Bio21 - Research Publications

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    Structure-activity relationship and target investigation of 2-aryl quinolines with nematocidal activity
    Shanley, HT ; Taki, AC ; Nguyen, N ; Wang, T ; Byrne, JJ ; Ang, C-S ; Leeming, MG ; Nie, S ; Williamson, N ; Zheng, Y ; Young, ND ; Korhonen, PK ; Hofmann, A ; Chang, BCH ; Wells, TNC ; Haberli, C ; Keiser, J ; Jabbar, A ; Sleebs, BE ; Gasser, RB (ELSEVIER SCI LTD, 2024-04)
    Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) - a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide.
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    Structure activity relationship and target prediction for ABX464 analogues in Caenorhabditis elegans
    Shanley, HT ; Taki, AC ; Nguyen, N ; Wang, T ; Byrne, JJ ; Ang, C-S ; Leeming, MG ; Nie, S ; Williamson, N ; Zheng, Y ; Young, ND ; Korhonen, PK ; Hofmann, A ; Wells, TNC ; Jabbar, A ; Sleebs, BE ; Gasser, RB (PERGAMON-ELSEVIER SCIENCE LTD, 2024-01-15)
    Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the 'Pandemic Response Box' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico-docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially.
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    The Proteome and Lipidome of Extracellular Vesicles from Haemonchus contortus to Underpin Explorations of Host-Parasite Cross-Talk
    Wang, T ; Koukoulis, TF ; Vella, LJ ; Su, H ; Purnianto, A ; Nie, S ; Ang, C-S ; Ma, G ; Korhonen, PK ; Taki, AC ; Williamson, NA ; Reid, GE ; Gasser, RB (MDPI, 2023-07)
    Many parasitic worms have a major adverse impact on human and animal populations worldwide due to the chronicity of their infections. There is a growing body of evidence indicating that extracellular vesicles (EVs) are intimately involved in modulating (suppressing) inflammatory/immune host responses and parasitism. As one of the most pathogenic nematodes of livestock animals, Haemonchus contortus is an ideal model system for EV exploration. Here, employing a multi-step enrichment process (in vitro culture, followed by ultracentrifugation, size exclusion and filtration), we enriched EVs from H. contortus and undertook the first comprehensive (qualitative and quantitative) multi-omic investigation of EV proteins and lipids using advanced liquid chromatography-mass spectrometry and informatics methods. We identified and quantified 561 proteins and 446 lipids in EVs and compared these molecules with those of adult worms. We identified unique molecules in EVs, such as proteins linked to lipid transportation and lipid species (i.e., sphingolipids) associated with signalling, indicating the involvement of these molecules in parasite-host cross-talk. This work provides a solid starting point to explore the functional roles of EV-specific proteins and lipids in modulating parasite-host cross-talk, and the prospect of finding ways of disrupting or interrupting this relationship to suppress or eliminate parasite infection.
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    Phosphoproteomic dysregulation in Huntington's disease mice is rescued by environmental enrichment
    Mees, I ; Li, S ; Tran, H ; Ang, C-S ; Williamson, NA ; Hannan, AJ ; Renoir, T (OXFORD UNIV PRESS, 2022-11-02)
    Huntington's disease is a fatal autosomal-dominant neurodegenerative disorder, characterized by neuronal cell dysfunction and loss, primarily in the striatum, cortex and hippocampus, causing motor, cognitive and psychiatric impairments. Unfortunately, no treatments are yet available to modify the progression of the disease. Recent evidence from Huntington's disease mouse models suggests that protein phosphorylation (catalysed by kinases and hydrolysed by phosphatases) might be dysregulated, making this major post-translational modification a potential area of interest to find novel therapeutic targets. Furthermore, environmental enrichment, used to model an active lifestyle in preclinical models, has been shown to alleviate Huntington's disease-related motor and cognitive symptoms. However, the molecular mechanisms leading to these therapeutic effects are still largely unknown. In this study, we applied a phosphoproteomics approach combined with proteomic analyses on brain samples from pre-motor symptomatic R6/1 Huntington's disease male mice and their wild-type littermates, after being housed either in environmental enrichment conditions, or in standard housing conditions from 4 to 8 weeks of age (n = 6 per group). We hypothesized that protein phosphorylation dysregulations occur prior to motor onset in this mouse model, in two highly affected brain regions, the striatum and hippocampus. Furthermore, we hypothesized that these phosphoproteome alterations are rescued by environmental enrichment. When comparing 8-week-old Huntington's disease mice and wild-type mice in standard housing conditions, our analysis revealed 229 differentially phosphorylated peptides in the striatum, compared with only 15 differentially phosphorylated peptides in the hippocampus (statistical thresholds fold discovery rate 0.05, fold change 1.5). At the same disease stage, minor differences were found in protein levels, with 24 and 22 proteins dysregulated in the striatum and hippocampus, respectively. Notably, we found no differences in striatal protein phosphorylation and protein expression when comparing Huntington's disease mice and their wild-type littermates in environmentally enriched conditions. In the hippocampus, only four peptides were differentially phosphorylated between the two genotypes under environmentally enriched conditions, and 22 proteins were differentially expressed. Together, our data indicates that protein phosphorylation dysregulations occur in the striatum of Huntington's disease mice, prior to motor symptoms, and that the kinases and phosphatases leading to these changes in protein phosphorylation might be viable drug targets to consider for this disorder. Furthermore, we show that an early environmental intervention was able to rescue the changes observed in protein expression and phosphorylation in the striatum of Huntington's disease mice and might underlie the beneficial effects of environmental enrichment, thus identifying novel therapeutic targets.
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    Proteomic analysis of Sarcoptes scabiei reveals that proteins differentially expressed between eggs and female adult stages are involved predominantly in genetic information processing, metabolism and/or host-parasite interactions
    Wang, T ; Gasser, RB ; Korhonen, PK ; Young, ND ; Ang, C-S ; Williamson, NA ; Ma, G ; Samarawickrama, GR ; Fernando, DD ; Fischer, K ; Taylan Ozkan, A (PUBLIC LIBRARY SCIENCE, 2022-12)
    Presently, there is a dearth of proteomic data for parasitic mites and their relationship with the host animals. Here, using a high throughput LC-MS/MS-based approach, we undertook the first comprehensive, large-scale proteomic investigation of egg and adult female stages of the scabies mite, Sarcoptes scabiei-one of the most important parasitic mites of humans and other animals worldwide. In total, 1,761 S. scabiei proteins were identified and quantified with high confidence. Bioinformatic analyses revealed differentially expressed proteins to be involved predominantly in biological pathways or processes including genetic information processing, energy (oxidative phosphorylation), nucleotide, amino acid, carbohydrate and/or lipid metabolism, and some adaptive processes. Selected, constitutively and highly expressed proteins, such as peptidases, scabies mite inactivated protease paralogues (SMIPPs) and muscle proteins (myosin and troponin), are proposed to be involved in key biological processes within S. scabiei, host-parasite interactions and/or the pathogenesis of scabies. These proteomic data will enable future molecular, biochemical and physiological investigations of early developmental stages of S. scabiei and the discovery of novel interventions, targeting the egg stage, given its non-susceptibility to acaricides currently approved for the treatment of scabies in humans.
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    Human Tim8a, Tim8b and Tim13 are auxiliary assembly factors of mature Complex IV
    Anderson, AJ ; Crameri, JJ ; Ang, C-S ; Malcolm, TR ; Kang, Y ; Baker, MJ ; Palmer, CS ; Sharpe, AJ ; Formosa, LE ; Ganio, K ; Baker, MJ ; McDevitt, CA ; Ryan, MT ; Maher, MJ ; Stojanovski, D (Wiley, 2023-06-05)
    Human Tim8a and Tim8b are paralogous intermembrane space proteins of the small TIM chaperone family. Yeast small TIMs function in the trafficking of proteins to the outer and inner mitochondrial membranes. This putative import function for hTim8a and hTim8b has been challenged in human models, but their precise molecular function(s) remains undefined. Likewise, the necessity for human cells to encode two Tim8 proteins and whether any potential redundancy exists is unclear. We demonstrate that hTim8a and hTim8b function in the assembly of cytochrome c oxidase (Complex IV). Using affinity enrichment mass spectrometry, we define the interaction network of hTim8a, hTim8b and hTim13, identifying subunits and assembly factors of the Complex IV COX2 module. hTim8‐deficient cells have a COX2 and COX3 module defect and exhibit an accumulation of the Complex IV S2 subcomplex. These data suggest that hTim8a and hTim8b function in assembly of Complex IV via interactions with intermediate‐assembly subcomplexes. We propose that hTim8–hTim13 complexes are auxiliary assembly factors involved in the formation of the Complex IV S3 subcomplex during assembly of mature Complex IV.
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    Structural insights into the multifunctionality of rabies virus P3 protein
    Sethi, A ; Rawlinson, SM ; Dubey, A ; Ang, C-S ; Choi, YH ; Yan, F ; Okada, K ; Rozario, AM ; Brice, AM ; Ito, N ; Williamson, NA ; Hatters, DM ; Bell, TDM ; Arthanari, H ; Moseley, GW ; Gooley, PR (NATL ACAD SCIENCES, 2023-04-04)
    Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.
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    Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses
    Chan, KL ; Gomez, J ; Cardinez, C ; Kumari, N ; Sparbier, CE ; Lam, EYN ; Yeung, MM ; Garciaz, S ; Kuzich, JA ; Ong, DM ; Brown, FC ; Chan, Y-C ; Vassiliadis, D ; Wainwright, EN ; Motazedian, A ; Gillespie, A ; Fennell, KA ; Lai, J ; House, IG ; Macpherson, L ; Ang, C-S ; Dawson, S-J ; Beavis, PA ; Wei, AH ; Burr, ML ; Dawson, MA (CELL PRESS, 2022-10-10)
    There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
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    N-Terminomic Changes in Neurons During Excitotoxicity Reveal Proteolytic Events Associated With Synaptic Dysfunctions and Potential Targets for Neuroprotection
    Ameen, SS ; Griem-Krey, N ; Dufour, A ; Hossain, MI ; Hoque, A ; Sturgeon, S ; Nandurkar, H ; Draxler, DF ; Medcalf, RL ; Kamaruddin, MA ; Lucet, IS ; Leeming, MG ; Liu, D ; Dhillon, A ; Lim, JP ; Basheer, F ; Zhu, H-J ; Bokhari, L ; Roulston, CL ; Paradkar, PN ; Kleifeld, O ; Clarkson, AN ; Wellendorph, P ; Ciccotosto, GD ; Williamson, NA ; Ang, C-S ; Cheng, H-C (ELSEVIER, 2023-05)
    Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.
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    Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction
    Fujihara, KM ; Zhang, BZ ; Jackson, TD ; Ogunkola, MO ; Nijagal, B ; Milne, J ; Sallman, DA ; Ang, C-S ; Nikolic, I ; Kearney, CJ ; Hogg, SJ ; Cabalag, CS ; Sutton, VR ; Watt, S ; Fujihara, AT ; Trapani, JA ; Simpson, KJ ; Stojanovski, D ; Leimkuhler, S ; Haupt, S ; Phillips, WA ; Clemons, NJ (AMER ASSOC ADVANCEMENT SCIENCE, 2022-09-16)
    The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, although the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limiting the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.