The Severe Acute Respiratory Syndrome (SARS) virus encodes several unusual RNA processing enzymes, including Nsp15, an endoribonuclease that preferentially cleaves 3? of uridylates through a Ribonuclease A-like mechanism. Crystal structures of Nsp15 confirmed that the Nsp15 active site is structurally similar to that of Ribonuclease A. These similarities and our molecular docking analysis lead us to hypothesize that previously characterized Ribonuclease A inhibitors will also inhibit the SARS-CoV Nsp15. Benzopurpurin B, C-467929, C-473872, N-36711, N-65828, N-103018 and Congo red were tested for effects on Nsp15 endoribonuclease activity. A real-time fluorescence assay revealed that the IC50 values for inhibiting Nsp15 were between 0.2 ?M and 40 ?M. Benzopurpurin B, C-473872, and Congo red are competitive inhibitors, according to kinetic studies and were demonstrated to bind SARS-CoV Nsp15 by a differential scanning fluorimetry assay. Benzopurpurin B also inhibited the Nsp15 orthologs from two other coronaviruses: mouse hepatitis virus (MHV) and infectious bronchitis virus. The three compounds reduced infectivity of MHV in L2 cells by 8 to 26 fold. The more effective drugs also caused a decrease in MHV RNA accumulation.

Combinations of sterically encumbered Lewis acids and Lewis bases are precluded from dative bond formation, failing to yield classical Lewis acid-base adducts. These unique systems are referred to as frustrated Lewis pairs (FLPs) and demonstrate a plethora of unique small molecule activations.Herein, the syntheses of phosphonium alkoxy- and aryloxyborate salts in addition to phosphonium thioborate salts are detailed. The scope of Lewis acid and base, alcohol and thiol, that are effective in this chemistry, is also detailed. The syntheses of novel borate and boronate esters of the form B(OR)3 and (C6R4O2)B(C6F5) are detailed and applied in metal-free heterolytic dihydrogen activation with phosphines. The further study of a variety of perfluoroboranes in the activation of H2 with tertiary phosphines is also detailed. Derivatization of triarylboranes, boronate esters, borinic esters and chloroboranes by reaction with one or two equivalents of alky- or aryldiazomethane is achieved yielding the corresponding RR’C insertion products. The solid-state structures of the free boranes, in addition to Lewis base adducts of a sampling of these species, are detailed. Reactivity of the resulting sterically encumbered boranes in imine hydrogenations, H2 and XeF2 activation are also detailed. Combinations of intermolecular frustrated Lewis pairs are found to react collaboratively to activate greenhouse gases such as carbon dioxide (CO2) and nitrous oxide (N2O), yielding the corresponding zwitterionic compounds R3P(CO2)BR2R’ and R3P(N2O)BR’3. Atom connectivity has been established via X-ray crystallographic studies and molecular structures and parameters are discussed. Subsequent exchange chemistry of both CO2 and N2O species with transition metal and other d-block Lewis acids are investigated and yield zwitterionic compounds and ion pairs which are otherwise unobtainable employing more conventional methods. Transition metal containing Lewis acids are employed in conjunction with tri(tert-butyl)phosphine for the FLP-mediated direct activation of N2O.

Combinations of sterically encumbered Lewis acids and Lewis bases are precluded from dative bond formation, failing to yield classical Lewis acid-base adducts. These unique systems are referred to as frustrated Lewis pairs (FLPs) and demonstrate a plethora of unique small molecule activations.Herein, the syntheses of phosphonium alkoxy- and aryloxyborate salts in addition to phosphonium thioborate salts are detailed. The scope of Lewis acid and base, alcohol and thiol, that are effective in this chemistry, is also detailed. The syntheses of novel borate and boronate esters of the form B(OR)3 and (C6R4O2)B(C6F5) are detailed and applied in metal-free heterolytic dihydrogen activation with phosphines. The further study of a variety of perfluoroboranes in the activation of H2 with tertiary phosphines is also detailed. Derivatization of triarylboranes, boronate esters, borinic esters and chloroboranes by reaction with one or two equivalents of alky- or aryldiazomethane is achieved yielding the corresponding RR’C insertion products. The solid-state structures of the free boranes, in addition to Lewis base adducts of a sampling of these species, are detailed. Reactivity of the resulting sterically encumbered boranes in imine hydrogenations, H2 and XeF2 activation are also detailed. Combinations of intermolecular frustrated Lewis pairs are found to react collaboratively to activate greenhouse gases such as carbon dioxide (CO2) and nitrous oxide (N2O), yielding the corresponding zwitterionic compounds R3P(CO2)BR2R’ and R3P(N2O)BR’3. Atom connectivity has been established via X-ray crystallographic studies and molecular structures and parameters are discussed. Subsequent exchange chemistry of both CO2 and N2O species with transition metal and other d-block Lewis acids are investigated and yield zwitterionic compounds and ion pairs which are otherwise unobtainable employing more conventional methods. Transition metal containing Lewis acids are employed in conjunction with tri(tert-butyl)phosphine for the FLP-mediated direct activation of N2O.

Throughout history, cancer has been severely plaguing mankind; the search for a cure to cancer had long been sought by scientists and still poses as one of the greatest challenges scientists have yet to overcome. Hypoxia in cells is a condition where there is little to no oxygen availability in its environment. In general, this event is detrimental since this can lead to cell necrosis or reoxygenation injuries. However, hypoxia, a prominent property of most solid tumors, activates the hypoxia-inducible factor (HIF-1) family of transcription factors that promotes angiogenesis. In this study, we describe the design and synthesis of small-molecule inhibitors of the HIF-1 pathway.

Indiana University-Purdue University Indianapolis (IUPUI) / Despite therapeutic advancements, prostate cancer remains the second most common cause of cancer-related mortality in men. Docetaxel is the first cytotoxic agent to show modest improvements in overall survival rate in patients with metastatic prostate cancer. Unfortunately, over half of these patients do not respond to treatment and ultimately all develop resistance. The mechanism mediating docetaxel resistance remains unknown. Survivin has a classical biological role in cancer, in fact survivin has been shown to be overexpressed in almost every solid tumor and is associated with drug resistance and clinically aggressive disease. In these studies I demonstrate that docetaxel resistant cells have overexpression of survivin compared to sensitive parental cells, knockdown of survivin decreases docetaxel resistance, and stable overexpression of survivin increases resistance to docetaxel. The data in these studies suggest that survivin is likely implicated in docetaxel resistance and treatment with a direct survivin inhibitor may sensitize resistant cells to docetaxel. To this end the evaluation and optimization of two different backbones of survivin inhibitors was performed. One such inhibitor identified is LQZ-7-3 which decreases survivin level via proteasome degradation, leads to apoptosis of cells, and showed efficacy in a prostate cancer xenograft model in vivo when given in an oral formulation. LQZ- 7-3 showed strong specificity to survivin versus other IAP family members at the protein level. Another inhibitor, LQZ-7F-1, demonstrated nanomolar inhibition of cancer cell growth and similar effects on survivin. Both compounds synergized with docetaxel in vitro warranting future in vivo efficacy studies as a combinatorial therapy. Overall, our findings indicate survivin is a significant contributor to docetaxel resistance in metastatic prostate cancer at the molecular level and survivin inhibitors may prove efficacious as a new therapy to sensitize cancer cells to chemotherapies.

Current dogma suggests modulating or preventing amyloid assembly will prove critical to the armamentarium of therapeutic interventions that will likely be required to overcome the multifaceted pathology associated with amyloid diseases. The work described in this dissertation reveals substantial gains in understanding key aspects relating to the anti-amylin amyloid activities associated with both individual and broad groups of small molecule amyloid inhibitors. A main observation was the important role that the catechol functional group plays in modulating and preventing amyloid formation. In this context, each chapter provides unique yet complementary mechanistic insight that delineates a wide range of anti-amyloid activities associated with preventing amylin amyloid formation by mainly catechol-containing structural scaffolds. Structure activity studies show that the catechol moiety present within baicalein, oleuropein and rosmarinic acid are critical for their anti-amyloid functions, including exerting cell rescue effects against amylin induced cytotoxicity. We also demonstrate that in general, autoxidation enhances the anti-amyloid potency associated with many catechol containing amyloid inhibitors that may be mechanistically linked to a covalent mode of action. For example, we demonstrate that the O-quinone form of baicalein conjugates with amylin via a Schiff base mechanism. In contrast, we also show that catechol mediated formation of protein denaturant resistant aggregates, which requires autoxidation and that also stems from a predicted covalent mode of action, does not necessarily correlate with the enhanced anti-amyloid activities that occur upon catechol autoxidation. Regardless of the chemical mechanism(s) that drive catechol mediated anti-amyloid activity in vitro, the observed cell rescue effects exhibited by catechol containing molecules against amylin amyloid induced cytotoxicity is congruent with several recent in vivo studies that indicate polyphenols prevent toxic amyloid deposition as well as decades of population based studies that show regular consumption of diets rich in polyphenols are linked to a reduce incidence of age-related neurodegenerative amyloid disease. Indeed, advances in structure based drug discovery against amyloid formation may provide new avenues to optimize various catechol containing scaffolds that could be readily leveraged into improving diagnostic tools or perhaps accelerate the effort of discovering anti-amyloid therapeutics. / Ph. D. / From causing dementia in diseases like Alzheimer’s disease (AD) to potentiating type 2 diabetes (T2D), amyloid diseases represent some of the most devastating and increasingly more common human diseases. Amyloids themselves mainly consist of an aggregated mass of a specific type of protein that is believed to be either directly or indirectly toxic. Currently, there are no known cures for preventing amyloid diseases, and so far, efforts to discover anti-amyloid therapeutics have been largely unsuccessful. Many studies indicate regular consumption of plant-based diets, like the Mediterranean diet, that includes foods such as olives, vegetables and red wine leads to reduced incidence of age related amyloid diseases. Guided by these data, scientists have begun to uncover specific molecules within these diets that are able to prevent amyloid formation. A main emphasis in this dissertation was to understand the details of how these molecules prevent toxic amyloid formation. The insights gained from these studies have elucidated key chemical structural features present within these molecules that convey unique effects on perturbing amyloid formation. Excitingly, we also found that the presence of oxygen within the air we breathe, interacts with and enhances the ability of these compounds to exhibit stronger anti-amyloid functions! These data can be used to engineer better amyloid inhibitors that could lead to drugs.

Bone metastases are a frequent clinical complication associated with cancer. The aim of this PhD thesis was to set up a model system for the study of tumour cell – bone cell interactions in vitro, ex vivo and in vivo and to use this system to test the efficacy of a novel therapeutic agent for the treatment of osteolytic bone disease. Co-culture or conditioned medium studies using human or mouse cancer cell lines were used to develop an in vitro model system of tumour cell – bone cell interactions. This showed that osteolytic tumour cells enhance osteoclast formation, fusion and resorption through the production of various factors that act directly on osteoclasts and their precursors. And in addition, that osteolytic tumour cells also enhance osteoclastogenesis indirectly via increasing the production of RANKL in osteoblasts. Other effects on osteoblasts included reductions in differentiation, migration and adhesion. Successful ex vivo and in vivo models for the study of tumour – induced osteolysis were created using adapted organ cultures and intratibial injection techniques respectively. IGF-1 and its receptor are known to play important roles in both bone metabolism and breast cancer. Therefore a study of the effects of IGF-1 receptor inhibition on tumour cell – bone cell interactions was performed. In vitro studies showed that the novel IGF-1 receptor tyrosine kinase inhibitor PQIP significantly inhibited IGF-1 and breast cancer enhanced osteoclast formation. Western blot analysis suggested this may be due to the inhibition of both IGF-1 and cancer conditioned medium induced PI3k/Akt activation. Moreover, treatment of osteoblasts with PQIP inhibited cancer cell conditioned medium induced increases in RANKL production. Ex vivo studies using human MDA-MB-231 – mouse calvarial organ co-cultures demonstrated that MDA-MB-231 cells caused osteolysis and this was completely prevented by PQIP without affecting cancer cell viability. Furthermore, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following mouse 4T1 breast cancer cell intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in breast cancer-induced osteoclast number and activity. Consistent with the significant inhibition of osteoblast differentiation, spreading, migration and bone nodule formation observed in vitro, PQIP also inhibited osteoblast number and bone formation in vivo. No inhibition of in vivo tumour volume was observed. These findings clearly suggest that oral PQIP treatment reduced the rate of cancer associated bone turnover. In conclusion, this thesis successfully demonstrates a model system for investigating tumour cell-bone cell interactions in vitro, ex vivo and in vivo. Using this model system I showed that pharmacologic inhibition of IGF-1 receptor kinase activity using PQIP inhibits osteoclast and osteoblast changes induced by breast cancer cells in vitro and in vivo and prevents osteolysis ex vivo and in vivo. This indicates that PQIP and its novel derivatives which are now in advanced clinical development may be of value in the treatment of osteolytic bone disease associated with breast cancer.

Small molecule libraries have become essential for the development of drug discovery campaigns and chemical genetics. The studies towards the synthesis of a small molecule library, based upon the cis-ring fused phenanthridine core I, will be described. The first section of this thesis examines the development and application of a novel intramolecular Heck cyclisation to the synthesis of core phenanthridine structure II, via precursor III (Chapter 2).The second section (Chapter 3) describes the extension of this methodology towards the development of a library of phenanthridines IV. This includes methodology designed to incorporate the key principles of diversity-oriented synthesis, namely appendage, stereochemical and skeletal diversity. The final part of this thesis (Chapter 4) describes the merging of these various methodologies to generate a small library of novel phenanthridine analogues. Preliminary biological evaluation of the phenanthridine library using whole organism zebrafish phenotyping, will also be discussed.

Photoreactive groups have been used in photoaffinity labelling of chemical macromolecules via the generation of highly reactive species upon short wave light irradiation. One of the most efficient photoreactive functional groups is trifluoromethyl aryldiazirine (TFMAD). This compound was synthesised as part of the work discussed in this thesis, making use of microwave irradiation to shorten reaction times (Chapter I). An investigation of properties allowed the development of three different applications for conjugation to biomolecules. The first application consisted of the development of an approach for generation of small-molecule microarrays, where a 2,000 compound library was immobilised onto the glass surface through carbene insertion. The microarray was then used to screen for potential binders to beta-transducin repeat containing protein (b-TrCP1) allowing the reduction of possible candidates to less than 25 compounds (Chapter II). The second application was the synthesis of two probes to allow the selective delivery of active compounds inside specific organelles or cells. The diazirine moiety was used as a rapid way to covalently capture a number of cargos. The approach allowed a peptoid and an anticancer drug to be conjugated to the two probes and their cell penetrability properties and therapeutic effect were studied, respectively (Chapter III). Finally, the insertion properties of TFMAD were used to develop approaches to attach DNA onto microspheres and the efficiency of this delivery system was evaluated (Chapter IV).

Melanocytes are pigment-producing cells that colour our hair, skin and eyes. Melanocytes are evolutionary conserved in vertebrates, and in addition to contributing to pigmentation and pattern formation, can contribute to background adaptation (zebrafish) and protection against harmful UV irradiation (humans). Many of the processes involved in melanocyte development – such as migration, proliferation and differentiation - are misregulated in melanoma. Here, I use chemical biology in zebrafish to identify targetable pathways in melanocyte development and regeneration, with a view to how these processes may be misregulated in melanoma and other pigmentation syndromes. We first wanted to address the potential for small molecules to regulate multiple stages of melanocyte development and differentiation. In Chapter 3, I describe my work involved in a small molecule screen for clinically active compounds that alter melanocyte biology (Colanesi et al., 2012). In this work we have identified small-molecules that affect melanocyte migration, differentiation, survival, morphology and number. This is important as it highlights new pathways essential for normal melanocyte development and consequently provides further tools in which to study melanocytes. Identifying the target of small molecules in vivo is a challenge in chemical biology. In Chapter 4, I describe my contributions to understanding how 5-nitrofuran compounds act in zebrafish (Zhou et al., 2012). My work has contributed to understanding the activity of 5-nitrofurans is dependent upon its nitrofuran ring structure. I have also helped confirm a conserved interaction between 5-nitrofurans and ALDH2, which may contribute to the off-target effects observed in the clinic. These results are important as they aid further understand of the 5-nitrofuran class of drugs and give evidence to support combination therapy of 5-nitrofurans with ALDH2 inhibitors as a way to overcome clinical side effects. Additionally I show that NFN1 treatment limits ensuing melanocyte regeneration thereby suggesting a role at the Melanocyte Stem Cell (MSC), which provides me with a key tool to study melanocyte regeneration in zebrafish. How tissue specific cell numbers are specified and maintained is a key question in developmental biology. In Chapter 5, I describe the identification of the MITF gene in the maintenance of cell cycle arrest in differentiated melanocytes (Taylor et al., 2011). We show that the human melanoma mutation MITF4TΔ2B promotes melanocyte division, thereby suggesting a role for melanocyte division in the pathogenesis of melanoma. This work is valuable because it highlights Mitf as a molecular rheostat that controls melanocyte proliferation and differentiation in living vertebrates, and helps us to understand the role of MITF in melanoma progression. Little is known about the pathways that control melanocyte stem cells in animals. To identify new melanocyte stem cell pathways, I used NFN1 as the basis for a small molecule screen for enhancers of melanocyte regeneration (Chapter 6). I find that chemical inhibition of Phosphatase of Regenerating Liver-3 (Prl-3) in zebrafish can enhance melanocyte regeneration. Importantly, I have found that there are an increased number of melanocyte progenitor cells in PRL3-inhibitor treated zebrafish. I propose that PRL-3 may control progenitor cell number in melanocyte regeneration. This is significant because it identifies PRL-3 as a novel molecular target controlling melanocyte progenitor cells, and identifies a new chemical tool with which to study melanocyte differentiation from a progenitor population. In the final chapter, I discuss how this work relates to the larger field of melanocyte developmental biology, and the new insight it provides into the fundamental processes of how organisms control cell number and pattern formation. In addition, I discuss how this work may have implications for understanding and treating melanocyte diseases, such as vitiligo (loss of melanocytes) and melanoma (cancer of the melanocyte).