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.
Trypanosoma brucei; quantitative
proteomics; peroxisome; glycosome
•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.
GPI; Trypanosoma brucei; Hydroxamic acid; Inhibitor; N-Deacetylase
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.
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.
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.
phosphorylation; SILAC; Trypanosoma brucei; quantitative proteomics; phosphoproteomics
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.
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 22.214.171.124) 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.
Glycosylphosphatidylinositol (GPI) biosynthesis; Zinc metalloenzyme inhibitor; Zinc-binding group; Branched monosaccharides, Phosphatidylinositol de-N-acetylase
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.
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.
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.
carbohydrates; glycosylphosphatidylinositol; lipid; mechanism-based drug design; metalloenzymes; 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.
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
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.
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.
short-chain dehydrogenase/reductases; Trypanosoma brucei; UDP-galactose-4′-epimerase; UDP-4-deoxy-4-fluoro-α-d-galactose
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.
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
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.