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Analyses of interactions between organophosphorus compounds and esterases and other targets for therapy in poisoning (OPEsterOX)

Project head: Nikolina Maček Hrvat

Croatian Science Foundation: Research project IP-2022-10-6885

Team members: Goran Šinko, Tena Čadež, Dora Kolić, Višnja Stepanić, Vesna Gabelica Marković, Zoran Radić

Duration: 48 months, 2023-2027

Abstract

Exposure to organophosphates (OP) from both pesticides and nerve agents leads to the covalent inhibition of the pivotal enzyme acetylcholinesterase (AChE) that leads to accumulation of neurotransmitter acetylcholine, which induces cholinergic crisis, that is, overstimulation of muscarinic and nicotinic membrane receptors in the central and peripheral nervous system. In severe cases, subsequent desensitisation of the receptors results in hypoxia, vasodepression, and respiratory arrest, followed by death. Standard therapy of OP poisoning that generally involves administration of anticholinergic atropine and an oxime reactivator of phosphylated AChE has limitations especially in the case of phosphoroamidates due to the structural-related mechanism of reactivation. An alternative therapy is the related enzyme butyrylcholinesterase (BChE) which binds OP compounds and as such acts as stoichiometric bioscavenger of OP. Therefore, this project utilizes known (e.g. tabun, methamidophos and fenamiphos), and new compounds (A-agents) to gain a better understanding of the mechanistic basis of cholinesterase family interactions and their limitations to find new effective leads for treatment. The biochemical mechanism of enzyme interactions will be comprehensively studied on a molecular level with in silico, in vitro, and ex vivo methods to define binding affinities, kinetic constants of inhibition and efficiency of reactivation of both enzymes. The possible mechanisms of toxicity of selected OPs will be studied at cell-culture level, and on mice exposed to OP. The neuroinflammatory effect of OP as w ell as the neuroprotective effectiveness of oxime therapy will be examined on the mouse brain by monitoring specific markers of astrogliosis, microgliosis and neuron viability. These comprehensive analyses will undoubtedly contribute to the knowledge of phosphoroamidate interactions and improved therapeutics in OP poisoning.

Publications
  • Maček Hrvat, Nikolina; Puljko, Borna; Sit, Rakesh K.; Ilić, Katarina; Kolić, Dora; Mlinac-Jerkovic, Kristina; Kalanj-Bognar, Svjetlana; Radić, Zoran; Sharpless, Barry K.; Taylor, Palmer et al. The ionizing zwitterionic oxime antidote attenuates gliosis in mice exposed to sarin. Chem-Biol Interact, 421 (2025), 111767, 8. doi: 10.1016/j.cbi.2025.111767
  • Čadež, Tena; Maček Hrvat, Nikolina; Šinko, Goran; Kalisiak, Jarosław;  Radić, Zoran; Fokin, Valery V.;  Sharpless, Karl Barry;  Taylor, Palmer;  Kovarik, Zrinka. Click-chemistry-derived oxime library reveals efficient reactivators of nerve agent-inhibited butyrylcholinesterase suitable for pseudo-catalytic bioscavenging. Arch Toxicol (2025) https://doi.org/10.1007/s00204-025-03985-6
  • Kovarik, Zrinka; Kolić, Dora; Čadež, Tena; Šinko, Goran; Tuksar, Petra; Zorbaz, Tamara; Curty, Christophe; Maček Hrvat, Nikolina. Outlining the A-series of organophosphorus compounds: Cholinesterase inhibition, reactivation, cytotoxicity, and acute toxicity in mice. Chem-Biol Interact, 421 (2025), 111762, 11. doi: 10.1016/j.cbi.2025.111762
  • Kolić, Dora; Kovarik, Zrinka. N-methyl-d-aspartate receptors: Structure, function, and role in organophosphorus compound poisoning. BioFactors, 50(5), 2024, 868–884.
  • Mlakić, Milena; Čadež, Tena; Šinko, Goran; Škorić, Irena; Kovarik, Zrinka. New Heterostilbene and Triazole Oximes as Potential CNS-Active and Cholinesterase-Targeted Therapeutics. Biomolecules, 14(6), 2024, 679(37)
Dissemination
  • 19th Medical Chemical Defense Conference, Munchen, Njemačka (2. – 3. 4. 2025.).
    Posterska prezentacija: The efficacy of centrally active RS oximes in reactivating phosphorylated acetylcholinesterase (N. Maček Hrvat)
    Posterska prezentacija: Assessment of nerve agent-induced toxicity in a cellular model (T. Čadež)
  • Mini-symposium “Multidisciplinarity in drug development”, Zagreb, Hrvatska (12. 2. 2025.)
    Pozvano predavanje: Targeting organophosphorus poisoning by pseudocatalytic detoxification system (T. Čadež)
  • 49th FEBS Congress: Bridging Continents to Advance Life Sciences, Istanbul, Turska, (5. – 9. 7. 2025.)
    Posterska prezentacija: Identifying cholinesterase inhibitors among commercially available pesticides by bioinformatic analysis and in vitro inhibition (Maček Hrvat, Nikolina; Stepanić, Višnja; Šinko, Goran; Čadež, Tena)
    Posterska prezentacija: Evaluation of compounds as multitarget therapeutics in neurological disorders (Čadež, Tena; Mlakić, Milena; Škorić, Irena; Maček Hrvat, Nikolina; Kovarik, Zrinka)
  • 59th Congress of the European Societies of Toxicology, Atena, Grčka (14. – 17. 9. 2025.)
    Posterska prezentacija Disruption of the plasma membrane organization and neuroinflammation can be triggered by toxic organophosphorus compounds (Maček Hrvat, Nikolina; Puljko, Borna; Ilić, Katarina; Mlinac-Jerković, Kristina; Kalanj-Bognar, Svjetlana)
    Posterska prezentacija Organophosphorus pesticides and neurotoxicity: Evaluation of human and animal cell model (Čadež, Tena; Maček Hrvat, Nikolina)
  • 10. hrvatski kongres neuroznanosti, Zagreb, Hrvatska (11. – 13. 9. 2025.)
    Kratka oralna prezentacija postera: Imidazolium oximes for the management of organophosphate compound neurotoxicity (D. Kolić, I. Primožič, Z. Kovarik, N. Maček Hrvat)
  • 48th FEBS Congress: Mining biochemistry for human health and well-being, Milano, Italija (29.06.2024 – 03.07.2024).
    Posterska prezentacija: New and old challenges in reversing organophosphorus toxicity (N. Maček Hrvat, D. Kolić, T. Čadež, Z. Kovarik).
  • EUROTOX2024: Toxicology – A Quest for safe Chemicals and Medicines, Copenhagen, Danska (08.09.2024 – 11.09.2024).
    Posterska prezentacija: Assessment of organophosphate-induced toxicity and evaluation of antidotal efficacy on cellular level (T. Čadež, Z. Kovarik).
  • ChEPON, 15th International Meeting on Cholinesterases, 9 th International Conference on Paraoxonases, Brdo pri Kranju, Slovenija (15.09.2024 – 18.09.2024).
    Posterske prezentacije: Outlining the A-series of organophosphorus compounds – cholinesterase inhibition, reactivation, cytotoxicity (Nikolina Maček Hrvat, Dora Kolić, Tena ČadeŽ, Goran Šinko, Zrinka Kovarik);
    Evaluation of resveratrol compounds as therapeutics in organophosphorus poisoning (Tena Čadez, Milena Mlakić, Nikolina Maček Hrvat, Irena Škorić, Zrinka Kovarik).
  • FEBS3+ Meeting: Exploring Molecular Frontiers, Pula, Hrvatska (25.09.2024 – 28.09.2024).
    Posterska prezentacija: Bioscavenging capacity of butyrylcholinesterase reduces Inhibition impact of organophosphate compounds in human blood (Dora Kolić, Goran Šinko, Ines Primožič, Zrinka Kovarik).
  • HKD simpozij: 8th Mini Symposium of Section of Medicinal and Pharmaceutical Chemistry, Zagreb, Hrvatska, (19. 11. 2024.).
    Predavanje: CNS-active ligands against neurotoxocity in organophosphate compound poisoning (Dora Kolić, Nikolina Maček Hrvat, Zoran Radić, Zrinka Kovarik).
Training
  •  Biocentar, Zagreb – workshops:
    ”Proizvodnja, pročišćavanje i karakterizacija proteina” (25.-27.11.2025.) – D. Kolić and P. Nakić
    Chemical codes – biostatistika u R-u, napredna razina (6.-7.11.2025.) – T. Čadež
    Chemical codes – biostatistika u R-u, osnovna razina (23.-29.10.2025.) – T. Čadež
  • Department of Neuroimaging, Institute of Psychiatry, Psychology & Neuroscience, King’s College London, UK (11.7.-12.9.2025.) – T. Čadež
  • Skaggs School of Pharmacy and Pharmaceutical Sciences of the UC San Diego in La Jolla,CA, USA (1.2.-16.3.2024.) – D. Kolić
  • The 2nd Course on Molecular Toxicology, Izmir, Turska (22.–24.5.2024.) – D. Kolić
Mentoring
  • Graduate thesis ˝Biosigurnost i učinkovitost oksima za reaktivaciju kolinesteraza nakon otrovanja organofosfatima˝ – Ema Krstulović (Faculty of Science, University of Zagreb)