The homologous recombination (HR) pathway of DNA repair is essential for the faithful repair of double-stranded DNA breaks (DSBs) in all organisms and as such helps maintain genomic stability. Furthermore, HR is instrumental in the cellular response to exogenous DNA damaging agents such as those used in the clinic for chemo- and radiotherapy. HR in humans is a complex, incompletely understood process involving numerous stages and diverse biochemical activities. Advancing our knowledge of the HR pathway in humans aids the understanding of how chemo- and radiotherapies act and may be used to develop novel therapeutic strategies. Recent studies have identified inhibition of HR as one of the mechanisms via which a number of recently developed chemotherapeutics have their effect. Accordingly, the clinical potential of HR inhibitors is under investigation. My work has centred around the identification of both novel HR proteins and novel, small molecule HR inhibitors. To further these aims, I have successfully employed high-throughput RNAi and small molecule screening strategies. RNAi screens are commonly used to identify genes involved in a given cellular process via genetic loss of function, whilst small molecule, cell based screens are a powerful tool in the drug discovery process.

Uma das várias formas da hemoglobina do peixe Leporinus Frederici (piava) não apresenta efeito Borh (variação da afinidade ao O2 com o pH). Purificação, caracterização e experimentos de cristalização foram conduzidos visando a determinação da estrutura através de difração de raios X, embora sem resultados positivos. O espectro óptico desta forma particular de hemoglobina foi medido no intervalo de 300-700 nm e subseqüentemente simulado, interpretado e comparado com o espectro da humana. Em outra área do trabalho experimental, três pequenas estruturas moleculares foram determinadas: uma é um intermediário na síntese de alcalóides, com um esqueleto sarpagina; outra é um dipeptídeo complexado com Cu2+; e a terceira é um complexo de picrato com Ce3+. As intensidades das reflexões foram medidas com um difratômetro automático de quatro ciclos CAD-4. As estruturas foram resolvidas por Patterson ou Métodos Diretos e foram refinadas por método de mínimo quadrado. Cetona, C19H21N3O, é um intermediário chave no caminho de reação para síntese de indoloquinolisidinas, pertence ao sistema, P21/C, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. As duas moléculas independentes são aproximadamente relacionadas por um centro de inversão, a principal diferença sendo relativa às configurações dos grupos nitril e metil. As moléculas enantioméricas estão mantidas por ligação de hidrogênio através do N(3)-O(1\') e N(3\')-O(1). A junção N(1)-C(6) é trans e o grupo CH2CN é axial. L-(triptofil)-L-glicinato-cobre(II). C13H13CuN3O3, um composto modelo para conseguir informações para interpretar os dados disponíveis para proteínas azuis, é ortorrômbico, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. O íon Cu2+ é coordenado por um oxigênio e dois nitrogênios de um dipeptídeo e com um oxigênio de um ligante simetricamente relacionado. A estrutura polimérica resultante está alinhada com o eixo b e tem uma estabilidade maior devido a uma ligação de hidrogênio entre o oxigênio carbonil de um dipeptídeo e o nitrogênio do triptofano do ligante vizinho. A coordenação é essencialmente quadrado planar. O complexo de picrato com Ce3+, CeO33N9C18H30, foi analisado dentro de um grande programa de pesquisa para estudar a química de coordenação dos lantanóides. Duas formas cristalinas são estudadas: uma é monoclínica, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ e a outra é monoc1ínica, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. A primeira é relativamente instável sobre a incidência de raios-X e embora a medida das intensidades apresentasse erros sistemáticos significantes, a estrutura pode ser resolvida. O número de coordenação dos dois complexos é 9 e os poliedros de coordenação são intermediários entre antiprisma quadrado monoencapuzado e prisma trigonal triencapuzado / One of the various forms of hemoglobin of the fish Leporinus Frederici (piava) does not present any Bohr effect (variation of the affinity to O2 with pH). Purification, characterization and crystallization experiments were conducted, aimed at the structure determination through X-ray diffraction, although with no positive results. The optical spectrum of this particular hemoglobin form was measured in the range 300-700 nm and subsequently simulated, interpreted and compared with the human hemoglobin spectra. In another area of experimental work, three small molecules structures were determinate: one is an intermediate in the synthesis of alkaloids, with a sarpagine backbone; another is a dipeptide complexes with Cu2+; and a third one is a complex of picrate with Ce3+. The intensities of the reflections were measured with an automatic four-circle difractometer CAD-4. The structures were solved by Patterson or Directs Methods, and were refined by the least squares methods. Ketone, C19H21N3O, is a key intermediate in the reaction pathway for synthesis of indoloquisidines, belongs to the monoclinic system, P21/c, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. The two independent molecule are approximately related by an inversion center, the main difference being the relative configurations of the nitril and methyl groups. The enantiomeric molecules are hydrogen bonded through N(3)-O(1\') and N(3\')-O(1). The junction N(l)-C(6) is trans and the group CH2CN is axial. L-(tryptophyl)-L-glycinate-copper(II), C13H13CuN3O3, a model compound to get information to interpret spectroscopic data available for blues proteins, is orthorhombic, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. The Cu-ion is coordinated by one oxygen and two nitrogen atoms of the one dípeptide and with an oxygen of a symmetrically related ligand. The resulting polymeric structure is aligned with the b-axis and is further stabilized by an H-bond between the carbonyl-oxygen of the one dipeptide and the tryptophan side-chain nitrogen of the neighboring ligand. The coordination is essentially square-planar. The complex of picrate with Ce3+, CeO33N9C18H30, was analyzed within a broader research program to study the chemistry of coordination of the lantanoids. Two crystalline forms are studied: one is monoclinic, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ and the other is monoclinic, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. The former is relatively unstable under the X-rays and although the measured intensities presented significant systematic errors, the structure could be solved. The coordination number of the two complexes is 9 and the coordination polyedra are intermediate between mono-coupled square antiprism and tri-coupled trigonal prism

G-quadruplexes are non-canonical nucleic acid secondary structures of increasing biological and medicinal interest due to their proposed physiological functions in transcription, replication, translation and telomere biology. Aberrant G4 formation and stabilisation have been linked to genome instability, cancer and other diseases. However, the specific genes and pathways involved are largely unknown, and the work within this thesis aims to investigate this. Stabilisation of G4s by small molecules can perturb G4-mediated processes and initial studies suggest that this approach has chemotherapeutic potential. I therefore also aimed to identify cell genotypes sensitive to G4-ligand treatment that may offer further therapeutic opportunities. To address these aims, I present the first unbiased genome-wide genetic screen in cells where genes were silenced via short-hairpin RNAs (shRNAs) whilst being treated with either PDS or PhenDC3, two independent G4-stabilising small molecules. I explored gene deficiencies that enhance cell death (sensitisation) or provide a growth advantage (resistance) in the presence of these G4-ligands. Additionally, I present a validation screen, comprising hits uncovered via genome-wide screening, and also the use of this in another cell line of different origin. Sensitivities were enriched in DNA replication, cell cycle, DNA damage repair, splicing and ubiquitin-mediated proteolysis proteins and pathways. Ultimately, I uncovered four synthetic lethalities BRCA1, TOP1, DDX42, GAR1, independent of cell line and ligand. These were validated with three G4-stabilising ligands (PDS, PhenDC3 and CX-5461) using an independent siRNA approach. The latter siRNA methodology was used to screen 12 PDS derivatives with improved medicinal chemistry properties and ultimately identified SA-100-128, as a lead compound. The mechanism behind synthetic lethality with G4-stabilising ligands was explored further for DDX42, which I show has in vitro affinity for both RNA- and DNA-G4s and may represent a previously unknown G4-helicase. Also within this thesis, gene deficiencies that provided a growth advantage to PDS and/or PhenDC3 as uncovered by genome-wide and focused screening were explored. These showed enrichment in transcription, chromatin and lysosome-associated genes. The resistance phenotype of three gene deficiencies, TAF1, DDX39A and ZNF217 was further supported by additional siRNA experiments. Overall, I satisfied the primary aims and established many novel synthetic lethal and resistance interactions that may represent new therapeutic possibilities. Additionally, the results expand our knowledge of G4-biology by identifying genes, functions and subcellular locations previously not known to involve or regulate G4s.

Methods for preparing an array of potential small molecule inhibitors of Bone Morphogenetic Protein Receptor 1b (BMPR1b) are described. Target molecules were prepared from two general classes: (1) N9-aryl-N6-ureidoadenines, and (2) dicarbamyl iodoacetamides. Recent data from the Peterson lab indicated that both classes might bind to BMPR1b and thus inhibit this key receptor. Docking studies performed using Sureflex Dock suggested the N9-aryl-N6-ureidoadenines would bind to the active site of BMPR1b. In addition antiproliferative activities of dicarbamyl iodoacetamides previously synthesized in the Peterson lab pointed to this moiety as an attractive target for structure activity relationship (SAR) development. Compounds were prepared in good to excellent yields and 40 derivatives were screened for antiproliferative activity. Of the N9-aryl-N6-ureidoadenine derivatives, N9-phenyl-N6-N-phenylureaadenine was most potent and exhibited selective activity against HeLa cells (IC50 = 11± 1 uM). Dicarbamyl iodoacetamide derivatives had similar activities compared to the previously reported compound (JRS-150).

Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.

Three classes of bifunctional compounds related to frustrated Lewis pair chemistry were studied. The first class, alkynyl-linked phosphonium borates, was strategically synthesized and the corresponding neutral alkynyl-linked phosphine boranes generated in solution. They were reacted with THF, alkenes and alkynes to undergo either ring-opening or multiple bond addition reactions, giving rise to zwitterionic macrocycles. In two select alkynyl-linked phosphonium borates, thermolysis resulted in unique rearrangements transforming the phosphino- and boryl-substituted alkynyl moieties into C4 chains. The alkynyl-linked phosphine boranes were further demonstrated to coordinate as η3-BCC ligands in Ni(0) complexes. The rigid nature of the coordination was confirmed by dimerization without cleavage of the Ni–B interaction upon the addition of acetonitrile or carbon monoxide. Moreover, reactions with Al-, Zn- and B-based Lewis acids prompted hydride transfer within the alkynyl-linked phosphonium borate and interesting functional group transfer reactions. The second class of the bifunctional systems, a series of gem-substituted bis-boranes, was subjected to reactions with tBu3P and CO2. The O-linked bis-borane was shown to coordinate the phosphino-carboxylate moiety with one B, while the methylene-linked bis-boranes were demonstrated to chelate the carboxyl group. The third bifunctional system class, vinyl-group tethered boranes, was examined to elucidate the mechanism of the frustrated Lewis pair addition reaction to olefins. Using a bis(pentafluorophenyl)alkylborane, the close proximity of the olefinic protons and the ortho-fluorine nuclei were evident by both NOE measurements and DFT calculations. Moreover, its reactions with phosphine bases suggested that an initial interaction between the highly electrophilic borane and the olefinic fragment precedes such frustrated Lewis pair addition reaction. Furthermore, a bis(pentafluorophenyl)alkoxyborane was synthesized and reacted with P-, N-, C- and H-based nucleophiles, demonstrating the wide range of Lewis bases that can be applied in olefin addition reactions with complementary regioselectivity.

Gold(I) and copper(I) imidazolium complexes were synthesized and probed for use as bulky Lewis acids in frustrated Lewis pairs (FLPs) with bulky phosphines and amines. Their reactivity with small molecules was investigated and the compounds were fully characterized by multinuclear NMR spectroscopy, elemental analysis and X-ray crystallography. Secondly, a new methylene-linked boron-sulfur Lewis acid was synthesized. Its thermodynamic properties were determined and its reactivity with terminal and internal alkynes was demonstrated. Adducts and heterocycles of this boron-sulfur system were fully characterized by multinuclear NMR spectroscopy, elemental analysis and X-ray crystallography. The application of these new systems for the activation of small molecules is described in this thesis.

Gold(I) and copper(I) imidazolium complexes were synthesized and probed for use as bulky Lewis acids in frustrated Lewis pairs (FLPs) with bulky phosphines and amines. Their reactivity with small molecules was investigated and the compounds were fully characterized by multinuclear NMR spectroscopy, elemental analysis and X-ray crystallography. Secondly, a new methylene-linked boron-sulfur Lewis acid was synthesized. Its thermodynamic properties were determined and its reactivity with terminal and internal alkynes was demonstrated. Adducts and heterocycles of this boron-sulfur system were fully characterized by multinuclear NMR spectroscopy, elemental analysis and X-ray crystallography. The application of these new systems for the activation of small molecules is described in this thesis.