Organic chemistry is a valuable tool for studying enzyme mechanisms. Upon incubation with a specific enzyme, synthetic substrate analogues labeled with heavy atoms or carrying extra functional groups can provide mechanistic insights. In the present work, new compounds were synthesised in order to study the mechanism and substrate selectivity of two enzymes: human γ-butyrobetaine hydroxylase and bacterial carboxymethylproline synthase. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses the stereospecific hydroxylation of γ-butyrobetaine, the final step of L-carnitine (L-Car) biosynthesis in mammals. Substrate analogues were synthesised to probe BBOX specificity in vitro. Some of those unnatural substrates were oxidised by BBOX and the products identified using a range of analytical techniques. 3-(2,2,2-Trimethylhydrazinium)propionate (THP) is a clinically used BBOX inhibitor. Under standard assay conditions, THP was oxidised by BBOX. NMR studies have identified the products of this reaction to be malonic acid semialdehyde, formaldehyde, dimethylamine and 3-amino-4-(methylamino)butanoic acid. The formation of 3-amino-4-(methylamino)butanoic acid suggests that BBOX can catalyse a Stevens type rearrangement involving N-N bond cleavage and C-C bond formation. The proposed structures and mechanisms were confirmed by mass spectrometric and NMR analyses using [¹³C]-labeled THP as well as synthetic standards of both enantiomers of 3-amino-4-(methylamino)butanoic acid. Although the structure of the rearrangement product was confirmed, the stereochemistry remains unknown. Altogether, these studies revealed the unprecedented nature of a BBOX-catalysed C–C bond formation reaction upon THP oxidation and may inspire the design of improved inhibitors for BBOX and other 2OG oxygenases. Pectobacterium carotovorum CarB and Streptomyces cattleya ThnE are two carboxymethylproline synthases (CMPS) that catalyse an early step in carbapenem antibiotics biosynthesis. CMPS produces (2S,5S)-carboxymethylproline (t-CMP) from malonyl-CoA and L-glutamate semi-aldehyde. L-Glutamate semi-aldehyde exists in equilibrium with L-5-hydroxyproline and L-pyrroline-5-carboxylate in solution (collectively abbreviated L-GHP). Because of the high stereoselectivity of t-CMP formation and the growing interest in novel carbapenem antibiotics, CMPS is potentially an interesting biocatalyst. A series of L-GHP analogues were synthesised and tested as CMPS substrates in an attempt to produce unnatural t-CMP derivatives enzymatically. Methyl-substituted L-GHP analogues were accepted by CMPS and the t-CMP products could be further carried through to the corresponding bicyclic carbapenams using CarA, a β-lactam synthetase. These results demonstrate the versatility of the early carbapenem biosynthetic pathway and the possibility of introducing structural diversity using synthetic substrate analogues. A crystal structure of S. cattleya ThnE was obtained in complex with L-proline and coenzyme A, giving the first insight into substrate binding. This structural information will potentially allow further rational mutagenesis studies aiming to broaden the range of unnatural L-GHP analogues accepted by CMPS.

Inflammation is a multifactorial response towards noxious stimuli, however appropriate regulation and resolution of inflammation is crucial for the prevention of chronic inflammatory diseases such as atherosclerosis. The endocannabinoid (eCB) system is an endogenous immunomodulatory system which consists of a series of lipophilic ligands that signal via two G-protein-coupled receptors. Cannabinoid receptor 1 (CB1R) is mainly expressed in the central nervous system and its activation has psychoactive effects. Cannabinoid receptor 2 (CB2R) is mainly expressed on leukocytes and receptor activation has anti-inflammatory actions in mouse models of atherosclerosis and chronic inflammatory pain. It is considered that CB2R activation is involved in modulation of the recruitment of inflammatory cells, especially monocytes/macrophages; however the exact mechanism of action has not been fully elucidated. We hypothesised that activation of CB2R modulates monocyte/ macrophage recruitment and signalling, thus providing a homeostatic mechanism to limit macrophage activation in inflammatory responses. The high lipophilicity of cannabinoid ligands and their lack of selectivity for CB2R over CB1R limits CB2R drug development. In collaboration with Dr Angela Russell, we used virtual screening and a CB2R cAMP assay that we validated to discover a novel CB2R agonist, 3-((2’-Cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6-b]indole, (DIAS2). In collaboration with Dr Russell’s group who did chemical synthesis, we extended this novel scaffold to include over 80 compounds. Using the same hCB2R cAMP screening assay we demonstrated that 16 compounds with the same scaffold are at active at CB2R in the nanomolar range. At least 3 compounds, including DIAS2, were found to be ≥ 300-fold selective for CB2R over CB1R in cAMP assays and radioligand binding studies. In the inflammatory model of zymosan-induced peritonitis, DIAS2 dose-dependently inhibited inflammatory monocyte recruitment by 50% at highest dose of 5 mg/kg with no effect on neutrophils. In further zymosan-induced peritonitis experiments 5 mg/kg of DIAS2 and a structurally-similar CB2R agonist from the same family of triazino-indoles inhibited monocyte recruitment while a different CB2R agonist (JWH-133) at 5 mg/kg did not inhibit monocyte recruitment. Analysis of peritoneal exudates showed that the inhibition of monocyte recruitment was not associated with changes in the levels of JE, MIP-1α and nitric oxide but was associated with increased levels of the chemokine KC. Using in vitro cell biology approaches, we demonstrated that 10μΜ dose of both DIAS2 and JWH-133 reduced forskolin-induced cAMP production in primary murine macrophages. Also 2.5 to 10 μΜ οf JWH-133 and HU-308 dose-dependently induced primary murine macrophage chemotaxis which could be blocked a CB2R antagonist (SR 144528, 1 μΜ) while DIAS2 at doses up to 10 μΜ was not a chemoattractant. Accordingly HU-308 and JWH-133 were at least 3-fold more efficacious than DIAS2 at recruiting β-arrestin to the murine CB2R. Moreover in studies with primary murine macrophages 10 μΜ dose of JWH-133 and HU-308 induced ERK1/2 and Akt phosphorylation within 30 minutes, while 2-AG (an endogenous eCB ligand) and DIAS2 at 10 μΜ had no such effect. In summary, we have discovered a novel family CB2R agonists and demonstrated that some devoid of chemotactic active CB2R agonists can reduce monocyte recruitment in vivo while other chemoattractant CB2R agonists have no in vivo anti-inflammatory effect. We propose that non-chemotactic CB2R agonists represent a new class of anti-inflammatory drugs with a novel mode of action.

Thesis advisor: Mary F. Roberts / Myo-inositol and its phosphorylated derivatives are found across all domains of life, and these molecules play crucial roles in a wide variety of cellular processes. While the biosynthesis of inositol is an evolutionarily conserved pathway, there are a wide variety of enzymes that use inositol and its derivatives as substrates. This thesis explores two such enzymes; a phosphatidylinositol- specific phospholipase C (PI-PLC) produced by <i>Staphylococcus aureus</i>, and AF2372, a dual action inositol monophosphatase/ fructose bisphosphatase produced by the <i>Archaeoglobus fulgidus</i>. At the outset of this work, the structure of the <i>S. aureus</i> PI-PLC was unknown, but some interesting biochemical properties about the enzyme had been observed. The structure of AF2372 had been reported, but a structure had not yet been solved in the presence of osmolytes known to thermoprotect the enzyme. Both the <i>S. aureus</i> PI-PLC and AF2372 catalyze the cleavage of phosphorylated inositol compounds, but share no mechanistic, structural, or taxonomical similarities. Protein crystallography is a powerful tool, and with it I have been able to study these two enzymes at a molecular level, providing insight into complex biological questions about each enzyme. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) requires conserved components that mediate RNA interference (RNAi) directed methylation of histone H3 on lysine 9 (H3K9). Fission yeast heterochromatin is mainly found at centromeres, telomeres, and the mating-type locus. At centromeres, transcripts from repetitive elements are processed to siRNAs and RNAi promotes chromatin modification by recruiting the Clr4 methyltransferase. RNAi is not required to maintain silent chromatin at the mating-type locus. This RNAi-directed form of centromeric heterochromatin provides an ideal system for in vivo screening to allow the identification of compounds that inhibit the activity of proteins involved in RNA silencing, chromatin modification and heterochromatin assembly in fission yeast and may inhibit conserved proteins in other organisms. A dominant selectable marker gene system at fission yeast centromeres that reports loss of heterochromatin integrity by increased resistance to G418 in 96-well plate format liquid cultures was developed. The resulting strain was used to screen a nontargeted chemically diverse compound library in vivo to identify compounds that disrupt the integrity of RNAi-directed heterochromatin. Two compounds, Emi1 and Emi14, were identified and found to cause a significant decrease in the level of H3K9 methylation on the outer repeats at fission yeast centromeres. Growth in the presence of Emi1 or Emi14 also caused a reduction in H3K9 methylation levels at the mating-type locus, suggesting that they do not act through RNAi. Consistent with this, Emi1 and Emi14 did not cause a decrease in centromeric siRNA levels. Analyses therefore suggest that Emi1 and Emi14 do not disrupt RNAi but that they inhibit downstream events in chromatin modification and heterochromatin assembly. Cells lacking RNAi due to loss of Dicer (dcr1Δ) or cells lacking the histone deacetylase (HDAC) Sir2 (sir2Δ) retain significant but lower levels of H3K9 methylation on the centromeric outer repeats. When dcr1Δ or sir2Δ cells were grown in the presence of Emi1 or Emi14 a further reduction in H3K9 methylation levels was observed on the outer repeats. This mimics the effect of combining clr3Δ with dcr1Δ or sir2Δ and suggests that Emi1 and Emi14 may interfere with SHREC function. SHREC is a chromatin remodelling complex that includes the HDAC Clr3 and the chromatin remodeler Mit1 and is known to contribute to heterochromatin integrity. Expression profiling performed on Emi1 and Emi14 treated cells confirmed the previous results. The changes in gene expression following Emi1 and Emi14 treatment were compared to known mutants defective in heterochromatin integrity. The profile of expression changes following Emi14 treatment was found to correlate with alterations in the expression pattern observed in cells with SHREC components deleted. No correlation with mutants lacking other HDACs or RNAi components was detected. Emi1 had a weaker correlation with defective SHREC function and thus may also partially inhibit the SHREC complex. Murine erythroleukemia (MEL) cells harbouring a silenced eGFP reporter transgene were used to assess whether Emi1 and Emi14 also affect silencing in mammalian cells. Emi1 was found to disrupt silencing at the eGFP reporter and this correlated with a decrease in H3K9 methylation. Structurally related analogues of Emi1 and Emi14 were selected and tested in the fission yeast assay. Interpretation of the obtained structure-activity relationships allowed identification of the chemical moieties key to Emi1 and Emi14 activity. Overall, an approach was developed to identify two novel small molecule inhibitors of a well-characterized chromatin modification pathway. The SHREC complex was identified as the putative target of these two compounds and structurally related active analogues were identified for them. Importantly, one of the compounds was also active in mammalian cells, highlighting the usefulness of this approach in identifying compounds that affect higher organisms.

The genome-wide HaploInsufficieny Profiling (HIPHOP) technique has been validated as a method to quantify the relative abundance of uniquely tagged yeast deletion strains using a microarray readout. The massive throughput of next generation sequencing presents a new technology for assessing HIPHOP profiles. I developed a new method called Barcode analysis by Sequencing (Bar-seq) that applies deep sequencing to genome-scale fitness. I show that Bar-seq outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex genome-scale fitness assay, Bar-seq quantitatively identifies drug-targets, exceeding the performance of the microarray assay. I also established that Bar-seq is well suited to a multiplex format and provides a dramatic increase in throughput. I used the genome-wide HIPHOP assay and other functional genomics tools to explore the mechanisms underlying drug-drug synergies. Drug combination therapy, and synergistic combinations in particular, have several advantages over monotherapies. Synergistic drug combinations allow the dose of each agent to be reduced, often with the benefit of diminishing side effects while maintaining efficacy and decreasing the chances of drug resistance. I used my yeast model to identify synergistic drug combinations and found that inhibitors of ergosterol biosynthesis are highly synergistic with several agents, including those targeting other points within the same pathway. I also devised a method that enriches for synergistic interactions during screening of compound combinations. This new synergy prediction method can aid in the rapid identification of anti-proliferative combinations and can be readily applied to other organisms for further characterization and/or confirmation. Finally, I examined synergistic combination HIPHOP profiles and identified Gene Ontology enrichments that are combination-specific.

The genome-wide HaploInsufficieny Profiling (HIPHOP) technique has been validated as a method to quantify the relative abundance of uniquely tagged yeast deletion strains using a microarray readout. The massive throughput of next generation sequencing presents a new technology for assessing HIPHOP profiles. I developed a new method called Barcode analysis by Sequencing (Bar-seq) that applies deep sequencing to genome-scale fitness. I show that Bar-seq outperforms the current benchmark barcode microarray assay in terms of both dynamic range and throughput. When applied to a complex genome-scale fitness assay, Bar-seq quantitatively identifies drug-targets, exceeding the performance of the microarray assay. I also established that Bar-seq is well suited to a multiplex format and provides a dramatic increase in throughput. I used the genome-wide HIPHOP assay and other functional genomics tools to explore the mechanisms underlying drug-drug synergies. Drug combination therapy, and synergistic combinations in particular, have several advantages over monotherapies. Synergistic drug combinations allow the dose of each agent to be reduced, often with the benefit of diminishing side effects while maintaining efficacy and decreasing the chances of drug resistance. I used my yeast model to identify synergistic drug combinations and found that inhibitors of ergosterol biosynthesis are highly synergistic with several agents, including those targeting other points within the same pathway. I also devised a method that enriches for synergistic interactions during screening of compound combinations. This new synergy prediction method can aid in the rapid identification of anti-proliferative combinations and can be readily applied to other organisms for further characterization and/or confirmation. Finally, I examined synergistic combination HIPHOP profiles and identified Gene Ontology enrichments that are combination-specific.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 133-142).

Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency. / Chemistry and Chemical Biology

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain brings promise for the treatment of neurological diseases and has yielded new insight into brain cancer. The complete repertoire of signaling pathways that governs these cells however remains largely uncharacterized. This thesis describes how chemical genetic approaches can be used to probe and better define the operational circuitry of the NSC. I describe the development of a small molecule chemical genetic screen of NSCs that uncovered an unappreciated precursor role of a number of neurotransmitter pathways commonly thought to operate primarily in the mature central nervous system (CNS). Given the similarities between stem cells and cancer, I then translated this knowledge to demonstrate that these neurotransmitter regulatory effects are also conserved within cultures of cancer stem cells. I then provide experimental and epidemiologically support for this hypothesis and suggest that neurotransmitter signals may also regulate the expansion of precursor cells that drive tumor growth in the brain. Specifically, I first evaluate the effects of neurochemicals in mouse models of brain tumors. I then outline a retrospective meta-analysis of brain tumor incidence rates in psychiatric patients presumed to be chronically taking neuromodulators similar to those identified in the initial screen. Lastly, by further exploring the phenotype and function of neurotransmitter pathways in purified populations of human NSCs, I determined that neurotransmitter pathway gene expression exists in a functionally heterogeneous phase-varying state that restricts the responsiveness of these populations to various stimuli. Taken together, this research provides novel insights into the phenotypic and functional landscape of neurotransmitter pathways in both normal and cancer-derived NSCs. In additional to a better fundamental understanding of NSC biology, these results suggest how clinically approved neuromodulators can be used to remodel the mature CNS and find application in the treatment of brain cancer.