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1.  Molecular control of irreversible bistability during trypanosome developmental commitment 
The Journal of Cell Biology  2015;211(2):455-468.
Phosphoproteomic and functional analysis of the developmental progression of Trypanosomes demonstrates that this transition shows bistability, with commitment to differentiation requiring new protein synthesis, and that the protein kinase NRK is a key regulator.
The life cycle of Trypanosoma brucei involves developmental transitions that allow survival, proliferation, and transmission of these parasites. One of these, the differentiation of growth-arrested stumpy forms in the mammalian blood into insect-stage procyclic forms, can be induced synchronously in vitro with cis-aconitate. Here, we show that this transition is an irreversible bistable switch, and we map the point of commitment to differentiation after exposure to cis-aconitate. This irreversibility implies that positive feedback mechanisms operate to allow commitment (i.e., the establishment of “memory” of exposure to the differentiation signal). Using the reversible translational inhibitor cycloheximide, we show that this signal memory requires new protein synthesis. We further performed stable isotope labeling by amino acids in cell culture to analyze synchronized parasite populations, establishing the protein and phosphorylation profile of parasites pre- and postcommitment, thereby defining the “commitment proteome.” Functional interrogation of this data set identified Nek-related kinase as the first-discovered protein kinase controlling the initiation of differentiation to procyclic forms.
PMCID: PMC4621835  PMID: 26483558
2.  Genomic and Proteomic Studies on the Mode of Action of Oxaboroles against the African Trypanosome 
PLoS Neglected Tropical Diseases  2015;9(12):e0004299.
SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class.
Author Summary
The mode of action of a new class of boron-containing chemicals (the oxaboroles), currently under development for the treatment of human African trypanosomiasis, is unknown. Here we identify a number of potential candidate proteins that could be involved either in the mode of action of these compounds or in the mechanism of resistance. This information could prove critical in protecting the compounds against resistance emerging in the field as well as opening up new avenues for drug discovery.
PMCID: PMC4689576  PMID: 26684831
3.  The Dictyostelium prestalk inducer differentiation-inducing factor-1 (DIF-1) triggers unexpectedly complex global phosphorylation changes 
Molecular Biology of the Cell  2015;26(4):805-820.
DIF-1, a polyketide, has been primarily characterized as a transcriptional regulator, but proteome-wide identification of peptides that change phosphorylation level in DIF-treated cell reveals many novel targets. Thus a small-molecule regulator of gene transcription plays additional roles as a multifunctional regulator of protein modification.
Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1–controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca2+/calmodulin–dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555.
PMCID: PMC4325849  PMID: 25518940
4.  TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei 
Nucleic Acids Research  2014;43(Database issue):D637-D644.
The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (, which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.
PMCID: PMC4384016  PMID: 25300491
5.  High-Confidence Glycosome Proteome for Procyclic Form Trypanosoma brucei by Epitope-Tag Organelle Enrichment and SILAC Proteomics 
Journal of Proteome Research  2014;13(6):2796-2806.
The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.
PMCID: PMC4052807  PMID: 24792668
Trypanosoma brucei; quantitative proteomics; peroxisome; glycosome
6.  Fragment screening reveals salicylic hydroxamic acid as an inhibitor of Trypanosoma brucei GPI GlcNAc-PI de-N-acetylase 
Carbohydrate Research  2014;387(100):54-58.
Graphical abstract
•First non-substrate analogue inhibitor of the trypanosome GPI pathway.•Active against recombinant enzyme and cell-free system.•Low molecular weight and good ligand efficiency.
The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity relationship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tolerated at the 4- and 5-positions.
PMCID: PMC3991331  PMID: 24589444
GPI; Trypanosoma brucei; Hydroxamic acid; Inhibitor; N-Deacetylase
7.  Probing the substrate specificity of Trypanosoma brucei GlcNAc-PI de-N-acetylase with synthetic substrate analogues† †Electronic supplementary information (ESI) available: Additional experimental procedures and characterisation data for the β-anomers 8 and 10 plus 1H and 13C NMR spectra of all the compounds. See DOI: 10.1039/c3ob42164c Click here for additional data file. Click here for additional data file.  
Organic & Biomolecular Chemistry  2014;12(12):1919-1934.
A series of substrates analogues of GlcNAc-PI de-N-acetylase were tested as substrates and inhibitors of the Trypanosoma brucei enzyme.
A series of synthetic analogues of 1-d-(2-amino-2-deoxy-α-d-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the d-myo-inositol, d-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The d-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the β-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors.
PMCID: PMC4326964  PMID: 24519084
8.  A Novel Allosteric Inhibitor of the Uridine Diphosphate N-Acetylglucosamine Pyrophosphorylase from Trypanosoma brucei 
ACS Chemical Biology  2013;8(9):1981-1987.
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi–bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP–inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.
PMCID: PMC3780468  PMID: 23834437
9.  Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and Bloodstream Form Lifecycle Stages of Trypanosoma brucei 
Journal of Proteome Research  2013;12(5):2233-2244.
We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3–4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the T. brucei kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in T. brucei is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism.
PMCID: PMC3646404  PMID: 23485197
phosphorylation; SILAC; Trypanosoma brucei; quantitative proteomics; phosphoproteomics
10.  Comparative SILAC Proteomic Analysis of Trypanosoma brucei Bloodstream and Procyclic Lifecycle Stages 
PLoS ONE  2012;7(5):e36619.
The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.
PMCID: PMC3344917  PMID: 22574199
11.  Synthesis of potential metal-binding group compounds to examine the zinc dependency of the GPI de-N-acetylase metalloenzyme in Trypanosoma brucei 
Carbohydrate Research  2011;346(6):708-714.
Graphical abstract
A small zinc-binding group (ZBG) library of deoxy-2-C-branched-monosaccharides, for example, 1,5-anhydroglucitols, consisting of either monodentate ligand binding carboxylic acids or bidentate ligand binding hydroxamic acids, were prepared to assess the zinc affinity of the putative metalloenzyme 2-acetamido-2-deoxy-α-d-glucopyranosyl-(1→6)-phosphatidylinositol de-N-acetylase (EC of glycosylphosphatidylinositol biosynthesis. The N-ureido thioglucoside was also synthesised and added to the ZBG library because a previous N-ureido analogue, synthesised by us, had inhibitory activity against the aforementioned de-N-acetylase, presumably via the N-ureido motif.
PMCID: PMC3125106  PMID: 21377660
Glycosylphosphatidylinositol (GPI) biosynthesis; Zinc metalloenzyme inhibitor; Zinc-binding group; Branched monosaccharides, Phosphatidylinositol de-N-acetylase
12.  A Multidimensional Strategy to Detect Polypharmacological Targets in the Absence of Structural and Sequence Homology 
PLoS Computational Biology  2010;6(1):e1000648.
Conventional drug design embraces the “one gene, one drug, one disease” philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.
Author Summary
Proteins play a critical role in human disease; bacteria, viruses, and parasites have unique proteins that can interfere with human health, and dysfunctional human proteins can likewise lead to illness. In order to find cures, scientists often try to identify small molecules (drugs) that can inhibit disease-causing proteins. The goal is to identify a molecule that can fit snugly into the pockets and grooves, or “active sites,” on the protein's surface. Unfortunately, drugs that inhibit a single disease-causing protein are problematic. A single protein can evolve to evade drug action. Additionally, when only one protein is targeted, drug potency is often diminished. Single drugs that simultaneously target multiple disease-causing proteins are much more effective. On the other hand, if scientists are not careful, the drugs they design might inhibit essential human proteins in addition to inhibiting their intended targets, leading to unexpected side effects. In our current work, we have developed a computer-based procedure that can be used to identify proteins with similar active sites. Once unexpected protein targets have been identified, scientists can modify drugs under development in order to increase the simultaneous inhibition of multiple disease-causing proteins while avoiding potential side effects by decreasing the inhibition of useful human proteins.
PMCID: PMC2799658  PMID: 20098496
13.  Inhibitors Incorporating Zinc-Binding Groups Target the GlcNAc-PI de-N-acetylase in Trypanosoma brucei, the Causative Agent of African Sleeping Sickness 
Chemical Biology & Drug Design  2012;79(3):270-278.
Disruption of glycosylphosphatidylinositol biosynthesis is genetically and chemically validated as a drug target against the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. The N-acetylglucosamine-phosphatidylinositol de-N-acetylase (deNAc) is a zinc metalloenzyme responsible for the second step of glycosylphosphatidylinositol biosynthesis. We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups. Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme. Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC50 values 0.1–1.5 mm, and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC50 = 19 ± 0.5 μm.
PMCID: PMC3473218  PMID: 22222041
carbohydrates; glycosylphosphatidylinositol; lipid; mechanism-based drug design; metalloenzymes; Trypanosoma brucei
14.  Casein kinase 1 isoform 2 is essential for bloodstream form Trypanosoma brucei☆ 
Induction of RNA interference targeted against casein kinase 1 isoform 2 (TbCK1.2, Tb927.5.800) in bloodstream form Trypanosoma brucei in vitro results in rapid cessation of growth, gross morphological changes, multinucleation and ultimately cell death. A null mutant of the highly homologous casein kinase 1 isoform 1 (Tb927.5.790) in bloodstream form T. brucei displays no growth or morphological phenotype in vitro. A truncated form of TbCK1.2 expressed in Escherichia coli as a GST fusion produces catalytically active recombinant protein, facilitating screening for small molecule inhibitors. These data show that TbCK1.2 is an attractive target for anti-trypanosomal drug discovery.
PMCID: PMC2697324  PMID: 19450734
LmCK1.2, Leishmania major casein kinase 1 isoform 2; TbCK1.2, Trypanosoma brucei casein kinase 1 isoform 2; TbCK1.1, Trypanosoma brucei casein kinase 1 isoform 1; RNAi, RNA interference; GST, glutathione-S-transferase; PKs, protein kinases; T. brucei, Trypanosoma brucei; L. Major, Leishmania major; RT-PCR, reverse transcriptase-polymerase chain reaction; Trypanosoma brucei; Protein kinase; Genetic validation; RNA interference; Drug target
15.  Galactose Starvation in a Bloodstream Form Trypanosoma brucei UDP-Glucose 4′-Epimerase Conditional Null Mutant 
Eukaryotic Cell  2006;5(11):1906-1913.
Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4′-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal β-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4′-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.
PMCID: PMC1694802  PMID: 17093269
16.  Trypanosoma brucei UDP-galactose-4′-epimerase in ternary complex with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose 
The structure of recombinant T. brucei UDP-galactose-4′-epimerase cocrystallized with NAD+ and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose has been determined at medium resolution. Comparisons with structures of human and E. coli UDP-galactose-4′-epimerase–ligand complexes reveal that the hexose moieties are able to adopt different orientations in the active site.
The structure of the NAD-dependent oxidoreductase UDP-galactose-4′-epimerase from Trypanosoma brucei in complex with cofactor and the substrate analogue UDP-4-deoxy-4-fluoro-α-d-galactose has been determined using diffraction data to 2.7 Å resolution. Despite the high level of sequence and structure conservation between the trypanosomatid enzyme and those from humans, yeast and bacteria, the binding of the 4-fluoro-α-d-galactose moiety is distinct from previously reported structures. Of particular note is the observation that when bound to the T. brucei enzyme, the galactose moiety of this fluoro-derivative is rotated approximately 180° with respect to the orientation of the hexose component of UDP-glucose when in complex with the human enzyme. The architecture of the catalytic centre is designed to effectively bind different orientations of the hexose, a finding that is consistent with a mechanism that requires the sugar to maintain a degree of flexibility within the active site.
PMCID: PMC2242870  PMID: 16946458
short-chain dehydrogenase/reductases; Trypanosoma brucei; UDP-galactose-4′-epimerase; UDP-4-deoxy-4-fluoro-α-d-galactose
17.  Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness 
Journal of Medicinal Chemistry  2010;53(13):5025-5032.
Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4′-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 μM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization.
PMCID: PMC2895357  PMID: 20527952
18.  Chemical Proteomic Analysis Reveals the Drugability of the Kinome of Trypanosoma brucei 
ACS Chemical Biology  2012;7(11):1858-1865.
The protozoan parasite Trypanosoma brucei is the causative agent of African sleeping sickness, and there is an urgent unmet need for improved treatments. Parasite protein kinases are attractive drug targets, provided that the host and parasite kinomes are sufficiently divergent to allow specific inhibition to be achieved. Current drug discovery efforts are hampered by the fact that comprehensive assay panels for parasite targets have not yet been developed. Here, we employ a kinase-focused chemoproteomics strategy that enables the simultaneous profiling of kinase inhibitor potencies against more than 50 endogenously expressed T. brucei kinases in parasite cell extracts. The data reveal that T. brucei kinases are sensitive to typical kinase inhibitors with nanomolar potency and demonstrate the potential for the development of species-specific inhibitors.
PMCID: PMC3621575  PMID: 22908928
19.  Genetic and structural validation of Aspergillus fumigatus UDP-N-acetylglucosamine pyrophosphorylase as an antifungal target 
Molecular Microbiology  2013;89(3):479-493.
The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.
PMCID: PMC3888555  PMID: 23750903

Results 1-19 (19)