Recent Results
Update: 10.07.2018

2017 - 2018


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., in press
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

Cross, M. , Rajan, S. Biberacher, S. Park, S.-Y., Coster, M.J., Długosz, E., Kim, J.-S., Gasser, R.B., Hofmann, A. (2017) Em. Topics Life Sci. 1, 675-683
Abstract
As opposed to organism-based drug screening approaches, protein-based strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors and thus afford a precise targeting. Capitalising on the increasing number of genome and transcriptome datasets, novel targets in pathogens for therapeutic intervention can be identified in a more rational manner as compared to conventional organism-based methodologies. Trehalose-6-phosphate phosphatases (TPPs) are structurally and functionally conserved enzymes of the trehalose biosynthesis pathway which play a critical role for pathogen survival, in particular in parasites. The absence of these enzymes as well as trehalose biosynthesis from mammalian hosts has recently given rise to increasing interest in TPPs as novel therapeutic targets for drugs and vaccines. Here, we summarise some key aspects of the current state of research towards novel therapeutics targeting in particular nematode TPPs.
DOI

Ma, G., Holland, C., Wang, T., Hofmann, A., Fan, C.-K., Maizels, R., Hotez, P.J., Gasser, R.B. (2018) Lancet Infect. Dis. 18, e14-24
Abstract
Parasitic nematodes of the genus Toxocara are socioeconomically important zoonotic pathogens. These parasites are usually directly transmitted to the human host via the faecal-oral route and can cause substantial clinical disease, known as toxocariasis, as well as associated complications including allergic and/or neurological disorders. Although it is estimated that tens of million people are exposed to or infected with Toxocara species, there is limited precise epidemiological information on the relationship between seropositivity and associated disease (toxocariasis) on a global scale. Some recent studies indicate that toxocariasis is having an increased human-health impact in some countries. To gain improved insights into human toxocariasis, this seminar reviews salient background on Toxocara and biology, summarises key aspects of the pathogenesis, diagnosis and treatment of toxocariasis, describes what is presently known about its geographic distribution and prevalence, and makes some recommendations regarding future research towards the prevention and control of this important disease.
PubMed | DOI

Preston, S., Jiao, Y., Baell, J.B., Keiser, J., Crawford, S., Koehler, A.V., Wang, T., Simpson, M.M., Kaplan, R.M., Cowley, K.J., Simpson, K.J., Hofmann, A., Jabbar, A., Gasser, R.B. (2017) Int. J. Parasitol. Drugs Drug Resist. 7, 286-294
Abstract
The discovery and development of novel anthelmintic classes is essential to sustain the control of socioeconomically important parasitic worms of humans and animals. With the aim of offering novel, lead-like scaffolds for drug discovery, Compounds Australia released the ‘Open Scaffolds’ collection containing 33,999 compounds, with extensive information available on the physicochemical properties of these chemicals. In the present study, we screened 14,464 prioritised compounds from the ‘Open Scaffolds’ collection against the exsheathed third-stage larvae (xL3s) of Haemonchus contortus using recently developed whole-organism screening assay. We identified a hit compound, called SN00797439, which was shown to reproducibly reduce xL3 motility by ≥70%; this compound induced a characteristic, “coiled” xL3 phenotype (IC50 = 3.46-5.93 μM), inhibited motility of fourth-stage larvae (L4s; IC50 = 0.31-12.5 μM) and caused considerable cuticular damage to L4s in vitro. When tested on other parasitic nematodes in vitro, SN00797439 was shown to inhibit (IC50 = 3-50 μM) adults of Ancylostoma ceylanicum (hookworm) and first-stage larvae of Trichuris muris (whipworm) and eventually kill (>90%) these stages. Furthermore, this compound completely inhibited the motility of female and male adults of Brugia malayi (50-100 μM) as well as microfilariae of both B. malayi and Dirofilaria immitis (heartworm). Overall, these results show that SN00797439 acts against genetically (evolutionarily) distant parasitic nematodes (i.e. H. contortus and A. ceylanicum [strongyloids] vs. B. malayi and D. immitis [filarioids] vs. T. muris [enoplid], and, thus, might offer a novel, lead-like scaffold for the development of a relatively broad-spectrum anthelmintic. Our future work will focus on assessing the activity of SN00797439 against other pathogens that cause neglected tropical diseases, optimising analogs with improved biological activities and characterising their targets.
PubMed | DOI

Wibowo, M., Wang, Q., Holst, J., White, J.M., Hofmann, A., Davis, R. (2017) J. Nat. Prod. 80, 1918-1925
Abstract
Seven new dihydro-β-agarofurans, celastrofurans A–G (1–7) along with two known secondary metabolites, 9β-benzoyloxy-1α-furoyloxydihydro-β-agarofuran (8) and (1R,2R,4R,5S,7R,9S,10R)-2-acetoxy-9-benzoyloxy-1-furoyloxydihydro-β-agarofuran (9) were obtained from the leaves of the Australian rainforest vine, Celastrus subspicata. The structures of the new compounds were determined by detailed spectroscopic (1D/2D NMR and MS) data analysis. The absolute configurations of compounds 1–4 were defined by ECD and single-crystal X-ray diffraction studies. All compounds were found to exhibit inhibitory activity on leucine transport in the human prostate cancer cell line LNCaP with IC50 values ranging from 7.0–98.9 μM. Dihydro-β-agarofurans 1–9 showed better potency than the L-type amino acid transporter (LAT) family inhibitor, 2-aminobicyclo[2.2.1]-heptane-2-carboxylic acid (BCH).
PubMed | DOI

Jiao, Y., Preston, S., Song, H., Jabbar, A., Liu, Y., Baell, J., Hofmann, A., Hutchinson, D., Wang, T., Koehler, A., Fisher, G., Andrews, K., Willis, P., Palmer, M., Wells, T., Wang, Q., Gasser, R.B. (2017) Parasites & Vectors 10, 272
Abstract
In this study, we tested five series of pyrazole-5-carboxamide compounds (n = 55) for activity against parasitic stages of the nematode Haemonchus contortus (barber’s pole worm). In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility and development of H. contortus. Amongst the 55 compounds, we identified two compounds (designated a-15 and a-17) that reproducibly inhibit xL3 motility as well as L4 motility and development, with IC50 values ranging between ~ 3.4 and 55.6 μM. We studied the effect of these two ‘hit’ compounds on mitochondrial function by measuring oxygen consumption. This assessment showed that xL3s exposed to each of these compounds consumed significantly less oxygen and had less mitochondrial activity than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. The present findings provide a sound basis for future work aiming to identify the targets of compounds a-15 and a-17, and to establish the modes of action of these chemicals in H. contortus.
PubMed | DOI

Dallaston, M.A., Rajan, S., Chekaiban, J., Wibowo, M., Cross, M., Coster, M.J., Davis, R.A., Hofmann, A. (2017) MedChemComm 8, 1318-1321
Abstract
The soluble mycobacterial carbonic anhydrases Rv3588c and Rv1284 belong to a different class of carbonic anhydrases than those found in humans, making them attractive drug targets by using the inherent differences in the folds of the different classes. By screening a natural product library, we identified naphthoquinone derivatives as a novel non-classical inhibitor scaffold of mycobacterial carbonic anhydrases that lack the sulfonamide/sulfamate group and thus did not affect human carbonic anhydrase II.
DOI

Türk, M., Schröder, R., Khuller, K., Hofmann, A., Berwanger, C., Ludolph, A.C., Dekomien, G., Müller, K., Weishaupt, J.H., Thiel, C.T., Clemen, C. (2017) Neurobiol. Aging 56, 213.e1-213.e5
Abstract
Mutations of the human VCP and WASH complex genes cause motor neuron and cognitive impairment disorders. Here, we analyzed a cohort of German patients with sporadic amyotrophic lateral sclerosis and frontotemporal lobar degeneration co-morbidity (ALS/FTD) for VCP and WASH complex gene mutations. Next generation panel sequencing of VCP, WASH1, FAM21C, CCDC53, SWIP, strumpellin, CAPZA1 and CAPZB genes was performed in 43 sporadic ALS/FTD patients. Subsequent analyses included Sanger sequencing, in silico analyses, real-time PCR, and CCDC53 immunoblotting. We identified one patient with the heterozygous variant c.26C>T in CAPZA1 and a second with the heterozygous variant c.2T>C in CCDC53. In silico analysis predicted structural changes in the N-terminus of CAPZα1, which may interfere with the CAPZα:CAPZβ dimerization. Though the translation initiation codon of CCDC53 is mutated, real-time PCR and immunoblotting did neither reveal any evidence for a CCDC53 haploinsufficiency nor for aberrant CCDC53 protein species. Thus, with exception of a putatively pathogenic heterozygous c.26C>T CAPZA1 mutation, our genetic analysis did not reveal mutations in VCP and the remaining WASH complex subunits.
PubMed | DOI

Cross, M., Rajan, S., Chekaiban, J., Saunders, J., Hamilton, C., Kim, J.-S., Coster, M. J., Gasser, R. B., Hofmann, A. (2017) Sci. Rep. 7, 2015
Abstract
Owing to the key role of trehalose in pathogenic organisms, there has recently been growing interest in trehalose metabolism for therapeutic purposes. Trehalose-6-phosphate phosphatase (TPP) is a key enzyme in the most prominent biosynthesis pathway (OtsAB). Here, we compare the enzyme characteristics of recombinant TPPs from five important nematode and bacterial pathogens, including three novel members of this protein family. Analysis of the kinetics of trehalose-6-phosphate hydrolysis reveals that all five enzymes display a burst-like kinetic behaviour which is characterised by a decrease of the enzymatic rate after the pre-steady state. The observed super-stoichiometric burst amplitudes can be explained by multiple global conformational changes by members of this enzyme family during substrate processing. In the search for specific TPP inhibitors, the trapping of the complex conformational transitions in TPPs during the catalytic cycle may present a worthwhile strategy to explore.
PubMed | DOI | Publisher

Cross, M., Lepage, R., Rajan, S., Biberacher, S., Young, N.D., Kim, B.-N., Coster, M.J., Gasser, R.B., Kim, J.-S., Hofmann, A. (2017) FASEB J. 31, 920-926
Abstract
The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics, since trehalose is an essential disaccharide in many pathogens, but neither is required nor synthesised in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis is likely an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms, and by way of a structure-based amino acid sequence alignment, we derive the topological structure of mono-enzyme trehalose-6-phosphate phosphatases and classify them into three groups. Comparison of the functional roles of amino acid residues located in the active site for TPPs belonging to different groups reveal nuanced variation. Since current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPP of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. Therefore, we suggest that homology-based inference of functionally important amino acids by sequence comparison for mono-enzyme TPPs should only be carried out for each of the three groups.
PubMed | DOI

Kim, B.-N., Shin, M., Ha, S.C., Park, S.Y., Seo, P.-W., Hofmann, A., Kim, J.-S. (2017) Sci. Rep. 7, 12303
Abstract
Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5’-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.
PubMed | DOI

Dilrukshi Herath, H.M.P., Preston, S., Hofmann, A., Davis, R.A., Koehler, A.V., Chang, B.B.-C., Jabbar, A., Gasser, R.B. (2017) Vet. Parasitol. 244, 172-175
Abstract
The control of parasitic roundworms (nematodes) is heavily reliant on the use of a limited number of anthelmintic drugs. However, drug resistance is now very widespread and no vaccines are available, such that the discovery of new chemical entities is crucial. Within this context, we screened a library of pure natural products (n = 400) against exsheathed third-stage (xL3) larvae of the parasitic nematode Haemonchus contortus using a whole-organism screening method. We identified two plant-derived rotenoids, namely deguelin and rotenone, with inhibitory activity on xL3 motility. Whereas rotenone was not investigated further because of its toxicity to mammals/vertebrates, dose response and cytotoxicity studies of deguelin showed potent and selective inhibitory activity on motility of xL3 larvae of H. contortus. Detailed future work needs to be conducted to explore the mode of action of this compound on H. contortus and related nematodes and to assess its potential as an anthelmintic candidate.
PubMed | DOI

Preston, S., Korhonen, P.K., Mouchiroud, L., Cornaglia, M., McGee, S.L., Young, N.D., Davis, R.A., Crawford, S., Nowell, C., Ansell, B.R.E., Fisher, G.M., Andrews, K.T., Chang, B., Gijs, M.A.M., Sternberg, P.W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R.B. (2017) FASEB J. 31, 4515-4532
Abstract
As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mito-ribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.
PubMed | DOI