Recent Results
Update: 19.12.2019

2019 - 2020


Wang, T., Ma, G., Ang, C.-S., Korhonen, P.K., Ströhlein, A., Young, N.D., Hofmanna, A., Chang, B.C.H., Williamson, N.A., Gasser, R.B. (2020) J. Proteomics 213, 103615
Abstract
Protein phosphorylation plays essential roles in many cellular processes. Despite recent progress in the genomics, transcriptomics and proteomics of socioeconomically important parasitic nematodes, there is scant phosphoproteomic data to underpin molecular biological discovery. Here, using the phosphopeptide enrichment-based LC-MS/MS and data-independent acquisition (DIA) quantitation, we characterised the first developmental phosphoproteome of the parasitic nematode Haemonchus contortus — one of the most pathogenic parasites of ruminant livestock. Totally, 1804 phosphorylated proteins with 4406 phosphorylation sites (‘phosphosites’) from different developmental stages/sexes were identified. Bioinformatic analyses of quantified ‘phosphosites’ exhibited distinctive stage- and sex-specific patterns during development, and identified a subset of phosphoproteins proposed to play crucial roles in processes such as spindle positioning, signal transduction and kinase activity. A sequence-based comparison of the phosphoproteome of H. contortus with those of two free-living nematode species (Caenorhabditis elegans and Pristionchus pacificus) suggested a limited number of common protein phosphorylation events among these species. Our findings infer active roles for protein phosphorylation in the adaptation of a parasitic nematode to a constantly changing external environment. The phosphoproteomic data set for H. contortus provides a basis to better understand phosphorylation and associated biological processes (e.g., regulation of signal transduction), and might enable the discovery of novel anthelmintic targets.
PubMed | DOI

Rostami, A., Ma, G., Wang, T., Koehler, A.V., Hofmann, A., Chang, B.C.H., Macpherson, C.N., Hotez, P.J., Gasser, R.B. (2019) Infect. Genet. Evol. 74, 104002
Abstract
Toxocariasis, a disease caused by infection with larvae of Toxocara canis, T. cati and/or congeners, represents clinical syndromes in humans including visceral and ocular larva migrans, neurotoxocariasis and covert/common toxocariasis. It is reported to be one of the most widespread public health and economically important zoonotic parasitic infections that humans share with dogs, wild canids, including foxes, and possibly other mammals. Humans become infected by accidental ingestion of embryonated Toxocara eggs or larvae from tissues from domestic or wild paratenic hosts. Most infections are asymptomatic, and human disease may go unnoticed, as clinical investigation is often not pursued and/or diagnostic testing not conducted. Sometimes toxocariasis can be associated with complications, such as allergic and/or neurological disorders, possibly including cognitive or developmental delays in children. There is no anti-toxocariasis vaccine, and chemotherapy in humans varies, depending on symptoms and location of larvae, and may include the administration of albendazole or mebendazole, together with anti-inflammatory corticosteroids. Some recent studies indicate that toxocariasis is having an increased, adverse impact on human health in some, particularly underprivileged, tropical and subtropical communities around the world. Although tens of millions of people, especially children, are expected to be exposed to, or infected by, Toxocara species, there is limited precise epidemiological data or information on the relationship between seropositivity and disease (toxocariasis) on a global scale. To gain an improved insight into this area, the present article reviews salient clinical aspects of human toxocariasis and the epidemiology of this disease, with particular reference to seroprevalence, and discusses future research and approaches/measures to understand and prevent/control this socioeconomically important, yet neglected zoonosis.
PubMed | DOI

Dilrukshi Herath, H.M.P., Preston, S., Jabbar, A., Garcia-Bustos, J., Taki, A.C., Addison, R.S., Hayes, S., Beattie, K.D., McGee, S.L., Martin, S.D., Ekins, M.G., Hooper, J.N.A., Chang, B.C.H., Hofmann, A., Davis, R.A., Gasser, R.B. (2019) Mar. Drugs 17, 598
Abstract
There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a resource with novel chemical entities. This study investigated 2,000 extracts from marine invertebrates collected from Australian waters for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus — a socioeconomically important parasitic nematode of livestock animals. In total, three extracts (Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4 development). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from M. unguiculata, the previously reported pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (IC50 = 26.6 ± 0.74 μM) and motility (IC50 = 39.4 ± 4.83 μM), although it had relatively low potency at inhibiting motility of xL3s (IC50 ≥ 100 μM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, the known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.
PubMed | DOI

Leow, C.Y., Willis, C., Leow, C.H., Hofmann, A., Jones, M.K. (2019) Mol. Biochem. Parasitol. 234, 111231
Abstract
Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently exists licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterisation of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin gene protein possesseses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalised to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognised by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular roles characterisation of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.
PubMed | DOI

De Ridder, W., Azmi, A., Clemen, C.S., Eichinger, L., Hofmann, A., Schröder, R., Johnson, K., Töpf, A., Straub, V., De Jonghe, P.,Maudsley, S., De Bleecker, J.L., Baets, J. (2019) Neurology, in press
Abstract
Objective
To assess the clinical, radiological, myopathological, and proteomic findings in the first patient suffering from a multisystem proteinopathy due to a homozygous p.Arg159His VCP mutation.
Methods
We studied a currently 36-year-old male index patient and his father both presenting with progressive limb-girdle weakness. Muscle involvement was assessed by MRI and muscle biopsies. We performed whole-exome sequencing and Sanger sequencing for segregation analysis of the identified p.Arg159His VCP mutation. To dissect biological disease signatures we applied state-of-the-art quantitative proteomics on muscle tissue of the index case, his father, three additional VCP-related myopathy patients and three control individuals.
Results
The index patient, homozygous for the known p.Arg159His mutation in VCP, manifested with a typical VCP-related myopathy phenotype, although with a markedly high creatine kinase value and a relatively early disease onset, and Paget disease of bone. The father exhibited a myopathy phenotype and discrete parkinsonism and multiple deceased family members on the maternal side of the pedigree displayed either a dementia, parkinsonism or myopathy phenotype. Bioinformatic interpretation of advanced quantitative proteomic data revealed the degenerative nature of the disease, with evidence suggesting selective failure of muscle regeneration and stress granule dyshomeostasis.
Conclusions
This is the first report of a patient showing a multisystem proteinopathy due to a homozygous VCP mutation. The severity of the phenotype is on the severe side of the spectrum, yet fundamental disease characteristics are preserved. Proteomic findings provide further insights in VCP-related pathomechanisms.
PubMed | DOI

Cross, M., York, M., Długosz, E., Straub, J.H., Biberacher, S., Dilrukshi Herath, H.M.P., Logan, S.A., Kim, J.-S., Gasser, R.B., Ryan, J.H., Hofmann, A. (2019) Sci. Rep. 9, 16165
Abstract
Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 μM and 0.56 μM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber’s pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 μM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.
PubMed | DOI

Mishra, A., Cross, M., Hofmann, A., Coster, M.J., Karim, A., Sattar, A. (2019) J. Comput. Biol. 26, 1470-1486
Abstract
Dipeptidyl peptidase–4 (DPP-4) is considered a major drug target for type 2 diabetes mellitus (T2DM). In addition to T2DM, a regulatory role of DPP-4 was also found in cardiovascular diseases. Existing DPP-4 inhibitors have been reported to have several adverse effects. Here, a computer-aided drug design approach and its use to detect a novel class of inhibitor for DPP-4 is reported. Through structure and pharmacophore-based screening we identified 13 hit compounds from a ∼4-million-compound library. Physical interactions of these hits with DPP-4 were studied using docking and explicit solvent molecular dynamics simulations. Later, MMPBSA binding energy was calculated for the ligand-protein simulation trajectories to determine the stability of compounds in the binding cavity. These compounds have a novel scaffold and exhibited a stable binding mode. ’Best-in-screen’ compounds (or their closest available analogues) were resourced and their inhibition of DPP-4 activity was experimentally validated using an in vitro enzyme activity assay in the presence of 100 μM and 10 μM compound. These assays identified a compound with a spirochromanone centre with 53% inhibition activity at 100 μM concentration. A further five spirochromanone compounds were synthesized and examined in silico and in vitro; again, one compound showed 53% inhibitory activity action at 100 μM. Overall, this study identified two novel ’spirochromanone’ compounds that lowered DPP-4 activity by more than ∼50% at 100 μM. This study also showed the impact of fast in silico drug design techniques utilizing virtual screening and molecular dynamics to identify novel scaffolds to bind and inhibit DPP-4. Spirochromanone motif identified here may be used to design molecules to achieve drug-like inhibitory action against DPP-4.
PubMed | DOI

Ebner, F., Balster, K., Janek, K., Niewienda, A., Malecki, P.H., Weiss, M.S., Sutherland, T.E., Heuser, A., Kühl, A.A., Zentek, J., Hofmann, A., Hartmann, S. (2019) bioRxive Preprint
Abstract
Previously, we reported significant immunomodulatory effects of the entire excretory-secretory (ES) proteins of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis in a rodent model of allergic hyperreactivity. In the present study, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and thus studied selected components for their immunomodulatory efficacy in an OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed significant similarities to mouse acidic mammalian chitinase (AMCase). In addition, the unique ability of T. suis chitinase to form dimers, as well as acidic surface patches within the dimerization region may contribute to the formation of cross-reactive antibodies to the mouse homologs. This hypothesis is supported by the observation that T. suis chitinase treatment induced cross-reactive antibodies to mouse AMCase and chitinase-like protein BRP-39 in the AHR model. In conclusion, a biologically active T. suis chitinase exhibits immunomodulatory properties despite its structural similarity to the mammalian counterpart.
DOI

Hofmann, A., Preston, S., Cross, M., Dilrukshi Herath, H.M.P., Simon, A., Gasser, R.B. (2019) BMC Bioinformatics 20, 262
Abstract
Background: Analyses of replicates in sets of discrete data, typically acquired in multi-well plate formats, is a recurring task in many contemporary areas in the Life Sciences. The availability of accessible cross-platform data analysis tools for such fundamental tasks in varied projects and environments is an important prerequisite to ensuring a reliable and timely turnaround as well as to provide practical analytical tools for student training.
Results: We have developed an easy-to-use, interactive software tool for the analysis of multiple data sets comprising replicates of discrete bivariate data points. For each dataset, the software identifies the replicate data points from a defined matrix layout and calculates their means and standard errors. The averaged values are then automatically fitted using either a linear or a logistic dose response function.
Conclusions: DRfit is a practical and convenient tool for the analysis of one or multiple sets of discrete data points acquired as replicates from multi-well plate assays. The design of the graphical user interface and the built-in analysis features make it a flexible and useful tool for a wide range of different assays.
PubMed | DOI

Hofmann, A., Coster, M.J., Taylor, P. (2019) J. Chem. Educ. 96, 1262-1267
Abstract
The interactive generation of chemical structure diagrams is an integral activity in the study of chemistry as well as in professional chemistry environments. For educational purposes, in particular, the existence of suitable software tools free of charge is of great importance. Albeit a number of free chemical drawing applications are currently available, there are often limitations as to the included basic drawing tools, ease of handling and installation, or time limits of free access. The Java desktop application cDraw is an interactive chemical drawing software for generation of publication-quality figures. The user interface has been designed with a clear focus on intuitive and convenient handling, and currently is offered in nine languages. The software allows drawing of 2D chemical structure diagrams using a basic set of built-in templates and bond generation mechanisms. Drawings can be exported as EPS, PDF, PNG, SVG, and TIFF images or copied through the clipboard. cDraw sessions can be stored in the XML-based CDML file format; additionally, there are basic import/export capabilities for the popular CDXML, CML, and SDF file formats. Molecules can be inserted using line notation (InChI, MCDL, and SMILES) and molecular meta information such as IUPAC name and Chemical Abstract Service number can be obtained from drawings via online NIH CACTUS queries. Due to multilanguage support and an intuitive user interface, the software should especially appeal to students in secondary and tertiary education.
DOI

Pedro, L., Cross, M., Hofmann, A., Mak, T., Quinn, R.J. (2019) Anal. Biochem. 575, 63-69
Abstract
The development of a high-performance liquid chromatography (HPLC)-based method for guanosine monophosphate kinase activity assays is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalysed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvivGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z’ factor of 0.6. For the best inhibitor, an IC50 curve was generated.
PubMed | DOI

Wibowo, M., Forster, P.I., Guymer, G.P., Hofmann, A., Davis, R.A. (2019) Molecules 24, 859
Abstract
An analytical method using UHPLC-MS was developed and applied to 16 crude CH2Cl2 extracts from Australian Celastraceae plants; the endemic plant materials were accessed from Griffith University's NatureBank resource and included bark, fruit, leaf, roots, twigs and mixed samples all of which were collected from Queensland, Australia. The generated UHPLC-MS data were analysed and dereplicated using the scientific databases Dictionary of Natural Products and SciFinder Scholar in order to potentially identify new dihydro-β-agarofurans from Celastraceae plants. These investigations led to the large-scale extraction and isolation work on a prioritised fruit sample that belonged to the rainforest plant, Denhamia celastroides. Chemical investigations resulted in the purification of four new natural products, denhaminols O-R (1-4), along with the related and known compound, denhaminol G (5). The structures of all the new compounds were determined via detailed analysis of NMR and MS data.
PubMed | DOI

Le, T.G., Kundu, A., Ghoshal, A., Nguyen, N.H., Preston, S., Jiao, Y., Ruan, B., Xue, L., Fei, H., Keiser, J., Hofmann, A., Chang, B.C.H., Garcia-Bustos, J., Wells, T.N.C., Palmer, M.J., Jabbar, A., Gasser, R.B., Baell, J.B. (2019) J. Med. Chem. 62, 1036-1053
Abstract
Recently, we discovered that the registered pesticide, tolfenpyrad (TFP), unexpectedly and potently inhibits the development of L4 larval stages of the parasitic nematode Haemonchus contortus with an IC50 value of 0.03 μM while displaying good selectivity, with an IC50 of 37.9 μM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure-activity relationships (SAR) for this chemical. Modifications of the left hand side (LHS), right hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29 and 33 were shown to be the most potent compounds of the series, with IC50 values at a sub-nanomolar levels of potency against the chemotherapeutically-relevant fourth larval (L4) stages of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes such as hookworms and whipworms.
PubMed | DOI

Ma, G., Wang, T., Korhonen, P.K., Nie, S., Reid, G.E., Ströhlein, A.J., Koehler, A.V., Chang, B.C.H., Hofmann, A., Young, N.D., Gasser, R.B. (2019) Parasites & Vectors 12, 32
Abstract
Background: Toxocara canis is quite closely related to Ascaris suum but its biology is more complex, involving a phase of arrested development (diapause or hypobiosis) in tissues as well as transplacental and transmammary transmission routes. In the present study, we explored and compared dauer-like signalling pathways of T. canis and A. suum to infer which components in these pathways might associate with, or regulate, this added complexity in T. canis.
Methods: Guided by information for Caenorhabditis elegans, we bioinformatically inferred and compared components of dauer-like signalling pathways in T. canis and A. suum using genomic and transcriptomic data sets. In these two ascaridoids, we also explored endogenous dafachronic acids (DAs), which are known to be critical in regulating larval developmental processes in C. elegans and other nematodes, by liquid chromatography-mass spectrometry (LC-MS).
Results: Orthologues of C. elegans dauer signalling genes were identified in T. canis (n = 55) and A. suum (n = 51), inferring the presence of a dauer-like signalling pathway in both species. Comparisons showed clear differences between C. elegans and these ascaridoids as well as between T. canis and A. suum, particularly in the transforming growth factor-β (TGF-β) and insulin-like signalling pathways. Specifically, in both A. suum and T. canis, there was a paucity of genes encoding SMAD transcription factor-related protein (daf-3, daf-5, daf-8 and daf-14) and insulin/insulin-like peptide (daf-28, ins-4, ins-6 and ins-7) homologues, suggesting an evolution and adaptation of the signalling pathway in these parasites. In T. canis, there were more orthologues coding for homologues of antagonist insulin-like peptides (Tc-ins-1 and Tc-ins-18), an insulin receptor substrate (Tc-ist-1) and a serine/threonine kinase (Tc-akt-1) than in A. suum, suggesting potentiated functional roles for these molecules in regulating larval diapause and reactivation. A relatively conserved machinery was proposed for DA synthesis in the two ascaridoids, and endogenous Δ4- and Δ7-DAs were detected in them by LC-MS analysis. Differential transcription analysis between T. canis and A. suum suggests that ins-17 and ins-18 homologues are specifically involved in regulating development and migration in T. canis larvae in host tissues.
Conclusion: The findings of this study provide a basis for functional explorations of insulin-like peptides, signalling hormones (i.e. DAs) and related nuclear receptors, proposed to link to development and/or parasite-host interactions in T. canis. Elucidating the functional roles of these molecules might contribute to the discovery of novel anthelmintic targets in ascaridoids.
PubMed | DOI

Jiao,Y.,Preston, S., Garcia-Bustos, J.F., Baell, J.B., Ventura, S.,Le, T., McNamara, N., Nguyen, N., Botteon, A., Ellis, S., Danne, J., Skinner, C., Koehler, A.V., Wang, T., Chang, B.C.H., Hofmann, A., Jabbar, A., Gasser, R.B. (2019) Int. J. Parasitol. Drugs Drug Resist. 9, 59-71
Abstract
In the present study, the anthelmintic activity of a human tyrosine kinase inhibitor, AG-1295, and 14 related tetrahydroquinoxaline analogues against Haemonchus contortus was explored. These compounds were screened against parasitic larvae — exsheathed third-stage (xL3) and fourth-stage (L4) — using a whole-organism screening assay. All compounds were shown to have inhibitory effects on larval motility, development and growth, and induced evisceration through the excretory pore in xL3s. The estimated IC50 values ranged from 3.5 to 52.0 μM for inhibition of larval motility or development. Cytotoxicity IC50 against human MCF10A cells was generally higher than 50 μM. Microscopic studies revealed that this eviscerated (Evi) phenotype occurs rapidly (< 20 min) and relates to a protrusion of internal tissues and organs (evisceration) through the excretory pore in xL3s; severe pathological damage in L4s as well as a suppression of larval growth in both stages were also observed. Using a relatively low concentration (12.5 μM) of compound m10, it was established that the inhibitor has to be present for a relatively short time (between 30 h and 42 h) during in vitro development from xL3 to L4, to induce the Evi phenotype. Increasing external osmotic pressure prevented evisceration and moulting, and xL3s remained unaffected by the test compound. These results point to a mode of action involving a dysregulation of morphogenetic processes during a critical time-frame, in agreement with the expected behaviour of a tyrosine kinase inhibitor, and suggest potential for development of this compound class as nematocidal drugs.
PubMed | DOI