Biochemistry and Pharmacology - Research Publications

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    Copper binding to the Alzheimer's disease amyloid precursor protein
    Kong, GK-W ; Miles, LA ; Crespi, GAN ; Morton, CJ ; Ng, HL ; Barnham, KJ ; McKinstry, WJ ; Cappai, R ; Parker, MW (SPRINGER, 2008-03-01)
    Alzheimer's disease is the fourth biggest killer in developed countries. Amyloid precursor protein (APP) plays a central role in the development of the disease, through the generation of a peptide called A beta by proteolysis of the precursor protein. APP can function as a metalloprotein and modulate copper transport via its extracellular copper binding domain (CuBD). Copper binding to this domain has been shown to reduce A beta levels and hence a molecular understanding of the interaction between metal and protein could lead to the development of novel therapeutics to treat the disease. We have recently determined the three-dimensional structures of apo and copper bound forms of CuBD. The structures provide a mechanism by which CuBD could readily transfer copper ions to other proteins. Importantly, the lack of significant conformational changes to CuBD on copper binding suggests a model in which copper binding affects the dimerisation state of APP leading to reduction in A beta production. We thus predict that disruption of APP dimers may be a novel therapeutic approach to treat Alzheimer's disease.
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    Bapineuzumab captures the N-terminus of the Alzheimer's disease amyloid-beta peptide in a helical conformation
    Miles, LA ; Crespi, GAN ; Doughty, L ; Parker, MW (NATURE PUBLISHING GROUP, 2013-02-18)
    Bapineuzumab is a humanized antibody developed by Pfizer and Johnson & Johnson targeting the amyloid (Aβ) plaques that underlie Alzheimer's disease neuropathology. Here we report the crystal structure of a Fab-Aβ peptide complex that reveals Bapineuzumab surprisingly captures Aβ in a monomeric helical conformation at the N-terminus. Microscale thermophoresis suggests that the Fab binds soluble Aβ(1-40) with a K(D) of 89 (±9) nM. The structure explains the antibody's exquisite selectivity for particular Aβ species and why it cannot recognize N-terminally modified or truncated Aβ peptides.
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    Small Molecule Binding to Alzheimer Risk Factor CD33 Promotes A beta Phagocytosis
    Miles, LA ; Hermans, SJ ; Crespi, GAN ; Gooi, JH ; Doughty, L ; Nero, TL ; Markulic, J ; Ebneth, A ; Wroblowski, B ; Oehlrich, D ; Trabanco, AA ; Rives, M-L ; Royaux, I ; Hancock, NC ; Parker, MW (CELL PRESS, 2019-09-27)
    Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-β (Aβ), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aβ peptide that is thought to cause AD.
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    Anti-A beta antibody target engagement: a response to Siemers et al.
    Watt, AD ; Crespi, GAN ; Down, RA ; Ascher, DB ; Gunn, A ; Perez, KA ; McLean, CA ; Villemagne, VL ; Parker, MW ; Barnham, KJ ; Miles, LA (SPRINGER, 2014-10-01)
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    Molecular basis for mid-region amyloid-beta capture by leading Alzheimer's disease immunotherapies
    Crespi, GAN ; Hermans, SJ ; Parker, MW ; Miles, LA (NATURE PUBLISHING GROUP, 2015-04-16)
    Solanezumab (Eli Lilly) and crenezumab (Genentech) are the leading clinical antibodies targeting Amyloid-β (Aβ) to be tested in multiple Phase III clinical trials for the prevention of Alzheimer's disease in at-risk individuals. Aβ capture by these clinical antibodies is explained here with the first reported mid-region Aβ-anti-Aβ complex crystal structure. Solanezumab accommodates a large Aβ epitope (960 Å(2) buried interface over residues 16 to 26) that forms extensive contacts and hydrogen bonds to the antibody, largely via main-chain Aβ atoms and a deeply buried Phe19-Phe20 dipeptide core. The conformation of Aβ captured is an intermediate between observed sheet and helical forms with intramolecular hydrogen bonds stabilising residues 20-26 in a helical conformation. Remarkably, Aβ-binding residues are almost perfectly conserved in crenezumab. The structure explains the observed shared cross reactivity of solanezumab and crenezumab with proteins abundant in plasma that exhibit this Phe-Phe dipeptide.
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    Promiscuous DNA-binding of a mutant zinc finger protein corrupts the transcriptome and diminishes cell viability
    Gillinder, KR ; Ilsley, MD ; Nebor, D ; Sachidanandam, R ; Lajoie, M ; Magor, GW ; Tallack, MR ; Bailey, T ; Landsberg, MJ ; Mackay, JP ; Parker, MW ; Miles, LA ; Graber, JH ; Peters, LL ; Bieker, JJ ; Perkins, AC (OXFORD UNIV PRESS, 2017-02-01)
    The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.
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    Ex vivo O-18-labeling mass spectrometry identifies a peripheral amyloid beta clearance pathway
    Portelius, E ; Mattsson, N ; Pannee, J ; Zetterberg, H ; Gisslen, M ; Vanderstichele, H ; Gkanatsiou, E ; Crespi, GAN ; Parker, MW ; Miles, LA ; Gobom, J ; Blennow, K (BIOMED CENTRAL LTD, 2017-02-20)
    BACKGROUND: Proteolytic degradation of amyloid β (Aβ) peptides has been intensely studied due to the central role of Aβ in Alzheimer's disease (AD) pathogenesis. While several enzymes have been shown to degrade Aβ peptides, the main pathway of Aβ degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aβ42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aβ42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis. METHOD: Using 18O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aβ in human CSF. RESULTS: The Aβ peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18O-labeling mass spectrometry identified proteolytic activities degrading Aβ into several short fragments, including abundant Aβ1-19 and 1-20. After antibiotic treatment, no degradation of Aβ was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aβ antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aβ degradation. CONCLUSION: 18O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aβ in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aβ antibody solanezumab.
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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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    Do current therapeutic anti-A beta antibodies for Alzheimer's disease engage the target?
    Watt, AD ; Crespi, GAN ; Down, RA ; Ascher, DB ; Gunn, A ; Perez, KA ; McLean, CA ; Villemagne, VL ; Parker, MW ; Barnham, KJ ; Miles, LA (SPRINGER, 2014-06-01)
    Reducing amyloid-β peptide (Aβ) burden at the pre-symptomatic stages of Alzheimer's disease (AD) is currently the advocated clinical strategy for treating this disease. The most developed method for targeting Aβ is the use of monoclonal antibodies including bapineuzumab, solanezumab and crenezumab. We have synthesized these antibodies and used surface plasmon resonance (SPR) and mass spectrometry to characterize and compare the ability of these antibodies to target Aβ in transgenic mouse tissue as well as human AD tissue. SPR analysis showed that the antibodies were able to bind Aβ with high affinity. All of the antibodies were able to bind Aβ in mouse tissue. However, significant differences were observed in human brain tissue. While bapineuzumab was able to capture a variety of N-terminally truncated Aβ species, the Aβ detected using solanezumab was barely above detection limits while crenezumab did not detect any Aβ. None of the antibodies were able to detect any Aβ species in human blood. Immunoprecipitation experiments using plasma from AD subjects showed that both solanezumab and crenezumab have extensive cross-reactivity with non-Aβ related proteins. Bapineuzumab demonstrated target engagement with brain Aβ, consistent with published clinical data. Solanezumab and crenezumab did not, most likely as a result of a lack of specificity due to cross-reactivity with other proteins containing epitope overlap. This lack of target engagement raises questions as to whether solanezumab and crenezumab are suitable drug candidates for the preventative clinical trials for AD.
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    Synthetic dityrosine-linked beta-amyloid dimers form stable, soluble, neurotoxic oligomers
    Kok, WM ; Cottam, JM ; Ciccotosto, GD ; Miles, LA ; Karas, JA ; Scanlon, DB ; Roberts, BR ; Parker, MW ; Cappai, R ; Barnham, KJ ; Hutton, CA (ROYAL SOC CHEMISTRY, 2013-01-01)