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Design, synthesis and evaluation of new antidotes in nerve agent and pesticide poisoning – CHOLINESTERASE

Project head: Zrinka Kovarik

TEAM MEMBERS

Funded by the Croatian Science Foundation

1 Oct 2014 – 30 Sept 2018

ABSTRACT
Our main goal is to develop new compounds to serve as more efficient antidotes and improve the treatment of highly toxic organophosphorus (OP) compound poisoning. OP compounds used as pesticides account for over 3,000,000 registered accidental or deliberate cases of poisoning per year worldwide. Furthermore, OPs known as nerve agents (soman, sarin, tabun, VX) present a threat in terrorist attacks and conflicts, as was the case recently in Syria. The main targets of OP compounds are cholinesterases: acetylcholinesterase (AChE), the essential enzyme in neurotransmission, and butyrylcholinesterase (BChE), its back-up enzyme. However, the antidotes currently in use, which act as reactivators of inhibited AChE, were empirically synthesized before the two enzyme’s crystal structures were resolved. Due to structural requirements, their binding affinity and reactivation rate have not been well-balanced. This project utilizes new compounds to gain a better understanding of the mechanistic basis of the limitations of reactivation and find new effective leads for further in vivo study. We combine several approaches, including: a study of the finely tuned interplay between these two sister enzymes, computational and experimental studies of cholinesterase interactions with a wide range of ligands defining favourable characteristic for potential new antidotes, in silico design of novel compounds that direct the subsequent synthesis of selected leads, and thorough in vitro and in vivo experimental evaluation guided by strict cost-benefit criteria. Such a comprehensive approach enables us to test a wide selection of candidates in a more reliable manner and obtain unambiguous data for further enhancements of the antidotal treatment. This also enables us to explore other possible OP treatments such as highly-effective bioscavengers. Many of the findings that would arise from this project should have an impact that reaches far beyond the level of cholinesterases.

SELECT PUBLICATIONS

  1. Z. Kovarik, J. Kalisiak, N. Maček Hrvat, M. Katalinić, T. Zorbaz, S. Žunec, C. Green, Z. Radić, V. V. Fokin, K. B. Sharpless, P. Taylor: Reversal of tabun toxicity enabled by a triazole-annulated oxime library – reactivators of acetylcholinesterase. Chemistry – A European Journal (2018) [IF = 5.317, Q1].
  2. T. Zorbaz, D. Malinak, N. Maraković, N. Maček Hrvat, A. Zandona, M. Novotny, A. Skarka, R. Andrys, M. Benkova, O. Soukup, M. Katalinić, K. Kuca, Z. Kovarik, K. Musilek: Pyridinium Oximes with Ortho-Positioned Chlorine Moiety Exhibit Improved Physicochemical Properties and Efficient Reactivation of Human Acetylcholinesterase Inhibited by Several Nerve Agents. Journal of Medicinal Chemistry 61 (2018) 10753–10766 [IF = 6.253, Q1].
  3. M. Katalinić, G. Šinko, N. Maček Hrvat, T. Zorbaz, A. Bosak, Z. Kovarik. Oxime-assisted reactivation of tabun-inhibited acetylcholinesterase analysed by active site mutations. Toxicology 104-113 (2018) 406–407 [IF = 3.265, Q2].
  4. T. Zorbaz, A. Braïki, N. Maraković, J. Renou, E. de la Mora, N. Maček Hrvat, M. Katalinić, I. Silman, J. L. Sussman, G. Mercey, C. Gomez, R. Mougeot, B. Pérez, R. Baati, F. Nachon, M. Weik, L. Jean, Z. Kovarik, P.-Y. Renard: Potent 3-hydroxy-2-pyridine aldoxime reactivators of organophosphate-inhibited cholinesterases with predicted bloodbrain barrier penetration, Chemistry – A European Journal (2018) [IF = 5.317, Q1].
  5. Maček Hrvat, T. Zorbaz, G. Šinko, Z. Kovarik: The estimation of oxime efficiency is affected by the experimental design of phosphylated acetylcholinesterase reactivation, Toxicology letters (2017) [IF=3.858, Q1].
  6. Bosak, A. Knezević, I. Gazić Smilović, G. Šinko, Z. Kovarik: Resorcinol-, catechol- and saligenin-based bronchodilating beta2-agonists as inhibitors of human cholinesterase activity, Journal of Enzyme Inhibition and Medicinal Chemistry 32 (2017) 789–797 [IF = 4.293, Q1].
  7. Maček Hrvat, S. Žunec, P. Taylor, Z. Radić, Z. Kovarik: HI-6 assisted catalytic scavenging of VX by acetylcholinesterase choline binding site mutants, Chemico-biological interactions 259 (2016) B 148-153 [IF=2.618, Q2].
  8. Maraković, A. Knežević, V. Vinković, Z. Kovarik, G. Šinko: Design and synthesis of N-substituted-2-hydroxyiminoacetamides and interactions with cholinesterases, Chemico-biological interactions 259 (2016) B 122–132 [IF=2.618, Q2].
  9. Katalinić, N. Maček Hrvat, K. Baumann, S. Morasi Piperčić, S. Makarić, S. Tomić, O. Jović, T. Hrenar, A. Miličević, D. Jelić, S. Žunec, 
I. Primožič, Z. Kovarik: A comprehensive evaluation of novel oximes in creation of butyrylcholinesterase-based nerve agent bioscavengers, Toxicology and Applied Pharmacology 310 (2016) 195–204 [IF = 3.791, Q1].

OTHER PUBLICATIONS

Available here.

PUBLIC/INVITED LECTURES

  1. T. Zorbaz: Novi pristup analizi oksima dizajniranih za zaštitu središnjeg živčanog sustava pri trovanju organofosfornim spojevima, 3 Dec 2018, organizer: IMI.
  2. Z. Kovarik: Pseudo-catalytic nerve agents scavenging by acetylcholinesterase assisted with aldoximes, 11.10.2018., organizatori: Cuban Society of Chemistry (SCQ) and Latin-American Federation of Chemical Associations (FLAQ), „33. Latin-American Congreee of Chemistry (33-CLAQ) and X Congress of Chemical Sciences, Technology and Inovation“ (QUIMICUBA 2018), Havana, Cuba.
  3. Z. Kovarik: Enhancement in pyridinium oxime-assisted reactivation of tabun-inhibited acetylcholinesterase achieved by active site mutations, 10.9.2018., organizer: University of Defence, Faculty of Military Health Sciences, Czech Republic, „The 13th International Meeting on Cholinesterases and the 7th International Conference on Paraoxonases“, Hradec Králové, Czech Republic.
  4. G. Šinko: Assessment of scoring functions for AChE-ligand interactions, 10.9.2018., organizator: University of Defence, Faculty of Military Health Sciences, Czech Republic, „The 13th International Meeting on Cholinesterases and the 7th International Conference on Paraoxonases“, Hradec Králové, Czech Republic.
  5. T. Zorbaz: Potent lipophilic reactivators of phosphorylated cholinesterases are not cytotoxic and are metabolically stable, 4.9.2018., organizer: Hungarian Society of Biochemistry, Slovene Society of Biochemistry, Croatian Society of Biochemistry and Molecular Biology, FEBS3+ conference „From molecules to living systems“, Siófok, Hungary, 2018.
  6. Z. Kovarik: Reactivation of inhibited acetylcholinesterase with newly synthesized chlorinated pyridinium aldoximes, 9.7.2018., organizer: FEBS, „ FEBS Open Bio, 43rd FEBS Congress“, Prague, Czech Republic.
  7. Z. Kovarik: Catalytic organophosphorus compounds scavenging by acetylcholinesterase assisted with aldoximes, 31 August 2017, organizer: St. John’s University, College of Pharmacy and Health Sciences, Jamaica, Queens, NY, USA
  8. N. Maraković: Development of new chiral 2-hydroxyiminoacetamide reactivators of phosphylated cholinesterases, 20 June 2017, organizer: Université de Rouen, Rouen, France
  9. M. Katalinić: Terapija kod otrovanja organofosfornim spojevima: od laboratorija do klinike i nazad, 13 June 2017, organizer: Croatian Society of Biochemistry and Molecular Biology
  10. M. Katalinić: Organophosphorus compound poisoning: from benchtop to clinic and back, 28 May 2017, organizer: „Skeletal muscle research – from cell to human 2017ˮ, Symposium and Workshop, Ljubljana, Slovenia
  11. T. Zorbaz: New uncharged potent reactivators of AChE and BChE inhibited by nerve agents, 5 April 2017, organizer: Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
  12. N. Maček Hrvat: Sva lica organofosfata, 25 November 2016, organizer: IMROH
  13. Z. Kovarik: Efficient detoxification of soman, tabun, and VX by oxime assisted reactivation of acetylcholinesterase mutants, 20 October 2016, organizer: „The XVth International Symposium on Cholinergic Mechanismsˮ, Marseille, France
  14. T. Zorbaz: The effects of organophosphorus compounds and novel antidotes on human neuronal cells, 18 October 2016, organizer: Société des Neurosciences, Marseille, France
  15. Z. Kovarik: Katalitička detoksikacija organofosfornih spojeva acetilkolinesterazom s aldoksimima, 12 July 2016, organiser: Ruđer Bošković Institute
  16. A. Bosak: Enzimi: što s njima, a što bez njih? 27 November 2015, organiser: IMROH
  17. M. Katalinić: Efficiency of imidazolium based oximes in reactivation of organophosphorus compound‐inhibited cholinesterases, 29 September 2015., organizator: „12th International Meeting on Cholinesterases and 6th Paraoxonase Conferenceˮ, Elche, Spain
  18. Z. Kovarik: Catalytic organophosphorus compounds scavenging by acetylcholinesterase assisted with aldoximes, 28 September 2015., organizator: „12th International Meeting on Cholinesterases and 6th Paraoxonase Conferenceˮ, Elche, Spain
  19. Z. Kovarik: Catalytic scavenging of nerve agents by acetylcholinesterase assisted with aldoximes, 19 May 2015., organizator: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA
  20. Z. Kovarik: Catalytic Organophosphorus Compounds Scavenging by Acetycholinesterase Assisted with Aldoximes, 22 April 2015, organizer: „24th Croatian Meeting of Chemists and Chemical Engineersˮ, Zagreb, Croatia
  21. Z. Kovarik: Kolinesteraze: ranjive mete ili novi štit u trovanju organofosfornim spojevima, 31 March 2015, organiser: IMROH
  22. Z. Kovarik: Rat i mir u svijetu kolinesteraza, 25 February 2015, organizer: Croatian Society of Natural Sciences
  23. A. Bosak: Metaproterenol, isoproterenol and their bisdimethylcarbamate derivatives as human cholinesterase inhibitors, 12 December 2014, organiser: IMROH
  24. Z. Radić: Novel (bio)molecules for new organophosphate intoxication treatments, 25 September 2014, organizer: „The Interplay of Biomolecules”, Congress of the Croatian Society of Biochemistry and Molecular Biology, Zadar, Croatia

WORKSHOPS

  1. Reactivators and Medical Countermeasures against Nerve Agents, 14 – 15 May 2018, Hotel Palace, Zagreb, Croatia. ABSTRACTS

AWARDS

  1. Zorbaz: FEBS Junior travel fellowship za sudjelovanje na FEBS3+ conference „From molecules to living systems”, Siófok, Hungary, 2018.
  2. Zorbaz: Grant to participate at the 13th International Meeting on Cholinesterases and the 7th International Conference on Paraoxonases, Hradec Králové, Czech Republic, 2018.
  3. T. Zorbaz: Poster Award at The 13th International Meeting on Cholinesterases and the 7th International Conference on Paraoxonases, Hradec Králové, Czech Republic, 2018. Link
  4. T. Zorbaz: Grant to participate at the 17th FEBS Young Scientists’ Forum (YSF) and 42nd FEBS Congress “From molecules to cells and back”, Jerusalem, Israel (7-14 Sep 2017)
  5. T. Zorbaz: Poster Award (3rd place) at the 16th Medical Chemical Defence Conference, Munich, Germany, 2017. Link
  6. T. Zorbaz: ISN/IBRO-PERC Young Investigator Poster Prize (International Society of Neurochemistry/International Brain Research Organization-Pan European Regional Committee), XVth International Symposium on Cholinergic Mechanisms, Marseille, France, 2016
  7. T. Zorbaz: Grant from the French Government for a one-month study visit to France in 2016
  8. M. Katalinić: winner of the „B.P. Doctor Young Investigator Award“, awarded to successful young researchers in the field of cholinesterase research, 2015
  9. M. Katalinić: Poster Award at the FEBS 3+ Meeting „Molecules of Life”, Portorož, Slovenia, 2015