Biochemistry and Pharmacology - Research Publications

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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 ; Markulic, J ; Parker, MW (SPRINGERNATURE, 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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    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, 2023-01-11)
    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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    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.
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    A dual role for the N-terminal domain of the IL-3 receptor in cell signalling
    Broughton, SE ; Hercus, TR ; Nero, TL ; Kan, WL ; Barry, EF ; Dottore, M ; Shing, KSCT ; Morton, CJ ; Dhagat, U ; Hardy, MP ; Wilson, NJ ; Downton, MT ; Schieber, C ; Hughes, TP ; Lopez, AF ; Parker, MW (NATURE PUBLISHING GROUP, 2018-01-26)
    The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.
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    Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites
    Braig, D ; Nero, TL ; Koch, H-G ; Kaiser, B ; Wang, X ; Thiele, JR ; Morton, CJ ; Zeller, J ; Kiefer, J ; Potempa, LA ; Mellett, NA ; Miles, LA ; Du, X-J ; Meikle, PJ ; Huber-Lang, M ; Stark, GB ; Parker, MW ; Peter, K ; Eisenhardt, SU (NATURE PUBLISHING GROUP, 2017-01-23)
    C-reactive protein (CRP) concentrations rise in response to tissue injury or infection. Circulating pentameric CRP (pCRP) localizes to damaged tissue where it leads to complement activation and further tissue damage. In-depth knowledge of the pCRP activation mechanism is essential to develop therapeutic strategies to minimize tissue injury. Here we demonstrate that pCRP by binding to cell-derived microvesicles undergoes a structural change without disrupting the pentameric symmetry (pCRP*). pCRP* constitutes the major CRP species in human-inflamed tissue and allows binding of complement factor 1q (C1q) and activation of the classical complement pathway. pCRP*-microvesicle complexes lead to enhanced recruitment of leukocytes to inflamed tissue. A small-molecule inhibitor of pCRP (1,6-bis(phosphocholine)-hexane), which blocks the pCRP-microvesicle interactions, abrogates these proinflammatory effects. Reducing inflammation-mediated tissue injury by therapeutic inhibition might improve the outcome of myocardial infarction, stroke and other inflammatory conditions.
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    Potent hepatitis C inhibitors bind directly to NS5A and reduce its affinity for RNA
    Ascher, DB ; Wielens, J ; Nero, TL ; Doughty, L ; Morton, CJ ; Parker, MW (NATURE PORTFOLIO, 2014-04-23)
    Hepatitis C virus (HCV) infection affects more than 170 million people. The high genetic variability of HCV and the rapid development of drug-resistant strains are driving the urgent search for new direct-acting antiviral agents. A new class of agents has recently been developed that are believed to target the HCV protein NS5A although precisely where they interact and how they affect function is unknown. Here we describe an in vitro assay based on microscale thermophoresis and demonstrate that two clinically relevant inhibitors bind tightly to NS5A domain 1 and inhibit RNA binding. Conversely, RNA binding inhibits compound binding. The compounds bind more weakly to known resistance mutants L31V and Y93H. The compounds do not affect NS5A dimerisation. We propose that current NS5A inhibitors act by favouring a dimeric structure of NS5A that does not bind RNA.