups for treating various diseases and evaluates these plants for efficacy and cytotoxicity. In the present study, ethnobotanical research was conducted with Central Anatolian villagers in Turkey. Folk concepts and etiologies surrounding diarrhea were analyzed, as were salient plant-based remedies for diarrhea. Reviewing the literature, 91 plant species were described as anti-diarrheal in all of Turkey. In Central Anatolia, villagers described 35 species. For continued research via bactericidal and bacteriostatic bioassays, 15 plants were selected. Methanolic and aqueous extracts of medicinally used plant parts were evaluated for inhibitory properties against 10 diarrhea-causing bacteria in the first bioassay, and later 21 bacteria in a second assay utilizing spectrophotometry. The cytotoxic properties were also evaluated in an Alamar Blue Assay using HepG-2, PC-3, and SkMEL-5 human cell lines. While several extracts showed bactericidal and bacteriostatic properties, the methanolic extract of R. canina galls inhibited the most bacteria at the lowest concentrations. They were not cytotoxic. Thus, R. canina methanolic gall extracts were selected for bio-assay guided fractionation. Antibacterial activity was maintained in the third fraction which was composed of almost pure ellagic acid. The bioassay was repeated with standard ellagic acid, and the polyphenol retained potency in inhibiting multiple bacterial strains. Several other extracts showed promise for safe, effective anti-bacterial remedies for diarrhea.

Ever since the advent of computer-aided drug design (CADD), in silico simulation methods have greatly accelerated the drug discovery process and lead to the discovery of numerous drug candidates. With the exponential growth of computational power, we nowadays simulate biologic systems at a scale unimaginable a decade ago and thus provides perspectives for drug design. In this dissertation research, combining in silico simulation methods like molecular docking and molecular dynamics (MD) simulation with organic synthesis, in vitro/in vivo experiments and clinical data mining, we developed new drug discovery strategies. These strategies were applied in our drug discovery projects and led to the discovery of inhibitors of microsomal prostaglandin E2 synthase 1 (mPGES-1) and butyrylcholinesterase (BChE) as potential drug candidates. Protein mPGES-1 is known as an ideal target for next generation of anti-inflammatory drugs without the side-effects of currently available anti-inflammatory drugs. Unfortunately, almost all the previously reported human mPGES-1 inhibitors are inactive (or possess very low activity) against mouse or rat mPGES-1 that prevents using well-established mouse/rat models of inflammation, pain, and other diseases for preclinical studies. It would be extremely challenging for the mPGES-1-based drug development to follow traditional drug discovery and development route. In order to solve this problem, we developed and applied Drug Repurposing Effort Applying Integrated Modeling-in vitro/vivo-Clinical Data Mining (DREAM-in-CDM) strategy in this project. With molecular dynamics simulation, we observed the process of how mPGES-1 adopts an alternative conformation to control the access of co-factor GSH (glutathione) and its impact on the function of the protein. Based on the simulation results, we not only found an explanation for the difference between the X-ray and CryoEM (cryogenic electron microscopy) structure of mPGES-1 but also used molecular docking method to identify FDA approved drug, lapatinib, as an mPGES-1 inhibitor by virtual screening and the subsequent in vitro experiments. By mining the available clinical trial data, we found solid evidence that lapatinib can be used to relieve various types of pain in cancer patients. Since lapatinib is very well tolerated, we expect lapatinib to be repurposed as a new treatment for cancer-related pain. BChE has been identified as an ideal drug target for the treatment of Alzheimer’s disease (AD) and heroin overdose. The selectivity of a therapeutically useful inhibitor for BChE over AChE is very important. Unfortunately, there is no good selective BChE inhibitor. With a robust and virtual screening strategy combining with in vitro experiments, we identified a series of compounds from the NCI compound depository as BChE inhibitors with novel scaffolds, high activity and selectivity at the same time. The most potent compound was re-synthesized and the enantiomers of the compound were separated for the first time. The binding mode of the most potent compound was also analyzed and the origin of its high activity and selectivity was revealed that will guide the development of BChE selective inhibitors in the future. In addition, a new tacrine-based BChE affinity chromatography resin was developed. The developed new resin has enabled us to more conveniently and efficiently purify the BChE proteins with improved high purity. In general, we have successfully developed new drug discovery strategies to identify novel inhibitors of different enzymes. With these newly developed strategies, we expect additional drug discoveries to be made in the foreseeable future.

No description available.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Obesity is a growing public health concern globally, particularly in developed countries such as the United States (US). More than 30% of Americans are considered obese. In the past 20 years in America, incidence of obesity has increased significantly (Mokdad et al., 2003). As a consequence, the Centers for Disease Control (CDC) anticipates that 1 in 3 American adults will be diabetic by 2050 (Boyle et al., 2001; Hedley et al., 2004). The most important cause of obesity is poor nutrition, absence of physical exercise and unfavourable lifestyle changes (James et al., 2001).

Estudios e investigaciones prueban que su uso es beneficioso para aliviar graves dolencias de salud, pero la legalidad de su uso aún se debate a nivel mundial. ¿Qué es el cannabis medicinal?

Part I. Development of an isoxylitone analog as an antiepileptic drug candidate. Delphinium denudatum is a medicinal plant traditionally used to treat a variety of conditions in Central Asia. Its interesting anticonvulsant effects were determined to be a property of the compound isoxylitone. Prior work from our group in collaboration with the Poulter group from Western University investigated this compound and generated a large number of isoxylitone analogs in order to optimize its antiepileptic activity. This led to the discovery of the prodrug 13 and the active form 15 shown below, which emerged as the most potent. In this work, the library of analogs was further expanded with 22 new compounds with several which matched the activity of 13 and 15, such as compounds 22 and 37, which led to valuable new insights on the activity of these analogs, and suggested other possible future improvements. In addition, efforts were continued regarding developing compound 15 as a clinical trial candidate. Optimization of the synthesis was performed to drastically reduce costs and waste of chemicals, as well as accelerating the duration of the synthesis. The purification of the final product was also greatly facilitated by the direct synthesis of 15, compared to the prior process of first preparing 13 and hydrolyzing the ester. Efforts were exerted to gather additional knowledge on the characteristics of the compound, with structural and conformational analysis via X-ray crystallography and NOE NMR as well as accelerated stability studies to test the viability of 15 in long-term storage under various conditions. All the information gathered throughout this work supported 15 and its sodium salt as excellent clinical trial candidates as treatments for epilepsy. Part II. Synthesis of SOX9 inhibitors as promoters of recovery from spinal cord injury. According to the World Health Organization, 250 to 500 thousand people develop a spinal cord injury each year with a large portion resulting in tetraplegia. A common misconception is that this is permanent because the damaged nerves cannot be repaired. In fact, nerves can and do regrow after being damaged, but cannot do so after spinal cord injuries due to formation of scar tissues which physically and chemically prevents the healing. The Brown group at Western University identified the SOX9 transcription factor as an important promoter of the formation of this scar and showed that SOX9 inhibitors could improve recovery and mobility in mice affected by spinal cord injuries. In collaboration with their group, previous work in our lab performed and SAR study on the lead compounds ZO2(1) and STL26 (2), shown below. The different sections of the molecule have been designated units A to D, to simplify discussion. Initial work by our group established an efficient method to prepare a library of analogs of the lead compounds. A number of compounds were prepared, which primarily investigated small amines as unit A and phenols with small aliphatic substituents as unit D. The initial SAR data confirmed the validity of STL26 as lead compound, as most alterations to the structure were detrimental to the SOX9 inhibitory activity. The objective of this work was to build on these preliminary SAR results, and expand the library of analogs. Larger substituents were introduced in unit A and D and showed that any group larger or smaller than diethylamide in unit A was detrimental to the activity, but that there seemed to be ample space to increase the size of the unit D isopropyl group. Analogs investigating unit B showed that adding substituents at most of the positions was detrimental, as well as changing the relative positions of unit A and B to be ortho or para to each other. However, the C4 on ring B seemed to be very tolerant to various electron donating or withdrawing functional groups. During this SAR study, a recurring theme was the awful solubility of the compounds in water, which heavily complicated their administration to mice during the bioassays. While none of the analogs tested proved superior to 2, the knowledge accrued during this work painted a clear path forward on which areas of the structure could be safely altered to improve solubility without negative impacts on SOX9 inhibition. Some additional efforts were put into obtaining an accurate three-dimensional structure of an active STL26 (2) analog, and information on the primary conformation in solution. Achieving these goals required the use of NOE NMR experiments and X-ray crystallography. One conformation was discovered to be strongly favoured as a result of an intramolecular hydrogen bond even in protic solvents. Subsequently, a small number of additional analogs were prepared containing modifications that would strongly favor or hinder the preferred conformation, in order to better understand its role in the inhibitory activity. The presence of this hydrogen bond appeared to be key to the activity of the compounds.

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

Over the past 200 years, much attention has been paid to natural products for their great contribution in the industry of drug development as many of them have been shown effective against various diseased conditions in humans by virtue of their structural diversity and biological potency. Therefore, they are undeniably a rich resource for the discovery of novel bioactive compounds. To date, many of the mainstay anticancer agents often lead to undesirable side effects and/or develop rapid emergence of drug resistance. Therefore, new therapeutic remedies are desperately needed. In fact, many active compounds are derived from macrocyclic natural products. The identification of their molecular targets may assist researchers to synthesize biological agents for combating particular diseased conditions. Cycloheptapeptides that modulate specific molecular pathways in suppressing the proliferation of cancer cells are potential candidates for anticancer therapeutics and/or chemopreventive agents. In the current research project, we have demonstrated that MV-A, a novel cycloheptapeptide with the unique amino acid DMCPA isolated from Maytenus variabilis (Loes.) C. Y. Cheng (Celastraceae), showed potent cytotoxic activities against a panel of human cancer cell lines, and is worthy for further investigation. Objectives--The objectives of this study were to i) evaluate the anticancer effect, ii) elucidate the mechanism of action, and iii) identify the binding target(s) of the natural cycloheptapeptide MV-A. Methods--We first carried out various kinds of cellular and animal studies for validating the in vitro and in vivo anticancer efficacy of MV-A. Next, we performed a number of bioassays to ascertain the inhibitory effect of MV-A on several major cancer-associated pathways, including apoptosis, cell cycle arrest, senescence and metastasis. The biochemical assays included sulforhodamine B colorimetric assay, flow cytometric analyses of apoptosis and cell cycle arrest, Western blotting, real-time polymerase chain reactions (qPCR) arrays, senescence-associated β-galactosidase staining, phospho-specific protein arrays, as well as migration and invasion staining experiments. Lastly, we also identified the potential protein targets of MV-A by biochemical means, particularly the drug affinity responsive target stability (DARTS) approach. Results--MV-A is a potent anti-proliferative agent against a variety of cancer cells. It inhibited the proliferation of the human colorectal carcinoma (CRC) HCT116 cells with an IC50 value of 2.28 nM. However, the application of MV-A at 2.68 nM did not induce significant apoptosis; rather it caused a notable cell-cycle arrest at the G1 phase. Moreover, the treatment with this compound (0.68 to 2.68 nM) led to a remarkable senescence in cancer cells as well as a mitigated cellular migration. Meanwhile, the expression levels of some components of the p16 cascade and PI3K-AKT pathway, so as several epithelial-to-mesenchymal transition (EMT) molecules were suppressed by MV-A. Furthermore, HSP90, calnexin, EF2, 14-3-3 and annexin A1 were identified as the direct binding targets of MV-A in our DARTS analysis.Conclusions--In the present study, our results indicated that the novel cycloheptapeptide MV-A inhibited proliferation and migration of CRC HCT116 cells via the induction of cellular senescence and modulation of multiple pathways, including the p16/Rb, PI3K-AKT and EMT signaling pathways. These results revealed a potential role of MV-A in cancer therapy. The direct binding targets of MV-A further uncovered its molecular actions against different diseased conditions. Our findings strongly support the development of MV-A as a therapeutic agent for combating cancerous pathologies, explicitly CRC.

A mini dissertation submitted in partial fulfillments for the degree of master of Environmental Education in the department of Comparative and Science Education at the University of Zululand, 2006. / Most tribes in South Africa still make use oftraditional medicines and use prescriptions ofherbs and other natural materials (Matsiliza and Barker 2001). Medicinal plants from Northern KwaZulu-Natal (KZN), particularly areas such as UIundi, Empangeni, Eshowe Mtubatuba and Nongoma are no exception and are a constituent of a traditional culture. The informal trade in indigenous plants for medicinal use (umuthi) is growing constantly (Mander, 1998). There are many reasons for this, including an expanding population coupled with modernisation of society that is hastened by the development of roads, improved communication methods and the migration of people from villages to cities. The prohibitive cost of modern medicines, together with the high rate of unemployment is forcing many people to revert to traditional medicines and even to gather and sell medicinal plants as a living. This results in over-exploitation of some plant species, sometimes to the brink ofextinction (Matsiliza and Barker 2001). This study aims at investigating the implications of plant harvests that are sold at bus ranks in northern KwaZuluNatal, and at identifYing target species for threatened plant programmes with the help of data gathered via direct interviews with medicinal plant sellers. It also aims at identifying and remedying knowledge gaps that may exist among plant gatherers about propagation techniques applicable to the medicinal plants they harvest.