Biochemistry and Pharmacology - Research Publications

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Now showing 1 - 10 of 2019
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    Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure
    Faysal, KMR ; Walsh, JC ; Renner, N ; Marquez, CL ; Shah, VB ; Tuckwell, AJ ; Christie, MP ; Parker, MW ; Turville, SG ; Towers, GJ ; James, LC ; Jacques, DA ; Bocking, T (eLIFE SCIENCES PUBL LTD, 2024-02-13)
    The HIV-1 capsid has emerged as a tractable target for antiretroviral therapy. Lenacapavir, developed by Gilead Sciences, is the first capsid-targeting drug approved for medical use. Here, we investigate the effect of lenacapavir on HIV capsid stability and uncoating. We employ a single particle approach that simultaneously measures capsid content release and lattice persistence. We demonstrate that lenacapavir's potent antiviral activity is predominantly due to lethal hyperstabilisation of the capsid lattice and resultant loss of compartmentalisation. This study highlights that disrupting capsid metastability is a powerful strategy for the development of novel antivirals.
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    Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis
    Patitucci, C ; Hernandez-Camacho, JD ; Vimont, E ; Yde, S ; Cokelaer, T ; Chaze, T ; Gianetto, QG ; Matondo, M ; Gazi, A ; Nemazanyy, I ; Stroud, DA ; Hock, DH ; Donnarumma, E ; Wai, T (NATURE PORTFOLIO, 2023-12-20)
    Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.
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    The broccoli-derived antioxidant sulforaphane changes the growth of gastrointestinal microbiota, allowing for the production of anti-inflammatory metabolites
    Marshall, SA ; Young, RB ; Lewis, JM ; Rutten, EL ; Gould, J ; Barlow, CK ; Giogha, C ; Marcelino, VR ; Fields, N ; Schittenhelm, RB ; Hartland, EL ; Scott, NE ; Forster, SC ; Gulliver, EL (ELSEVIER, 2023-08)
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    Multi-targeted loss of the antigen presentation molecule MR1 during HSV-1 and HSV-2 infection.
    Samer, C ; McWilliam, HEG ; McSharry, BP ; Velusamy, T ; Burchfield, JG ; Stanton, RJ ; Tscharke, DC ; Rossjohn, J ; Villadangos, JA ; Abendroth, A ; Slobedman, B (Elsevier BV, 2024-02-16)
    The major histocompatibility complex (MHC), Class-I-related (MR1) molecule presents microbiome-synthesized metabolites to Mucosal-associated invariant T (MAIT) cells, present at sites of herpes simplex virus (HSV) infection. During HSV type 1 (HSV-1) infection there is a profound and rapid loss of MR1, in part due to expression of unique short 3 protein. Here we show that virion host shutoff RNase protein downregulates MR1 protein, through loss of MR1 transcripts. Furthermore, a third viral protein, infected cell protein 22, also downregulates MR1, but not classical MHC-I molecules. This occurs early in the MR1 trafficking pathway through proteasomal degradation. Finally, HSV-2 infection results in the loss of MR1 transcripts, and intracellular and surface MR1 protein, comparable to that seen during HSV-1 infection. Thus HSV coordinates a multifaceted attack on the MR1 antigen presentation pathway, potentially protecting infected cells from MAIT cell T cell receptor-mediated detection at sites of primary infection and reactivation.
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    Fetal drug exposure after maternally administered CFTR modulators Elexacaftor/Tezacaftor/Ivacaftor in a rat model.
    Li, D ; Zhu, Y ; Donnelley, M ; Parsons, D ; Habgood, MD ; Schneider-Futschik, EK (Elsevier BV, 2024-02)
    BACKGROUND: The potential effects of the very effective cystic fibrosis triple combination drug, Elexacaftor/Tezacaftor/Ivacaftor (ETI) in pregnancy on prenatal development of offspring remain largely unknown. RESEARCH QUESTION: We aimed to investigate the fetal tissue distribution pattern of maternally administered ETI by placental transfer in the rat fetuses. STUDY DESIGN AND METHODS: Sprague Dawley pregnant rats were administered ETI (6.7 mg/kg/d elexacaftor + 3.5 mg/kg/d tezacaftor + 25 mg/kg/d ivacaftor) traced with [3 H]-ivacaftor in single dose acute experiments (intraperitoneal injection) or treated orally with ETI (the same dose) for 7 days in sub-chronic experiments. Fetal tissue samples were collected at embryonic day (E) 19 and analyzed using liquid scintillation counting for acute experiments or liquid chromatography-mass spectrometry for sub-chronic experiments. RESULTS: On day E19, after acute exposure, the entry of ivacaftor into fetal brain (brain/plasma concentration ratios <50%) was significantly lower than to other tissues (>100%). However, after sub-chronic exposure, the entry of all 3 components into the developing brain was comparably extensive as into other tissues (tissue/plasma ratios, 260 - 1000%). Each component of ETI accumulated in different fetal tissues to approximately equal extent. Inter-litter differences on fetal drug distribution were found in cortex for ivacaftor, muscle for tezacaftor and cortex and mid/hindbrain for elexacaftor. Fetal plasma concentrations of ETI (ng/mL) were variable between litters. The entry of ivacaftor and tezacaftor into adult brain appeared to be restricted (<100%). INTERPRETATION: Fetal rats are exposed to maternally ingested ETI after sub-chronic exposure, potentially impacting fetal development. The brain entry data highlights the need for attention be paid to any long-term potential effects ETI exposure could have on normal brain development.
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    Nuclear export of circular RNA.
    Ngo, LH ; Bert, AG ; Dredge, BK ; Williams, T ; Murphy, V ; Li, W ; Hamilton, WB ; Carey, KT ; Toubia, J ; Pillman, KA ; Liu, D ; Desogus, J ; Chao, JA ; Deans, AJ ; Goodall, GJ ; Wickramasinghe, VO (Springer Science and Business Media LLC, 2024-03)
    Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.
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    Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist
    Abdul-Ridha, A ; de Zhang, LA ; Betrie, AH ; Deluigi, M ; Vaid, TM ; Whitehead, A ; Zhang, Y ; Davis, B ; Harris, R ; Simmonite, H ; Hubbard, RE ; Gooley, PR ; Plu''ckthun, A ; Bathgate, RAD ; Chalmers, DK ; Scott, DJ (AMER CHEMICAL SOC, 2024-01-18)
    α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.
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    Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin
    Segeritz, P ; Kolesnik, K ; Scott, DJ ; Collins, DJ (ROYAL SOC CHEMISTRY, 2024-03-12)
    Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.
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    Vasculature is getting Hip(po): Hippo in vascular development and disease
    Kobayashi, S ; Cox, AG ; Harvey, KF ; Hogan, BM (CELL PRESS, 2023-12-04)
    The Hippo signaling pathway regulates developmental organ growth, regeneration, and cell fate decisions. Although the role of the Hippo pathway, and its transcriptional effectors YAP and TAZ, has been well documented in many cell types and species, only recently have the roles for this pathway come to light in vascular development and disease. Experiments in mice, zebrafish, and in vitro have uncovered roles for the Hippo pathway, YAP, and TAZ in vasculogenesis, angiogenesis, and lymphangiogenesis. In addition, the Hippo pathway has been implicated in vascular cancers and cardiovascular diseases, thus identifying it as a potential therapeutic target for the treatment of these conditions. However, despite recent advances, Hippo's role in the vasculature is still underappreciated compared with its role in epithelial tissues. In this review, we appraise our current understanding of the Hippo pathway in blood and lymphatic vessel development and highlight the current knowledge gaps and opportunities for further research.
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    Structural Insights into SARS-CoV-2 Nonstructural Protein 1 Interaction with Human Cyclophilin and FKBP1 to Regulate Interferon Production
    Vankadari, N ; Ghosal, D (AMER CHEMICAL SOC, 2024-01-19)
    The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 coronavirus and the perpetual rise of new variants warrant investigation of the molecular and structural details of the infection process and modulation of the host defense by viral proteins. This Letter reports the combined experimental and computational approaches to provide key insights into the structural and functional basis of Nsp1's association with different cyclophilins and FKBPs in regulating COVID-19 infection. We demonstrated the real-time stability and functional dynamics of the Nsp1-CypA/FKBP1A complex and investigated the repurposing of potential inhibitors that could block these interactions. Overall, we provided insights into the inhibitory role Nsp1 in downstream interferon production, a key aspect for host defense that prevents the SARS-CoV-2 or related family of corona virus infection.