Recent Results
Update: 14.10.2022

2021 - 2022


Taki, A.C., Wang, T., Nguyen, N.N., Ang, C.-S., Leeming, M.G., Nie, S., Byrne, J.J., Young, N.D., Zheng, Y., Ma, G., Korhonen, P.K., Koehler, A.V., Williamson, N.A., Hofmann., A., Chang, B.C.H., Häberli, C., Keiser, J., Jabbar, A., Sleebs, B.E., Gasser, R.B. (2022) Front. Pharmacol. 13, 1014804
Abstract
Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber’s pole worm, and in other socioeconomically important parasitic nematodes. The “hit-to-target” workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.
DOI

Zheng, Y., Ma, G., Wang, T., Hofmann, A., Song, J., Gasser, R.B., Young, N.D. (2022) Int. J. Parasitol. 52, 581-590
Abstract
The ubiquitin-mediated pathway has been comprehensively explored in the free-living nematode Caenorhabditis elegans, but very little is known about this pathway in parasitic nematodes. Here, we inferred the ubiquitination pathway for an economically significant and pathogenic nematode – Haemonchus contortus – using abundant resources available for C. elegans. We identified 215 genes encoding ubiquitin (Ub; n = 3 genes), ubiquitin-activating enzyme (E1; one), -conjugating enzymes (E2s; 21), ligases (E3s; 157) and deubiquitinating enzymes (DUBs; 33). With reference to C. elegans, Ub, E1 and E2 were relatively conserved in sequence and structure, and E3s and DUBs were divergent, likely reflecting functional and biological uniqueness in H. contortus. Most genes encoding ubiquitination pathway components exhibit high transcription in the egg compared with other stages, indicating marked protein homeostasis in this early developmental stage. The ubiquitination pathway model constructed for H. contortus provides a foundation to explore the ubiquitin-proteasome system, crosstalk between autophagy and the proteasome system, and the parasite-host interactions. Selected E3 and DUB proteins which are very divergent in sequence and structure from host homologues or entirely unique to H. contortus and related parasitic nematodes may represent possible anthelmintic targets.
PubMed | DOI

Seo, P.-W., Hofmann, A., Kim, J.-H., Hwangbo, S.-A., Kim, J.-H., Kim, J.-W., Huynh, T.Y.L., Choy, H.E., Kim, S.-J., Lee, J., Lee, J.-O., Jin, K.S., Park, S.-Y., Kim, J.-S. (2022) Int. J. Biol. Macromol. 208, 381-389
Abstract
Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.
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Korhonen, P., Kinkar, L., Young, N.D., Cai, H., Lightowlers, M.W., Gauci1, C., Jabbar, A., Chang, B.C.H., Wang, T., Hofmann, A., Koehler, A.V., Li, J., Li, J., Qiangba, G., Xie, J., Wang, D., Yin, J., Jenkins, D.J., Saarma, U., Laurimäe, T., Rostami-Nejad, M., Andresiuk, V., Irshadullah, M., Mirhendi, H., Sharbatkhori, M., Ponce Gordo, F., Simsek, S., Casulli, A., Zait, H., Luiz de la Rue, M., Romig, T., Wassermann, M., Atoyan, H.A., Aghayan, S.A., Hasmik, G., Yang, B., Gasser, R.B. (2022) Commun. Biol. 5, 199
Abstract
Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most significant translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosusE. granulosus, and the development of improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.
PubMed | DOI

Campos, T.L., Korhonen, P.K., Hofmann, A., Gasser, R.B., Young, N.D. (2022) Biotechnol. Advances 54, 107822
Abstract
The availability of high-quality genomes and advances in functional genomics have enabled large-scale studies of essential genes in model eukaryotes, including the ‘elegant worm’ (Caenorhabditis elegans; Nematoda) and the ‘vinegar fly’ (Drosophila melanogaster; Arthropoda). However, this is not the case for other, much less-studied organisms, such as socioeconomically important parasites, for which functional genomic platforms usually do not exist. Thus, there is a need to develop innovative techniques or approaches for the prediction, identification and investigation of essential genes. A key approach that could enable the prediction of such genes is machine learning (ML). Here, we undertake an historical review of experimental and computational approaches employed for the characterisation of essential genes in eukaryotes, with a particular focus on model ecdysozoans (C. elegans and D. melanogaster), and discuss the possible applicability of ML-approaches to organisms such as socioeco­nomically important parasites. We highlight some recent results showing that high-performance ML, combined with feature engineering, allows a reliable prediction of essential genes from extensive, publicly available ‘omic data sets, with major potential to prioritise such genes (with statistical confidence) for subsequent functional genomic validation. These findings could ‘open the door’ to fundamental and applied research areas. Evidence of some commonality in the essential gene-complement between these two organisms indicates that an ML–engineering approach could find broader applicability to ecdysozoans such as parasitic nematodes or arthro­pods, provided that suitably large and informative data sets become/are available for proper feature engineering, and for the robust training and validation of algorithms. This area warrants detailed exploration to, for example, facilitate the identification and characterisation of essential molecules as novel targets for drugs and vaccines against parasitic diseases. This focus is particularly important, given the substantial impact that such diseases have worldwide, and the current challenges associated with their prevention and control and with drug resis­tance in parasite populations.
PubMed | DOI

Ramage, K.S., Taki, A.C., Lum, K.Y., Hayes, S., Byrne, J.J., Wang, T., Hofmann, A., Ekins, M.G., White, J.M., Jabbar, A., Davis, R.A., Gasser, R.B. (2021) Mar. Drugs 19, 698
Abstract
High-throughput screening of the NatureBank marine extract library (n = 7,616) using a phenotypic assay for the parasitic nematode Haemonchus contortus identified an active extract derived from the Australian marine sponge Citronia sp. bioassay-guided fractionation of the CH2Cl2/MeOH extract from Citronia sp. resulted in the purification of two known hexachlorinated peptides, dysidenin (1) and dysideathiazole (2). Compound 1 inhibited the growth/development of H. contortus larvae and induced multiple phenotypic changes, including a lethal, evisceration (Evi) phenotype and/or somatic cell/tissue destruction. This is the first report of anthelmintic activity for these rare and unique polychlorinated peptides.
PubMed | DOI

Taki, A.C., Byrne, J.J., Jabbar, A., Lum, K.Y., Hayes, S., Addison, R.S., Ramage, K.S., Hofmann, A., Ekins, M.G., Wang, T., Chang, B.C.H., Davis, R.A., Gasser, R.B. (2021) Molecules 26, 5846
Abstract
Widespread resistance in parasitic nematodes to most classes of anthelmintic drugs demands the discovery and development of novel compounds with distinct mechanisms of action to complement strategic or integrated parasite control programs. Products from nature – which assume a diverse ‘chemical space’ – have significant potential as a source of anthelmintic compounds. In the present study, we screened a collection of extracts (n = 7,616) derived from marine invertebrates sampled from Australian waters in a high throughput bioassay for in vitro anti-parasitic activity against the barber’s pole worm (Haemonchus contortus) – an economically important parasitic nematode of livestock animals. In this high throughput screen (HTS), we identified 58 active extracts that reduced larval motility by ≥70% (at 90 h), equating to an overall “hit rate” of ≈0.8%. Of these 58 extracts, 16 also inhibited larval development by ≥80% (at 168 h) and/or induced ‘non-wildtype’ (abnormal) larval phenotypes with reference to ‘wild-type’ (normal) larvae not exposed to extract (negative controls). Most active extracts (54 of 58) originated from sponges, three from chordates (tunicates) and one from a coral; these extracts represented 37 distinct species/taxa of 23 families. An analysis of samples by 1H NMR fingerprinting was utilised to dereplicate hits and to prioritise a set of 29 sponge samples for future chemical investigation. Overall, these results indicate that a range of sponge species from Australian waters represents a rich source of natural compounds with nematocidal or nematostatic properties. Our plan now is to focus on in-depth chemical investigations of the sample set prioritised herein.
PubMed | DOI

Riehl, J., Rijal, R., Nitz, L., Clemen, C.S., Hofmann, A., Eichinger, L. (2021) Frontiers Cell Develop. Biol. 9, 2618
Abstract
The abundant homohexameric AAA+ ATPase p97 (also known as valosin-containing protein, VCP) is highly conserved from Dictyostelium discoideum to human and a pivotal factor of cellular protein homeostasis as it catalyses the unfolding of proteins. Owing to its fundamental function in protein quality control pathways, it is regulated by more than 30 cofactors, including the UBXD protein family, whose members all carry an Ubiquitin Regulatory X (UBX) domain that enables binding to p97. One member of this latter protein family is the largely uncharacterised UBXD9 protein.
Here, we analysed protein-protein interactions of D. discoideum UBXD9 with p97 using a series of N- and C-terminal truncation constructs and probed the UBXD9 interactome in D. discoideum.
Pull-down assays revealed that the UBX domain (amino acids 384–466) is necessary and sufficient for p97 interactions and that the N-terminal extension of the UBX domain, which folds into a β0–α1–α0 lariat structure, is required for the dissociation of p97 hexamers. Functionally, this finding is reflected by strongly reduced ATPase activity of p97 upon addition of full length UBXD9 or UBXD9261–573. Results from Blue Native PAGE as well as structural model prediction suggest that hexamers of UBXD9 or UBXD9261–573 interact with p97 hexamers and disrupt the p97 subunit interactions via insertion of a helical lariat structure, presumably by destabilising the p97 D1:D1' intermolecular interface. It is thus proposed that UBXD9 regulates p97 activity in vivo by shifting the quaternary structure equilibrium from hexamers to monomers.
Using three independent approaches, we further identified novel interaction partners of UBXD9, including glutamine synthetase type III as well as several actin-binding proteins. These findings suggest a role of UBXD9 in the organisation of the actin cytoskeleton, and are in line with the hypothesised oligomerisation-dependent mechanism of p97 regulation.
PubMed | DOI

Clemen, C.S., Schmidt, A., Winter, L., Canneva, F., Wittig, I., Becker, L., Coras, R., Berwanger, C., The German Mouse Clinic Consortium, Hofmann, A., Eggers, B., Marcus, K., Gailus-Durner, V., Fuchs, H., Hrabe de Angelis, M., Krüger, M., von Hörsten, S., Eichinger, L., Schröder, R. (2022) Neuropath. Appl. Neuro. 48, e12750
Abstract
Aims: We investigated N471D WASH complex subunit strumpellin knock-in and WASH complex subunit strumpellin knock-out mice as models for hereditary spastic paraplegia type 8.
Methods: We generated heterozygous and homozygous N471D WASH complex subunit strumpellin knock-in mice, which underwent a comprehensive clinical, morphological, and laboratory parameter screen and gait analysis. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous WASH complex subunit strumpellin knock-out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting.
Results: Homozygous N471D WASH complex subunit strumpellin knock-in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels, and increased white blood cell counts. Gait analysis revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, hematology, and gait parameters of heterozygous animals also deviated from the values expected for healthy individuals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock-in mice. WASH complex subunit strumpellin-related protein interaction partners and complexes showed no change in abundancies. Heterozygous knock-out mice showing normal WASH complex subunit strumpellin levels could not be bred to homozygosity.
Conclusions: While biallelic ablation of WASH complex subunit strumpellin was prenatally lethal, expression of N471D mutated WASH complex subunit strumpellin led to several mild clinical and laboratory parameter abnormalities, but not to a SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 in the brain of knock-in animals suggest mechanistic links for hereditary spastic paraplegia type 8 through their roles in neurodegeneration and protein quality control, respectively.
PubMed | DOI

Amani, P., Habibpour, R., Karami, L., Hofmann, A. (2021) ACS Chem. Neurosci. 12, 2218-2232
Abstract
Chemoinformatics appraisal and molecular docking were employed to investigate 225 complexes of 75 schizophrenia antipsychotics with the dopamine receptor subtypes D2R, D3R, and D4R. Considering the effective noncovalent interactions in the subtype-D2 receptor selectivity of antipsychotics, this study evaluated the possible physicochemical properties of ligands underlying the design of safer and more effective antipsychotics. The pan-assay interference compounds (PAINs) include about 25% of typical antipsychotics and 5% of atypicals. Popular antipsychotics like haloperidol, clozapine, risperidone, and aripiprazole are not PAINs. They have stronger interactions with D2R and D4R, but their interactions with D3R are slightly weaker, which is similar to the behavior of dopamine. In contrast to typical antipsychotics, atypical antipsychotics exhibit more noncovalent interactions with D4R than with D2R. These results suggest that selectivity to D2R and D4R comes from the synergy between hydrophobic and hydrogen-bonding interactions through their concomitant occurrence in the form of a hydrogen-bonding site adorned with hydrophobic contacts in antipsychotic–receptor complexes. All the antipsychotics had more synergic interactions with D2R and D4R in comparison with D3R. The atypical antipsychotics made a good distinction between the subtype D2 receptors with high selectivity to D4R. Among the popular antipsychotics, haloperidol, clozapine, and risperidone have hydrophobic–hydrogen-bonding synergy with D4R, while aripiprazole profits with D2R. The most important residue participating in the synergic interactions was threonine for D2R and cysteine for D4R. This work could be useful in informing and guiding future drug discovery and development studies aimed at receptor-specific antipsychotics.
PubMed | DOI

Kim, J-H, Hofmann, A, Kim, J-S (2021) Proteins 89, 1030-1038
Abstract
Carbohydrates play a major role in infection strategies of various enteric pathogens. In Campylobacter jejuni, the most common cause of gastroenteritis, uniquely modified heptoses found in surface carbohydrates are synthesized by specific pathways. Owing to the importance of such pathways for the infectious potential of pathogens and/or their virulence, these biosynthesis pathways present potential targets for therapeutic intervention. Here, we determined the crystal structure of GDP-6-OMe-4-keto–L-xylo-heptose reductase (MlghC), an enzyme within the L-gluco-heptose synthesis pathway of C. jejuni strain NCTC 11168. This enzyme lacks the canonical tyrosine residue of the conserved catalytic Ser-Lys-Tyr triad commonly found among functionally related reductases. Despite adopting the overall two-domain fold shared with other short-chain dehydrogenase/reductase family members, subtle structural differences in the interface between the cofactor- and substrate-binding domains explain the absence of epimerase activity and different substrate specificity of this reductase. Modeling of the product-bound complex based on the crystal structure presented here suggests that a tyrosine residue unique to MlghC replaces the missing canonical residue of the catalytic triad.
PubMed | DOI

Liu, G.-H., Korhonen, P.K., Young, N.D., Lu, J., Wang, T., Fu, Y.-T., Koehler, A.V., Hofmann, A., Chang, B.C.H., Wang, S., Li, N., Lin, C.-Y., Zhang, H., Xiangli, L., Lin, L., Liu, W.-M., Li, H.-W., Gasser, R.B., Zhu, X.-Q. (2021) Genomics 113, 1272-1280
Abstract
Here, we present a draft genome of the tapeworm Dipylidium caninum (family Dipylidiidae) and compare it with other cestode genomes. This draft genome of D. caninum is 110 Mb in size, has a repeat content of approximately 13.4% and is predicted to encode ~10,000 protein-coding genes. We inferred excretory/secretory molecules (representing the secretome), other key groups of proteins (including peptidases, kinases, phosphatases, GTPases, receptors, transporters and ion-channels) and predicted potential intervention targets for future evaluation. Using 144 shared single-copy orthologous sequences, we investigated the genetic relationships of cestodes for which nuclear genomes are available. This study provides first insights into the molecular genetics and biology of D. caninum and a new resource for molecular ecological studies and future comparative molecular-genetic and genomic explorations of this and other flatworms.
PubMed | DOI

Seo, P-W., Park, S.-Y., Hofmann, A., Kim, J.-S. (2021) Acta Crystallogr. D 77, 618–627
Abstract
Peptidoglycan comprises repeating units of N-acetylmuramic acid (NAM), N-acetylglucosamine (NAG), and short cross-linking peptides. After conversion of UDP-N-acetylglucosamine (UNAG) into UDP-N-acetylmuramic acid (UNAM) by the MurA and MurB enzymes, an amino acid is added to UNAM by the UDP-N-acetylmuramic acid L-alanine ligase (MurC). As peptidoglycan is an essential component of the bacterial cell wall, the enzymes involved in its biosynthesis represent promising targets for the development of novel antibacterial drugs. Here, we report the crystal structure of Mycobacterium bovis MurC (MbMurC) which exhibits a three-domain architecture for binding of UNAM, ATP, and an amino acid as substrates with a nickel ion at the domain interface. The ATP-binding loop adopts a conformation not seen in other MurCs. In the UNAG-bound structure of MbMurC, the substrate mimic interacts with the UDP-binding domain of MbMurC, which does not invoke rearrangement of three domains. Interestingly, the glycine-rich loop of the UDP-binding domain of MbMurC interacts through hydrogen bonds with the glucose moiety, but not with the ligand’s pyrophosphate moiety. These findings suggest that UNAG analogs might serve as potential candidates for neutralizing the catalytic activity of bacterial MurC.
PubMed | DOI

Herath, H.M.P.D., Taki, A.C., Sleebs, B.E., Hofmann, A., Nguyen, N., Preston, S., Davis, R.A., Jabbar, A., Gasser, R.B. (2021) Adv. Parasitol. vol. 111, 203-251
Abstract
Widespread resistance to currently-used anthelmintics represents a major obstacle to controlling parasitic nematodes of livestock animals. Given the reliance on anthelmintics in many control regimens, there is a need for the continued discovery and development of new nematocides. Enabling such a focus are: (i) the major chemical diversity of natural products; (ii) the availability of curated, drug-like extract-, fraction- and/or compound-libraries from natural sources; (iii) the utility and practicality of well-established whole-worm bioassays for Haemonchus contortus – an important parasitic nematodes of livestock – to screen natural product libraries; and (iv) the availability of advanced chromatographic (HPLC), spectroscopic (NMR) and spectrometric (MS) techniques for bioassay-guided fractionation and structural elucidation. This context provides a sound basis for the identification and characterisation of anthelmintic candidates from natural sources. This chapter provides a background on the importance and impact of helminth infections/diseases, parasite control and aspects of drug discovery, and reviews recent work focused on (i) screening well-defined compound libraries to establish the methods needed for large-scale screening of natural extract libraries; (ii) discovering plant and marine extracts with nematocidal or nematostatic activity, and purifying bioactive compounds and assessing their potential for further development; and (iii) synthesising analogues of selected purified natural compounds for the identification of possible ‘lead’ candidates. The chapter describes some lessons learned from this work and proposes future areas of focus for drug discovery. Collectively, the findings from this recent work show potential for selected natural product scaffolds as candidates for future development. Developing such candidates via future chemical optimisation, efficacy and safety evaluations, broad spectrum activity assessments, and target identification represents an exciting prospect and, if successful, could pave the way to subsequent pre-clinical and clinical evaluations.
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. (2021) J. Immunol. Res. 2021, 6234836
Abstract
Immunomodulation of airway hyperreactivity by excretory-secretory (ES) products of the first larval stage (L1) of the gastrointestinal nematode Trichuris suis is reported by us and others. Here, we aimed to identify the proteins accounting for the modulatory effects of the T. suis L1 ES proteins and studied six selected T. suis L1 proteins for their immunomodulatory efficacy in a murine OVA-induced allergic airway disease model. In particular, an enzymatically active T. suis chitinase mediated amelioration of clinical signs of airway hyperreactivity, primarily associated with suppression of eosinophil recruitment into the lung, the associated chemokines, and increased numbers of RELMα+ interstitial lung macrophages. While there is no indication of T. suis chitinase directly interfering with dendritic cell activation or antigen presentation to CD4 T cells, treatment of allergic mice with the worm chitinase influenced the hosts’ own chitinase activity in the inflamed lung. The three-dimensional structure of the T. suis chitinase as determined by high-resolution X-ray crystallography revealed high similarities to mouse acidic mammalian chitinase (AMCase) but a unique ability of T. suis chitinase to form dimers. Our data indicate that the structural similarities between the parasite and host chitinase contribute to the disease-ameliorating effect of the helminth-derived chitinase on allergic lung inflammation.
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