Biochemistry and Pharmacology - Research Publications

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    Bromodomain and extraterminal protein-targeted probe enables tumour visualisation in vivo using positron emission tomography
    Dickmann, CGF ; McDonald, AFF ; Huynh, N ; Rigopoulos, A ; Liu, Z ; Guo, N ; Osellame, LDD ; Gorman, MAA ; Parker, MWW ; Gan, HKK ; Scott, AMM ; Ackermann, U ; Burvenich, IJG ; White, JMM (ROYAL SOC CHEMISTRY, 2023-02-14)
    Bromodomain and extraterminal (BET) proteins, a family of epigenetic regulators, have emerged as important oncology drug targets. BET proteins have not been targeted for molecular imaging of cancer. Here, we report the development of a novel molecule radiolabelled with positron emitting fluorine-18, [18F]BiPET-2, and its in vitro and preclinical evaluation in glioblastoma models.
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    Covalent Inhibition by a Natural Product-Inspired Latent Electrophile
    Byun, DPP ; Ritchie, J ; Jung, Y ; Holewinski, R ; Kim, H-R ; Tagirasa, R ; Ivanic, J ; Weekley, CMM ; Parker, MWW ; Andresson, T ; Yoo, E (AMER CHEMICAL SOC, 2023-05-15)
    Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.
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    Translating the biology of B common receptor- engaging cytokines into clinical medicine
    Pant, H ; Hercus, TR ; Tumes, DJ ; Yip, KH ; Parker, MW ; Owczarek, CM ; Lopez, AF ; Huston, DP (MOSBY-ELSEVIER, 2023-02)
    The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called β-common or βc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.
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    Structural biology of cell surface receptors implicated in Alzheimer's disease.
    Hermans, SJ ; Nero, TL ; Morton, CJ ; Gooi, JH ; Crespi, GAN ; Hancock, NC ; Gao, C ; Ishii, K ; Markulić, J ; Parker, MW (Springer Science and Business Media LLC, 2022-02)
    Alzheimer's disease is a common and devastating age-related disease with no effective disease-modifying treatments. Human genetics has implicated a wide range of cell surface receptors as playing a role in the disease, many of which are involved in the production or clearance of neurotoxins in the brain. Amyloid precursor protein, a membrane-bound signaling molecule, is at the very heart of the disease: hereditary mutations in its gene are associated with a greatly increased risk of getting the disease. A proteolytic breakdown product of amyloid precursor protein, the neurotoxic Aβ peptide, has been the target for many drug discovery efforts. Antibodies have been designed to target Aβ production with some success, although they have not proved efficacious in clinical trials with regards to cognitive benefits to date. Many of the recently identified genes associated with late-onset Alzheimer's disease risk are integral to the innate immune system. Some of these genes code for microglial proteins, such as the strongest genetic risk factor for the disease, namely APOE, and the cell surface receptors CD33 and TREM2 which are involved in clearance of the Aβ peptide from the brain. In this review, we show how structural biology has provided key insights into the normal functioning of these cell surface receptors and provided a framework for developing novel treatments to combat Alzheimer's disease.
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    Cholesterol-dependent cytolysins: The outstanding questions
    Johnstone, BA ; Joseph, R ; Christie, MP ; Morton, CJ ; McGuiness, C ; Walsh, JC ; Bocking, T ; Tweten, RK ; Parker, MW (WILEY, 2022-07-14)
    The cholesterol-dependent cytolysins (CDCs) are a major family of bacterial pore-forming proteins secreted as virulence factors by Gram-positive bacterial species. CDCs are produced as soluble, monomeric proteins that bind specifically to cholesterol-rich membranes, where they oligomerize into ring-shaped pores of more than 30 monomers. Understanding the details of the steps the toxin undergoes in converting from monomer to a membrane-spanning pore is a continuing challenge. In this review we summarize what we know about CDCs and highlight the remaining outstanding questions that require answers to obtain a complete picture of how these toxins kill cells.
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    A novel phosphocholine-mimetic inhibits a pro-inflammatory conformational change in C-reactive protein
    Zeller, J ; Shing, KSCT ; Nero, TL ; McFadyen, JD ; Krippner, G ; Bogner, B ; Kreuzaler, S ; Kiefer, J ; Horner, VK ; Braig, D ; Danish, H ; Baratchi, S ; Fricke, M ; Wang, X ; Kather, MG ; Kammerer, B ; Woollard, KJ ; Sharma, P ; Morton, CJ ; Pietersz, G ; Parker, MW ; Peter, K ; Eisenhardt, SU (WILEY, 2022-12-05)
    C-reactive protein (CRP) is an early-stage acute phase protein and highly upregulated in response to inflammatory reactions. We recently identified a novel mechanism that leads to a conformational change from the native, functionally relatively inert, pentameric CRP (pCRP) structure to a pentameric CRP intermediate (pCRP*) and ultimately to the monomeric CRP (mCRP) form, both exhibiting highly pro-inflammatory effects. This transition in the inflammatory profile of CRP is mediated by binding of pCRP to activated/damaged cell membranes via exposed phosphocholine lipid head groups. We designed a tool compound as a low molecular weight CRP inhibitor using the structure of phosphocholine as a template. X-ray crystallography revealed specific binding to the phosphocholine binding pockets of pCRP. We provide in vitro and in vivo proof-of-concept data demonstrating that the low molecular weight tool compound inhibits CRP-driven exacerbation of local inflammatory responses, while potentially preserving pathogen-defense functions of CRP. The inhibition of the conformational change generating pro-inflammatory CRP isoforms via phosphocholine-mimicking compounds represents a promising, potentially broadly applicable anti-inflammatory therapy.
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    Heparin Inhibits SARS-CoV-2 Replication in Human Nasal Epithelial Cells
    Lee, LYY ; Suryadinata, R ; McCafferty, C ; Ignjatovic, V ; Purcell, DFJ ; Robinson, P ; Morton, CJ ; Parker, MW ; Anderson, GP ; Monagle, P ; Subbarao, K ; Neil, JA (MDPI, 2022-12-01)
    SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies.
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    Single-molecule analysis of the entire perfringolysin O pore formation pathway
    Mc Guinness, C ; Walsh, JC ; Bayly-Jones, C ; Dunstone, MA ; Christie, MP ; Morton, CJ ; Parker, MW ; Bocking, T (eLIFE SCIENCES PUBL LTD, 2022-08-24)
    The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
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    Inhibitors of the Oncogenic PA2G4-MYCN Protein-Protein Interface
    Massudi, H ; Luo, J-S ; Holien, JK ; Gadde, S ; Krishan, S ; Herath, M ; Koach, J ; Stevenson, BW ; Gorman, MA ; Venkat, P ; Mayoh, C ; Luo, X-Q ; Parker, MW ; Cheung, BB ; Marshall, GM (MDPI, 2023-03-01)
    MYCN is a major oncogenic driver for neuroblastoma tumorigenesis, yet there are no direct MYCN inhibitors. We have previously identified PA2G4 as a direct protein-binding partner of MYCN and drive neuroblastoma tumorigenesis. A small molecule known to bind PA2G4, WS6, significantly decreased tumorigenicity in TH-MYCN neuroblastoma mice, along with the inhibition of PA2G4 and MYCN interactions. Here, we identified a number of novel WS6 analogues, with 80% structural similarity, and used surface plasmon resonance assays to determine their binding affinity. Analogues #5333 and #5338 showed direct binding towards human recombinant PA2G4. Importantly, #5333 and #5338 demonstrated a 70-fold lower toxicity for normal human myofibroblasts compared to WS6. Structure-activity relationship analysis showed that a 2,3 dimethylphenol was the most suitable substituent at the R1 position. Replacing the trifluoromethyl group on the phenyl ring at the R2 position, with a bromine or hydrogen atom, increased the difference between efficacy against neuroblastoma cells and normal myofibroblast toxicity. The WS6 analogues inhibited neuroblastoma cell phenotype in vitro, in part through effects on apoptosis, while their anti-cancer effects required both PA2G4 and MYCN expression. Collectively, chemical inhibition of PA2G4-MYCN binding by WS6 analogues represents a first-in-class drug discovery which may have implications for other MYCN-driven cancers.
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    C-reactive protein, immunothrombosis and venous thromboembolism
    Dix, C ; Zeller, J ; Stevens, H ; Eisenhardt, SU ; Shing, KSCT ; Nero, TL ; Morton, CJ ; Parker, MW ; Peter, K ; McFadyen, JD (FRONTIERS MEDIA SA, 2022-09-13)
    C-reactive protein (CRP) is a member of the highly conserved pentraxin superfamily of proteins and is often used in clinical practice as a marker of infection and inflammation. There is now increasing evidence that CRP is not only a marker of inflammation, but also that destabilized isoforms of CRP possess pro-inflammatory and pro-thrombotic properties. CRP circulates as a functionally inert pentameric form (pCRP), which relaxes its conformation to pCRP* after binding to phosphocholine-enriched membranes and then dissociates to monomeric CRP (mCRP). with the latter two being destabilized isoforms possessing highly pro-inflammatory features. pCRP* and mCRP have significant biological effects in regulating many of the aspects central to pathogenesis of atherothrombosis and venous thromboembolism (VTE), by directly activating platelets and triggering the classical complement pathway. Importantly, it is now well appreciated that VTE is a consequence of thromboinflammation. Accordingly, acute VTE is known to be associated with classical inflammatory responses and elevations of CRP, and indeed VTE risk is elevated in conditions associated with inflammation, such as inflammatory bowel disease, COVID-19 and sepsis. Although the clinical data regarding the utility of CRP as a biomarker in predicting VTE remains modest, and in some cases conflicting, the clinical utility of CRP appears to be improved in subsets of the population such as in predicting VTE recurrence, in cancer-associated thrombosis and in those with COVID-19. Therefore, given the known biological function of CRP in amplifying inflammation and tissue damage, this raises the prospect that CRP may play a role in promoting VTE formation in the context of concurrent inflammation. However, further investigation is required to unravel whether CRP plays a direct role in the pathogenesis of VTE, the utility of which will be in developing novel prophylactic or therapeutic strategies to target thromboinflammation.