Recent Results
Update: 09.04.2019

2018 - 2019


Hofmann, A., Coster, M.J., Taylor, P. (2019) J. Chem. Educ., in press
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., Stroehlein, 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

Dilrukshi Herath, H.M.P., Preston, S., Jabbar, A., Garcia-Bustos, J.F., Addison, R.S., Hayes, S., Ralid, T., Wang, T., Koehler, A.V., Chang, B.C.H., Hofmann, A., Davis, R.A., Gasser, R.B. (2019) Int. J. Parasitol. Drugs Drug Resist. 9, 72-79
Abstract
Due to the widespread occurrence and spread of anthelmintic resistance, there is a need to develop new drugs against resistant parasitic nematodes of livestock animals. The Nobel Prize-winning discovery and development of the anti-parasitic drugs avermectin and artemisinin have inspired the exploration of components in natural products as anthelmintics. In the present study, we screened 7,500 plant extracts for in vitro-activity against the barber's pole worm, Haemonchus contortus, a highly significant pathogen of ruminants, and then characterised and defined the anthelmintic fractions and chemicals derived from two plants (Cryptocarya novoguineensis and Piper methysticum). Subsequently, chemicals with anthelemintic activity were purified from fractions by high-performance liquid chromatographic (HLPC) separation. Four α-pyrones (goniothalamin, dihydrokavain, desmethoxyyangonin and yangonin) were purified from fractions from the two plants, the styryl-pyrone goniothalamin from C. novoguineensis, and the kavalactones dihydrokavain, desmethoxyyangonin and yangonin from P. methysticum. The three kavalactones induced a lethal non-wild-type (protrusion) phenotype in treated exheathed L3s (xL3s), and desmethoxyyangonin and yangonin had moderate potencies (IC50 values of 31.7 ± 0.230 μM and 23.7 ± 2.05 μM, respectively) at inhibiting the development of fourth-stage larvae (L4s). Although goniothalamin had limited potency (200—300 μM) at inhibiting L4 development, it was the only compound that reduced L4 motility (IC50 of 6.25—12.5 μM). Each of the four chemicals purified here affected H. contortus in an irreversible manner. These findings suggest that structure-activity relationship studies of α-pyrones are worth pursuing to assess their potential as anthelmintics.
PubMed | DOI

Hofmann, A., Cross, M., Karow, M.A., Straub, J.H., Clemen, C.S., Eichinger, L. (2019) Biochem. Mol. Biol. Edu. 47, 207-210
Abstract
The Java software jBar consists of a graphical user interface that allows the user to customise and assemble an included script for R. Based on this, the scripted R pipeline calculates means and standard errors/deviations for replicates of numerical bivariate data and generates presentations in the form of bar graphs. A two-sided Student’s t-test is carried out against a user-selected reference and p-values are calculated. The user can enter the data conveniently through the built-in spreadsheet and configure the R pipeline in the graphical user interface. The configured R script is written into a file and then executed. Bar graphs can be generated as static PNG, PDF and SVG files or as interactive HTML widgets.
PubMed | DOI

Le, T., Kundu, A., Ghoshal, A., Nguyen, N., Preston, S., Jiao, Y., Ruan, B., Xue, L., Huang, F., Keiser, J., Hofmann, A., Chang, B., Garcia-Bustos, J., Jabbar, A., Wells, T.N.C., Palmer, M.J., Gasser, R.B., Baell, J.B. (2018) J. Med. Chem. 61, 10875-10894
Abstract
A phenotypic screen of a diverse library of small molecules for inhibition of the development of larvae of the parasitic nematode Haemonchus contortus led to the identification of a 1-methyl-1H-pyrazole-5-carboxamide derivative with an IC50 of 0.29 μM. Medicinal chemistry optimization targeted modifications on the left-hand side, middle section and right-hand side of the scaffold in order to elucidate the structure-activity relationship (SAR). Strong SAR allowed for the iterative and directed assembly of a focus set of 64 analogues, from which compound 60 was identified as the most potent compound, inhibiting the development of the fourth larval (L4) stage with an IC50 of 0.01 μM. In contrast, only 18% inhibition of mammal epithelial cell viability was observed, even at concentrations as high as 50 μM.
PubMed | DOI

Song, L., Rijal, R., Karow, M., Stumpf, M., Hahn, O., Park, L., Insall, R., Schröder, R., Hofmann, A., Clemen, C.S., Eichinger, L. (2018) Dis. Model Mech. 11, dmm033449
Abstract
Hereditary Spastic Paraplegias (HSP) are genetically diverse and clinically characterized by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str), a member of the WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homologue) complex, have been shown to cause HSP type 8 (SPG8). To investigate the molecular functions of wild type and N417D Str, we generated Dictyostelium Str- cells and ectopically expressed Strwt -GFP or StrN471D in Str- and wt cells. Overexpression of both proteins apparently caused a defect in cell division as we observed a clear increase in multinucleate cells. Real time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str- cells, but Western blots showed a two-fold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known as well as new Str interacting proteins and also proteins that did no longer bind to StrN471D. On the cellular level, Str- cells displayed defects in cell growth, phagocytosis, macro-pinocytosis, exocytosis and lysosomal function. Expression of Strwt-GFP in Str- cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit Str in the endo-lysosomal system and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the N471D mutation causes a partial loss of Str function in the endo-lysosomal system and thus, provide a basis for a better understanding of the molecular mechanism of SPG8.
PubMed | DOI

Dilrukshi Herath, H.M.P., Song, H., Preston, S., Jabbar, A., Wang, T., McGeed, S.L., Hofmann, A., Garcia-Bustosa, J., Chang, B.C.H., Koehler, A.V., Liu, Y., Ma, Q., Zhang, P., Zhao, Q., Wang, Q., Gasser, R.B. (2018) Int. J. Parasitol. Drugs Drug Resist. 8, 379-385
Abstract
Widespread resistance of parasitic nematodes necessitates the development of new anthelmintics. Due to cost and time concerns for developing a new drug, repurposing of known chemicals seems to be a promising approach in antiparasitic drug development. In the present study, we tested a library (n= 600) of analogues derived from various pesticides against exsheathed third stage larvae of Haemonchus contortus using a whole-organism phenotypic screening technique which tests the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from arylpyrroles and fipronil. All seven compounds, selected based on their high potency and/or limited cytotoxicity, are arylpyrroles reduced the motility of fourth stage larvae with a significant potency. Since the parent compounds of active arylpyrrole analogues are uncouplers of oxidative phosphorylation, we tested the effect of analogues on oxygen consumption using Seahorse XF24 flux analyser. The larvae treated with test compounds showed a significant increase in oxygen consumption compared to the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study might provide a basis for repurposing such natural product derivatives as anthelmintic candidates following substantial medicinal chemical optimisation.
PubMed | DOI

Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R.B., Kim, J.-S., Coster, M.J., Hofmann, A. (2018) FASEB J. 32, 5470-5482
Abstract
The opportunistic bacterium Pseudomonas aeruginosa has been recognised as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas that is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intra-cellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of two TPP genes with either chromosomal or extra-chromosomal location. Both TPPs were produced as recombinant proteins and characterisation of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of trehalose 6-phosphate. The three-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers which possess the overall fold of halo-acid dehydrogenases.
PubMed | DOI

Ma, G., Wang, T., Korhonen, P.K., Ang, C.-S., Williamson, N.A., Young, N.D., Ströhlein, A.J., Hall, R.S., Koehler, A.V., Hofmann, A., Gasser, R.B. (2018) Int. J. Parasitol. 48, 763-772
Abstract
In this study, we explored the molecular alterations in the developmental switch from the third (L3) to the fourth larval (L4) stage of Haemonchus contortus in vitro using an integrated transcriptomic, proteomic and bioinformatic approach. Totals of 9,754 messenger RNAs (mRNAs), 88 microRNAs (miRNAs) and 1,591 proteins were identified, and 6,686 miRNA-mRNA pairs inferred in all larval stages studied. Approximately 16% of the transcriptome (representing all three larval stages) was represented by proteins, and there were positive correlations (r = 0.39 to 0.44) between mRNA transcription and protein expression in the three distinct developmental stages of the parasite. Of the predicted targets, 1,019 (27.0%) mRNA transcripts were expressed as proteins, and there was a negative correlation (r = -0.60 to -0.50) in the differential mRNA transcription and protein expression between developmental stages upon pairwise comparison. The changes in transcription (mRNA and miRNA) and protein expression from the free-living to parasitic life cycle phase of H. contortus related to enrichments in biological pathways associated with metabolism (e.g., carbohydrate and lipid degradation, and amino acid metabolism), environmental information processing (signal transduction, signalling molecules and interaction) and/or genetic information processing (transcription and translation). Specifically, fatty acid degradation, steroid hormone biosynthesis, Rap1 signalling pathway were suppressed, whereas transcription, translation and protein processing in the endoplasmic reticulum were upregulated during the transition from the free-living L3 to the parasitic xL3 and L4 of the parasite in vitro. Dominant post-transcriptional regulation was inferred to elicit these changes, and particular miRNAs (e.g., hco-miR-34 and hco-miR-252) appear to play roles in stress responses and/or environmental adaptations during developmental transitions of H. contortus. Taken together, these integrated results provide a comprehensive insight into the developmental biology of this important parasite at the molecular level in vitro. The approach described here for H. contortus can be readily applied to other parasitic nematodes.
PubMed | DOI

Wibowo, M., Wang, Q., Holst, J., White, J.M., Hofmann, A., Davis, R.A. (2018) Phytochemistry 148, 71-77
Abstract
Phytochemical studies of the roots of the Australian plant, Maytenus bilocularis, resulted in the identification of six new dihydro-β-agarofuran sesquiterpenoids, bilocularins D-G (4-9), along with three known natural products, namely 1α,2α,6β,15-tetraacetoxy-9β-benzoyloxydihydro-β-agarofuran (10), pristimerin (11), and celastrol (12). The structures of all compounds were elucidated by analysis of 1D/2D NMR and MS data. The absolute configuration of bilocularin D (4) was defined by X-ray crystallography analysis. Compounds 4, 7, and 10 inhibited leucine transport in the human prostate cancer cell line LNCaP with IC50 values ranging from 2.5-27.9 μM, which were more potent that the L-type amino acid transporters (LATs) family inhibitor 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH). Bilocularins D-F (4-6) are the first examples of dihydro-β-agarofurans bearing a hydroxyacetate group.
PubMed | DOI