Small molecules play important roles in therapeutics and drug discovery. Significant progress has been made by the chemical biology community to discover small-molecule probes to explore biological processes and to treat disease. This thesis describes both the discovery of novel probes for the Hedgehog (Hh) pathway and the application of small molecules in identifying cancer dependencies. / Chemistry and Chemical Biology

Advances in genome and proteome research have led to a dramatic increase in the number of macromolecular targets of interest to the life sciences. A solution-phase method to simultaneously reveal all ligand-target binding pairs from a single solution containing libraries of ligands and targets could significantly increase the efficiency and effectiveness of target-oriented screening efforts. Here, we describe interaction-dependent PCR (IDPCR), a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes identifying the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein-ligand pairs out of 67,599 possible sequences.

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This thesis examines the use of anionic nitrogen chelate ligands in coordination-driven self-assembly and small molecule activation. The two classes of anionic nitrogen chelate ligands that were explored are β-diiminate and 4,5-diazafluorenide derivatives. Chapter 2 deals with Pd β-diiminate chemistry. Chloro-bridged dimers served as versatile starting materials, and their reactivity toward pyridine and arylboronic acids was explored. An unusual transmetallation reaction with arylboronic acids triggered the self-assembly of tetrapallada-macrocycles. The formation of the self-assembled tetrapallada-macrocycles is through the generation of new Pd-C bonds. Chapter 3 deals with 4,5-diazafluorenide as an actor ligand in CO2 activation. A reversible formal insertion of CO2 into a remote ligand C-H bond was discovered. A variety of spectator metal centres were used to tune the reactivity of the actor ligand toward CO2. The spectator metal centre could even be replaced entirely with an organic group allowing for the first metal-free reversible tandem CO2 and C-H activation. Chapter 4 deals with the reactivity of dinuclear Rh 4,5-diazafluorenide-9-carboxylate complexes with dihydrogen in an attempt to reduce the trapped CO2 moiety. A series of stepwise stoichiometric reactions with H2, NMR experiments at low temperatures with added PPh3 or CO2, along with 13C-labelling experiments were conducted in an attempt to identify the products of this reaction and gain some mechanistic insight. Chapter 5 deals with using ambidentate 4,5-diazafluorene derivatives to synthesize linkage isomers, heterobimetallic complexes, and self-assembled macrocycles. The synthesis a new ligand family, 3,6-substituted 4,5-diazafluorene ligands is presented, along with coordination chemistry towards a {RuCp*}+ fragment. Finally in Chapter 6 the coordination chemistry of 3,6-diaryl substituted 4,5-diazafluorene derivatives was explored with the goal of generating low-coordinate species for the activation of small molecules, especially N2. The synthesis of the first trialkylborohydride complex of vanadium is presented.

Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication. There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not. CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59. These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.

A series of small molecules was synthesized to probe three protein targets in order to elucidate the key small molecule-protein interactions required for potency. Triclosan is an antibacterial compound that has surfaced as a potential environmental hazard and is hypothesized to cause perturbations in the thyroid hormone response of frogs. Using a C-fin assay and a GH3 cell line, our work suggests that triclosan itself may not in fact be the cause of the observed endocrine disruptions. Instead, methyl triclosan (a result of biological methylation during waste water treatment) was shown to disrupt the thyroid hormone response in tadpoles. Secondly, a set of probes was designed based on a cyclopentane scaffold derived from the known neuraminidase inhibitor peramivir. Kinetic assays using both a recombinant neuraminidase protein and an inactivated sample of influenza virus showed that the guanidine group contributes a 10 fold increase in potency while the α-hydroxyl group was observed to have little to no effect. This result suggests that future neuraminidase drug design based on a cyclopentane scaffold may forgo the use of both the guanidinium group and the hydroxyl group to potentially increase the oral availability of these drugs while sacrificing little in the way of potency. Finally, a series of truncated analogues related to the western half of the natural product didemnaketal A was synthesized. These compounds will be used as probes to better understand the mechanism of didemnaketal-mediated protease inhibition. It is hypothesized that a more rigid structure (due to molecular gearing enforced by the presence of additional methyl groups, relative to previously examined analogues) will increase the potency of these molecules toward HIV-1 protease and may lead to new information for designing next-generation dissociative inhibitors. Work was also begun toward the total synthesis of the natural product itself. / Graduate

The oncogenic transcription factor STAT3 is aberrantly activated in over 70% of human tumours, including Multiple myeloma (MM). The present studies use both genetic and chemical tools to validate STAT3 as a therapeutic target, and demonstrate the anti-MM activity of a novel small molecule STAT3 inhibitor, BP-4-018. We show that shRNA-mediated STAT3 knockdown induces apoptosis in human myeloma cell lines (HMCLs). We translate these findings to a therapeutically relevant setting by demonstrating the broad anti-MM activity of BP-4-018 against HCMLs and primary patient samples, and demonstrate that BP-4-018 remains active against HMCLs co-cultured with bone marrow stroma. Inhibiting STAT3 via shRNA knockdown and BP-4-018 suppresses STAT3 transcriptional activity and down-regulates anti-apoptotic and proliferative STAT3 target genes. Finally, we show that BP-4-018 has activity in vivo, both alone and combined with subtherapeutic doses of bortezomib, without significant toxicities. Taken together, these data support the utility of STAT3 inhibitors for MM treatment.

The oncogenic transcription factor STAT3 is aberrantly activated in over 70% of human tumours, including Multiple myeloma (MM). The present studies use both genetic and chemical tools to validate STAT3 as a therapeutic target, and demonstrate the anti-MM activity of a novel small molecule STAT3 inhibitor, BP-4-018. We show that shRNA-mediated STAT3 knockdown induces apoptosis in human myeloma cell lines (HMCLs). We translate these findings to a therapeutically relevant setting by demonstrating the broad anti-MM activity of BP-4-018 against HCMLs and primary patient samples, and demonstrate that BP-4-018 remains active against HMCLs co-cultured with bone marrow stroma. Inhibiting STAT3 via shRNA knockdown and BP-4-018 suppresses STAT3 transcriptional activity and down-regulates anti-apoptotic and proliferative STAT3 target genes. Finally, we show that BP-4-018 has activity in vivo, both alone and combined with subtherapeutic doses of bortezomib, without significant toxicities. Taken together, these data support the utility of STAT3 inhibitors for MM treatment.

The chemistry and physical properties of actinide complexes has become increasingly significant and relevant since the dawn of the nuclear age. In addition to increasing the potency of nuclear power and the safety and disposal of its subsequent waste products, exploration of the chemistry of actinide complexes provides a fascinating insight into the increased complexity and divergence of reactivity of these complexes when compared to transition metal complexes. Chapter One provides a brief introduction to the chemistry of actinides and in particular, the major focus of this work, of thorium. This is followed by a survey of examples of rare examples of thorium complexes with a formal oxidation state other than Th (IV). Following this is a review of selected examples of thorium (IV) complexes exhibiting unusual reactivity surveying thorium hydride and alkyl complexes initially. This progresses into reviewing the chemistry of thorium complexes containing multiple bonds to non-metal atoms, beginning with carbon atoms and then progressing to atoms in the chalcogen and pnictogen groups. The introduction finishes with an investigation into the properties of the terphenolate ligands used in this study, including examples of unusual complexes that they have been shown to stabilise. In Chapter Two, an exploration into the catalytic activity of fairly simple actinide amide catalysts, N”2Th (IV) {k2-N(SiMe3)SiMe2CH2, N”2U (IV) {k2-N(SiMe3)SiMe2CH2} and UN”3, upon terminal acetylenes is presented. The chapter begins with a brief introduction summarising the previous reactivity observed in the catalysis of terminal acetylenes, with particular focus on actinide-based catalyst mediated reactions. The catalytic results on a variety of terminal acetylenes with different steric and electronic properties is then reported upon. It is found that high conversions and selectivities can be achieved upon optimisation of the catalytic process. It was also found that the different catalysts and substrates favoured different products, with selective oligomerisation and cyclotrimerisation reactions observed. The differing reactivities lend support to the role of f-electrons upon the catalytic route of the reaction. Conclusions are discussed at the end of the chapter. In Chapter Three, the synthesis and characterisation of heteroleptic terphenolate thorium chloride complexes and their subsequent reactivity was investigated. The synthesis and characterisation of ThCl2(OTerMes)2DME and ThCl2(OTerMes)2(H2O)3 are initially described. The reactivity of these complexes favoured transmetallation of the terphenolate ligands, with the complexes; [Li(OTerMes)THF]2, [Li(OTerMes)]2THF, μ3- (TerMesO)μ3-(CH2SiMe3)3Li4, LiAlH2(OTerMes)2, [(THF)K(OTerMes)]2, MgCl(OTerMes)(THF)2, MgBr(OTerMes)(THF)2 and Fe(OTerMes)2(py)2 synthesised and characterised from reactions attempting to transform the ancillary chlorido-ligands. The reactivity of ThCl2(OTerMes)2DME was found to not be solely transmetallation of the terphenolate ligands as elucidated by the synthesis and characterisation of [Th(OTerMes)2(Cl)2(4,4’- bipyridyl)1.5]∞ and [MgTh2μ2-Cl2μ3-Cl(OTerMes)2(C4H7)2μ-η3:η3(C4H7)H]. The synthesis of [MgTh2μ2-Cl2μ3-Cl(OTerMes)2(C4H7)2μ-η3:η3(C4H7)H] was found to proceed via a reductive elimination route with concomitant formation of a terphenolate transmetallation product Mg(OTerMes)2(THF)2. The formation of[Th(OTerMes)2(Cl)2(4,4’- bipyridyl)1.5]∞ was achieved via reaction with the Lewis base 4-4’ bipyridine. Reactions attempting to form heteroleptic uranium terphenolate complexes were also detailed. Conclusions are discussed at the end of the chapter. In Chapter Four, the synthesis and characterisation of heteroleptic terphenolate thorium borohydride complexes and their subsequent reactivity was investigated. It was found that the conversion of ThCl2(OTerMes)2DME to Th(BH4)2(OTerMes)2DME proceeded smoothly using a precedented reaction route. In contrast to ThCl2(OTerMes)2DME, reaction with a Lewis acid was found to result in abstraction of the solvating DME molecule, resulting in the synthesis and characterisation of Th(BH4)2(OTerMes)2. In similarity to ThCl2(OTerMes)2DME, Th(BH4)2(OTerMes)2DME was found to react with a Lewis base (4-4’ bipyridine) to form Th(BH4)2(OTerMes)2(4,4’ bipyridine)∞. However, despite the increased robustness and versatility of the borohydride complexes, transmetallation of the terphenolate complexes remained an issue as shown by the synthesis and characterisation of Mg(OTerMes)((μ-H)3BH)THF)2. Th(BH4)2(OTerMes)2 was found to be able to facilitate small molecule activation in a variety of substrates, encompassing CO, CO2 and CS2 amongst others. In most cases this small molecule activation favoured the formation of BMe3, with the concomitant formation of HB(OTerMes)2 in the case of CO2 and CS2. Attempts at catalysis of isonitriles and terminal acetylenes by Th(BH4)2(OTerMes)2 are presented with mixed results. Conclusions are discussed at the end of the chapter. In Chapter Five, investigations into the effects of changing the donor atom of the terphenyl moiety were probed. The chapter began by examining the differing properties of a phosphorous atom acting as a ligating atom, as opposed to the oxygen atom seen in Chapters Three and Four. The chapter continued by detailing the result of reactions attempting to synthesise and characterise terphenyl phosphino-actinide complexes. It was found that in the case of actinides with easily accessible lower oxidation states, i.e. U (IV), that reductive elimination was favoured, culminating in the isolation of (TerMesPH)2. Following this result attempts were made to modify the ligand system in an attempt to divert the reaction away from this product, in the hope of isolating a phosphino-actinide complex. Reactions attempting to ligate the terphenyl moiety via the aryl α-carbon to thorium were also detailed, resulting in radicular degeneration and the isolation of nBuTerTrip and ClTerTrip. Conclusions are discussed at the end of the chapter. Experimental and characterising data are provided in Chapter Six.

Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.