1,721,350 research outputs found
Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively
Full text linkHypoxia plays a critical role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated the by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybridscreen, is an asparaginyl hydroxylase that negatively regulates transcriptionalactivation by HIF. This study aimed to define mechanisms that govern transitions of FIH between nucleus and the cytoplasm. We report that FIH accumulates in thenucleus within a short time window upon hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by “coupling” with HIF1α for importin β1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Mechanistic studies on 2-oxoglutarate dependent oxygenases
Full text linkThe first identfied 2-oxoglutarate (2OG) dependent oxygenase was a collagen modifying enzyme in the work by Hutton et al. in 1967. Subsequent work has revealed that 2OG dependent oxygenases are a large family with diverse biological roles. With small molecule substrates, these enzymes catalyse a wide range of oxidative reactions, including those that form part of antibiotic biosynthetic pathways. The currently accepted consensus mechanism for catalysis by 2OG-dependent oxygenases is based on crystallographic data, kinetics and on quantum chemical calculations. The consensus mechanism involves oxidative decarboxylation of 2OG by reaction with an oxygen molecule producing CO2, succinate and a reactive oxidising species that reacts with the `prime' substrate. Deacetoxycephalosporin C synthase (DAOCS) is a 2OG-dependent oxygenase involved in cephalosporin biosynthesis. The mechanism of DAOCS is of particular interest because it has recently been proposed to be different from the consensus mechanism. The new mechanism proposal from Valeg ard et al. is primarily based on high-resolution crystallographic data with support from steady-state kinetic experiments and quantum-chemical calculations. The work in discussed in this thesis aimed to test the proposal of Valegård et al. by using a combination of spectroscopic and spectrometric methods analysing enzyme-substrate interactions. Substrate binding was investigated using both protein-observe (Chapter 3) and ligand-observe (Chapter 4.1 and 4.2) methods. Preliminary UV-visible data on enzyme-substrates complex formation was also obtained. The strength of substrate and cosubstrate binding was characterised through dissociation constant measurement. An activity assay (Chapter 2) that allows for direct and simultaneous monitoring of 2OG decarboxylation and penicillin ring expansion was optimised. Both the ligand-observe and protein-observe binding experiments as well as the preliminary UV-visible data indicate that the formation of a ternary complex between DAOCS, 2OG and the penicillin substrate is viable. The activity assay conclusively showed that in the presence of unnatural substrates, such as penicillin G, 2OG oxidation is significantly uncoupled from penicillin oxidation. Uncoupled turnover does not occur in the presence of the natural substrate, penicillin N, which is an aspect that should be considered in the analysis of the steady-state kinetic data. Overall, the results provide evidence that, the consensus mechanism for 2OG-dependent oxygenases is viable for DAOCS, at least in the presence of the natural substrate, penicillin N. It is possible that in the presence of an unnatural substrate, the catalytic process undergoes a more complex mechanism, possibly with the direct involvement of reducing agents in the system
Structural and kinetic studies on beta-lactamase mechanism and inhibition
Full text linkBeta-lactamases constitute one of the most prevalent identified mechanisms of bacterial resistance to the beta-lactam antibiotics. These enzymes are broadly divided into two mechanistic subclasses, the serine-beta-lactamases and the metallo-beta-lactamases. The metallo-beta-lactamases constitute an important subclass of beta-lactamases on account of their ability to hydrolyse almost all classes of beta-lactam, in particular the carbapenems, which are considered to be beta-lactams of last resort. At present inhibitors of the serine-beta-lactamases are clinically available for the treatment of resistant bacterial infections; these include clavulanic acid and related compounds, and more recently avibactam. However, there are currently no clinically validated inhibitors of the metallo-beta-lactamases.
Structural and mechanistic study of the beta-lactamases will aid in the development of novel inhibitor scaffolds with clinical potential in the treatment of resistant bacterial infections. In this work, the ability of the metallo-beta-lactamases to promiscuously bind and employ transition metals, other than their native zinc, in catalysis is described in the context of ferrous iron. The effects of metal substitution on enzyme structure, mechanism and susceptibility to inhibitors are investigated. These studies demonstrate that the metallo-beta-lactamases are able to employ iron, a transition metal of relatively high bioavailability, in catalysis with only small changes in catalytic efficiency.
In an increasing number of cases, bacteria are found to exhibit resistance to beta-lactams mediated by both serine- and metallo-beta-lactamases, simultaneously. With this likely to be an important issue in the future, the utility of developing molecules with the capability to inhibit both serine- and metallo-beta-lactamases cannot be understated. In the work described herein, cyclic boronic acids were explored as a chemotype with inhibitory activity against the two mechanistic classes of beta-lactamase and are shown to be potent inhibitors of both. Structural characterisation of the cyclic boronic acids in complex with beta-lactamases reveals that their inhibitory activity likely derives from their mimicking of a tetrahedral anionic intermediate common to both mechanistic classes of beta-lactamase. The activity of these molecules against clinically derived resistant bacterial strains is also explored.
While the metallo-beta-lactamases are largely studied on account of their involvement in antibiotic resistance, the metallo-beta-lactamase protein fold can facilitate a diverse range of chemistry from small molecule hydrolysis, to DNA and RNA processing, to oxidoreductase reactions. Moreover, a number of human enzymes share the metallo-beta-lactamase fold. Thus the study of these human enzymes and development of activity assays for their functions will likely prove useful in the discovery of bacterial metallo-beta-lactamase inhibitors with low toxicity in human patients. In this thesis, efforts made in the development of a novel high throughput assay for the unusual metallo-beta-lactamase persulfide dioxygenase ETHE1 via the fluorescent detection of sulfite are described.
Overall, the work of this thesis explores the mechanism of beta-lactamases as a framework for the discovery of novel beta-lactamase inhibitors, and makes efforts in the development of novel assays for human metallo-beta-lactamase fold enzymes to expedite the development of such molecules.</p
A potent and selective inhibitor of a histone demethylase
Full text linkPost-translational modifications (PTMs) to histone proteins play important roles in the regulation of gene expression. Histone lysine demethylases (KDMs) remove methyl groups from Nϵ-amino methylated lysine residues. There are two classes of human KDMs; the flavin adenosine dinucleotide-dependent demethylases (the KDM1 subfamily) and the 2-oxoglutarate- (2OG) dependent Jumonji C (JmjC) demethylases (KDM2â7). Misregulation and mutation of histone demethylases are associated with multiple human diseases including cancer. Hence potent and selective inhibitors are of interest for investigations into the biological roles of KDMs and their relevance as targets for drug discovery. JmjC KDM inhibitors have been reported in the literature but there are few subfamily selective examples. The aim of the work described in this thesis was to design and synthesise potent and subfamily selective inhibitors of human JmjC KDMs for use as chemical probes.
An aminomethyl pyridine-4-carboxylate series that was derived from a high throughput screening hit delivered a selective, submicromolar KDM2A inhibitor. X-ray crystallographic analyses demonstrated that the aminomethyl pyridine moiety bound the active site iron revealing a novel JmjC KDM inhibitor scaffold.
A known 2,2'-bipyridine-4-carboxylate scaffold was used as a starting point for the identification of a second, novel JmjC KDM inhibitor series that contains a triazolopyridine moiety as a replacement for the bipyridine system. The triazolopyridine core was shown to bind in the 2OG binding site of KDM4A by X-ray crystallography. Optimisation of the triazole substituent gave a selective inhibitor of KDM2A/7B that is significantly more potent (KDM2A IC50: 58 nM, KDM7B IC50: 150 nM) than reported KDM2/7 subfamily selective inhibitors. As for many other JmjC KDM inhibitors, this compound was not found to be efficacious in a cellular assay. A variety of strategies were pursued with the aim of improving the cellular efficacy. However, only a modest effect was observed in the cellular assay (less than 25% inhibition at 100 μM). A biotinylated analogue was immobilised onto streptavidin-coated beads for chemoproteomics experiments and was found to interact with KDM2A in cell lysate.
Overall, this work resulted in the identification of two new scaffolds for JmjC KDM inhibition and in a potent and selective inhibitor of KDM2A/7B.</p
Structural, functional and evolutionary studies on prolyl-hydroxylases
Full text linkThis thesis studies the prolyl-hydroxylase family of 2-oxoglutarate dependent oxygenases from structural, functional and evolutionary perspectives. The role of prolyl-hydroxylation was first identified in collagen, wherein hydroxyproline was found to stabilise the collagen triple helix. In the 1960s, the presence of hydroxyproline in collagen was found to be a result of enzyme catalysed protein modification. An enzyme, now known as collagen prolyl-4-hydroxylase (CP4H), was found to be completely dependent on Fe(II), 2-oxoglutarate (2OG) and molecular oxygen for catalysis, and was the inaugural member of enzyme family known as the Fe(II) and 2OG-dependent oxygenases (2OG oxygenases), the members of which have since expanded dramatically – more than 60 2OG oxygenases are predicted to exist in humans alone. It was not until the 21st century that hydroxyproline was found to play roles in human biology beyond its well-characterised role in collagen stabilisation. In animals, cells adapt to low oxygen conditions (hypoxia) via the upregulation of hundreds of target genes as governed by the hypoxia-inducible transcription factor (HIF). The mammalian hypoxic sensing system was discovered to be regulated by a conserved family of hypoxia-inducible factor prolyl-hydroxylases (PHDs or EGLNs), which catalyse the prolyl-4-hydroxylation of a conserved proline residue in HIF-α under normoxic conditions, so targeting HIF-α for proteasomal degradation via the von Hippel-Lindau (pVHL) E3 ubiquitin ligase pathway. As a result, the PHDs are current therapeutic targets for the treatment of anemia and ischemia-related diseases. Thus, hydroxyproline also plays a critical role in mammalian oxygen sensing. However, the discovery also raised the question of the evolutionary origin of these enzymes and what roles, if any, they may play in other organisms. This thesis begins by describing the identification and biochemical characterisation of the first homologue of the human PHDs in prokaryotes, specifically, in Pseudomonas species, which contains pathogens such as P. aeruginosa. Pseudomonas PHD (PPHD) was discovered to catalyse the prolyl-hydroxylation of a conserved region of elongation factor Tu (EF-Tu), a translational GTPase universally conserved in prokaryotes and known for its critical role in bacterial translation. A crystal structure of PPHD, the first of a prokaryotic prolyl-hydroxylase, was then determined, revealing a striking structural homology of PPHD to the human PHDs. The further determination of crystal structures of Pseudomonas EF-Tu and a PPHD:EF-Tu protein-protein complex, the first of any 2OG oxygenase in complex with its full-length protein substrate, provides important insights into the substrate recognition mechanisms of both the CP4Hs and the PHDs and reveals an evolutionarily conserved pathway of substrate recognition that extends to prokaryotes and will be useful in the design of selective inhibitors of the PHDs. Differences were investigated between the PHDs and a recently discovered subfamily of eukaryotic prolyl-3-hydroxylases, which catalyse the hydroxylation of a conserved proline residue in the small ribosomal subunit S23 (RPS23) and have been implicated in translation accuracy and the stress response. Crystal structures of the RPS23 hydroxylases human OGFOD1 and yeast Tpa1 in complex with 2OG-mimetic inhibitors provide insight into their evolutionary origins. Analyses of the structures will be useful for targeting either OGFOD1 or the PHDs for human therapy. The thesis then describes work on human CP4H, a 240 kDa α2β2 heterotetramer. A novel expression and purification protocol is described for the CP4H complex in addition to the first known reports of its crystallisation and diffraction. Further, the foundations of a high-throughput inhibition assay of the human CP4Hs is presented and will be of immediate interest for assaying inhibitors of the human PHDs in clinical trials, some of which are also predicted to inhibit the CP4Hs. In closing, the thesis attempts to synthesise the results presented in order to provide further insight into the question of the ancestral origins of the prolyl-hydroxylases, a family of enzymes whose range of functions and biological roles likely will continue to expand
Mechanistic and inhibition studies on gamma-butyrobetaine hydroxylase
Full text linkCarnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or 19F NMR, respectively. Furthermore, the use of 19F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols
Studies on an N-terminal nucleophile hydrolase and enzymes of clavulanic acid biosynthesis
No full text(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure
Chemical and biological studies on human oxygenases
Full text linkAs depicted in Chapter I, 2-oxoglutarate- (2OG) dependent oxygenases are ubiquitous in living systems and display a wide range of cellular functions, spanning metabolism, transcription, and translation. Although functionally diverse, the 2OG oxygenases share a high degree of structural similarities between their catalytic sites. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity presents a rather challenging task for the development of selective small molecules for functional studies in vivo. The non-selective metal chelator 8-hydroxyquinoline (8HQ) was used as a template for the generation of tool compound I for the KDM4 subfamily of histone demethylases via application of the Betti reaction. Structural analogue II was used as the corresponding negative control (Figure A). These compounds were characterised in vitro against a range of 2OG oxygenases and subsequently used for studies in cells. I displays selectivity for KDM4 and increases the level of the H3K9me3 histone mark in cells. It has an effect on the post-translational modification pattern of histone H3, but not other histones, and reduces the viability of lung cancer cells, but not normal lung cells, derived from the same patient. I also stabilises hypoxia-inducable factor HIF in cells via a mechanism which seems to be independent from prolyl hydroxylase inhibition. This work is described in Chapters II and III. The chemical biology research in epigenetics is complemented by qualitative analysis conducted in the social sciences at Said Business School. With a global view on how innovation occurs and may actively be fostered, Chapter IV focuses on the potential of epigenetics in drug discovery and how this process may actively be promoted within the framework of open innovation. Areas of focus include considerations of incremental and disruptive technology; how to claim, demarcate, and control the market; how knowledge brokering occurs; and insights about process, management, organisation, and culture of open innovation. In contrast to the open-skies approach adopted for the development of a tool compound in Chapters II and III, a focused-library approach was taken for the generation of a tool compound for the OGFOD1 ribosomal prolyl hydroxylase. The development of a suitable in vitro activity assay for OGFOD1 in Chapter V enabled the development of lead compound III in Chapter VI. III is selective for OGFOD1 against the structurally closely related prolyl hydroxylase PHD2
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