Cruchaga, C. (Carlos)

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    Selective excision of chain-terminating nucleotides by HIV-1 reverse transcriptase with phosphonoformate as substrate
    (American Society for Biochemistry and Molecular Biology, 2006) Rouzaut, A. (Ana); Cruchaga, C. (Carlos); Anso, E. (Elena); Martinez-Irujo, J.J. (Juan José)
    A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.
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    A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer
    (Portland Press, 2007) Rouzaut, A. (Ana); Cruchaga, C. (Carlos); Font, M. (María); Anso, E. (Elena); Martinez-Irujo, J.J. (Juan José); Martino, V.S. (Virginia S.)
    Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.
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    Mendelian randomization implies no direct causal association between leukocyte telomere length and amyotrophic lateral sclerosis
    (2020) Boeve, B.F. (Bradley F.); Blesa, R. (Rafael); Petersen, R.C. (Ronald C.); Schlachetzki, J.C.M. (Johannes C. M.); Thompson, E. (Elizabeth); Scarpini, E. (Elio); Graf-Radford, N.R. (Neill R.); Benussi, L. (Luisa); Ortega-Cubero, S. (Sara); Riemenschneider, M. (Matthias); Baker, M. (Matt); Giaccone, G. (Giorgio); Tagliavini, F. (Fabrizio); Spillantini, M.G. (Maria Grazia); Piguet, O. (Olivier); Ibach, B. (Bernd); Dickson, D.W. (Dennis W.); Hjermind, L.E. (Lena E.); Forloni, G. (Gianluigi); Nilsson, C. (Christer); Pijnenburg, Y.A.L. (Yolande A. L.); Serpente, M. (Maria); Grifths, T.D. (Timothy D.); Morris, C.M. (Christopher M.); Pickering-Brown, S. (Stuart); Seelaar, H. (Harro); Landqvist-Waldö, M. (Maria); Binetti, G. (Giuliano); Rosen, H. (Howard); Albani, D. (Diego); Perneczky, R. (Robert); Heutink, P. (Peter); Brice, A. (Alexis); Mann, D.M.A. (David M.A.); Trojanowski, J.Q. (John Q.); Ferrari, R. (Rafaele); Ferrucci, L. (Luigi); Padovani, A. (Alessandro); Danek, A. (Adrian); Rossor, M.N. (Martin N.); Leber, I. (Isabelle); Grafman, J. (Jordan); Sorrentino, P. (Paolo); Graf, C. (Caroline); Novelli, V. (Valeria); Dobson-Stone, C. (Carol); Gu, W. (Wei); Cruchaga, C. (Carlos); Hernandez, D.G. (Dena G.); Fenoglio, C. (Chiara); Deramecourt, V. (Vincent); Anfossi, M. (Maria); Zhao, H. (Huashuo); Franceschi, M. (Massimo); Borroni, B. (Barbara); Grossman, M. (Murray); Kapogiannis, D. (Dimitrios); Van-der-Zee, J. (Julie); Bernardi, L. (Livia); Vercelletto, M. (Martine); Rogaeva, E. (Ekaterina); Hodges, J.R. (John R.); Richardson, A. (Anna); Ghidoni, R. (Roberta); Mayhaus, M. (Manuel); Scheltens, P. (Philip); McKeith, I.G. (Ian G.); Rollin, A. (Adeline); Frangipane, F. (Francesca); Pastor, P. (Pau); Wassermann, E.M. (Eric M.); Rainero, I. (Innocenzo); Nalls, M.A. (Michael A.); Rohrer, J.D. (Jonathan D.); Van-Swieten, J.C. (John C.); Josephs, K.A. (Keith A.); Diehl-Schmid, J. (Janine); Momeni, P. (Parastoo); Fox, N.C. (Nick C.); Jaros, E. (Evelyn); Uphill, J. (James); Rossi, G. (Giacomina); Piaceri, I. (Irene); Lebouvier, T. (Thibaud); Halliday, G.M. (Glenda M.); Bagnoli, S. (Silvia); Schofeld, P.R. (Peter R.); Lleó, A. (Alberto); Cruts, M. (Marc); Nacmias, B. (Benedetta); Puca, A.A. (Annibale A.); Ramasamy, A. (Adaikalavan); Singleton, A.B. (Andrew B.); Mead, S. (Simon); Alexopoulos, P. (Panagiotis); Baborie, A. (Atik); Haan, E. (Eric); Miller, B.L. (Bruce L.); Pasquier, F. (Florence); Wang, T. (Ting); Rowe, J.B. (James B.); Razquin, C. (Cristina); Van-Deerlin, V.M. (Vivianna M.); Milan, G. (Graziella ); Bartley, L. (Lauren); Collinge, J. (John); Pinessi, L. (Lorenzo); Hernández, I. (Isabel); Rademakers, R. (Rosa); Sorbi, S. (Sandro); Attems, J. (Johannes); Capozzo, R. (Rosa); Morris, H.R. (Huw R.); Hardy, J. (John); Nielsen, J.E. (Jørgen E.); Kristiansen, M. (Mark); Smirne, N. (Nicoletta); Galimberti, D. (Daniela); Maletta, R. (Rafaele); Zeng, P. (Ping); Knopman, D. (David); Ruiz, A. (Agustín); Karydas, A.M. (Anna M.); Pichler, S. (Sabrina); Brooks, W.S. (William S.); Nilsson, K. (Karin); Dopper, E.G.P. (Elise G. P.); Rubino, E. (Elisa); Clarimon, J. (Jordi); Hannequin, D. (Didier); Huey, E.D. (Edward D.); Alonso, E. (Elena); Pietrini, P. (Pietro); Cappa, S.F. (Stefano F.); Van-Broeckhoven, C. (Christine); Gerhard, A. (Alexander); Rollinson, S. (Sara); Thomas, A.J. (Alan J.); Conidi, M.E. (Maria Elena); Tierney, M.C. (Michael C.); Bruni, A.C. (Amalia C.); Rizzu, P. (Patrizia); Gallo, M. (Maura); Cairns, N.J. (Nigel J.); Chiang, H.H. (Huei Hsin); Kurz, A. (Alexander); Kwok, J.B.J. (John B. J.); Gao, Y. (Yixin); Parisi, J.E. (Joseph E.); Seely, W.W. (William W.); Yu, X. (Xinghao); St-George Hyslop, P. (Peter); Boada, M. (Mercè); Snowden, J.S. (Julie S.); Gasparoni, G. (Gilles); Warren, J.D. (Jason D.); Postiglione, A. (Alfredo); Logroscino, G. (Giancarlo); Mackenzie, I.R.A. (Ian R.A.); Cupidi, C. (Chiara); Golfer, V. (Véronique)
    We employed Mendelian randomization (MR) to evaluate the causal relationship between leukocyte telomere length (LTL) and amyotrophic lateral sclerosis (ALS) with summary statistics from genomewide association studies (n= ~ 38,000 for LTL and ~ 81,000 for ALS in the European population; n= ~ 23,000 for LTL and ~ 4,100 for ALS in the Asian population). We further evaluated mediation roles of lipids in the pathway from LTL to ALS. The odds ratio per standard deviation decrease of LTL on ALS was 1.10 (95% CI 0.93–1.31, p = 0.274) in the European population and 0.75 (95% CI 0.53–1.07, p = 0.116) in the Asian population. This null association was also detected between LTL and frontotemporal dementia in the European population. However, we found that an indirect efect of LTL on ALS might be mediated by low density lipoprotein (LDL) or total cholesterol (TC) in the European population. These results were robust against extensive sensitivity analyses. Overall, our MR study did not support the direct causal association between LTL and the ALS risk in neither population, but provided suggestive evidence for the mediation role of LDL or TC on the infuence of LTL and ALS in the European population.