Archives: Projekti

IMI projekti

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

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

 

022-0222412-2403 Oligoelements in Biological Matrices and Multielement Profile Quality Control

THE MINISTRY OF SCIENCE, EDUCATION AND SPORTS OF THE REPUBLIC OF CROATIA (2007-2010)

Principal Investigator: Nikola Ivičić

SUMMARY
The accurate trace element (TE) analysis of various biological matrices (hair, blood, mails, other tissue) depends upon the complete decomposition of such a matrice. Under the standard working conditions, such destruction is possible with teperatures below 600 C, but many of the metabolicaly important ET would start evaporating above 400 C. Difficulties associated with specific problems, and this problems only amounts with the multielement profile (MP) simultaneus ET analysis. The hallmarks of quality ET analysis are the proper decoposition of the biological matrice in a several different  ways, analyses by the different analytical methods and comparison of the results against the certified standards. In this Project it means comparison of the analytical results acquired by the (1) differential pulsed anodic stripping voltammetry (DPASV), (2) electro thermal atomic absorption spectrometry (ET-AAS), and  (3) inductvely coupled plasma mass spectrometry (ICP-MS). Every of them has it advantages and drawbaks: DPASV is very sensitive but slow and critically dependent upon the buffer system; ET-AAS depends upon the complete destruction of the biological matrice and requires a separate lamp for every ET under the consideration; whereas ICP-MS  is the method of choice today. Since we at IMI dont hawe an ICP-MS, we collaborate with the Center for Biotic Medicine (CBM), Moscow, Russia. In our previous reserch we were able to compare all three methods for quality control of molybdenum analysis in the human blood. Our working hypothesis is that the potential differences in the MP of ET in various diseases would be first reflected un the metabolic changes of the less well studied ET of very low concentration in our body. Therefore, we envisage introduction of the new methods for analyzing lanthanum (La), molybdenum (Mo), scandium (Sc), and tin (Sn),beyound those already in use for Pb, Cd, Zn, and Cu in the biological samples collected in the other two Projects of this Programme. The mercury would be analyzed in collaboration with the EERC, Grand Forks, ND USA supported by the NIH research grant on idiorrythmic Hg exposure for the assessment of Hg toxicity in continuous low and sporadic high Hg exposure. The DPASV ET analytical results would be compared with those gained by the ET-AAS and ICP-MS.

022-0222148-2139 Therapeutic Effect of Newly-synthesized Compounds in Organophosphorus Poisoning

MINISTRY OF SCIENCE, EDUCATION AND SPORTS OF THE REPUBLIC OF CROATIA (2007-2014)

Principal Investigator: Božica Radić (until 31 September 2010), Ana Lucić Vrdoljak (since 1 January 2011)

SUMMARY
General toxicological effect of organophosphorus compounds (OPc) is based on irreversible inhibition of the hydrolytic enzymes; vitally important acetylcholinestrase (AChE) and butyrylcholinesterase (BuChE). These compounds primarily affect the cholinergic nerve system. Besides their wide and beneficial use in agriculture, public health, and in human and veterinary medicine, they are used as harmful nerve agents in chemical warfare. Despite numerous research efforts, there is no efficient antidote for OPc poisoning, especially by nerve agents. Therefore, the ultimate goal of our research is to contribute to the development of new, satisfactorily effective antidotes.
It has been known that compounds containing an oxime group (oximes) and primarily nucleophilic properties reactivate the inhibited AchE, and protect its inhibition. That is the main reason for synthetyzation of the new classes of oximes (imidazolium, imidazolium-pyridinium, pyridinium oximes), non-oxime compounds but also of other compounds that could protect or reduce the effects of OPc poisoning through some other mechanisms that cannot be attributed to AChE protection or reactivation (e.g. adamantane derivatives which are the antagonists for N-methyl-D-aspartate receptors). The research deals with basic toxicity of each new compound, their cytotoxicity, genotoxicity as well as with rapidity of achieving therapeutic levels in the blood, distribution and retention within, and elimination from the body. The research investigates possible synergistic effects of these new compounds when applied according to a modified therapeutic protocol (related to pretreatment, treatment, supplemental therapy), which has been a proven method to identify the most effective antidote. In order to determine whether the oxidative stress is involved in the mechanism of OPc toxic effects, we will examine the biomarkers of oxidative damage of lipids (malondyaldehide) and proteins (antioxidation enzymes), as well as of relieving effects of OPc poisoning by antioxidants. A part of the research will investigate the properties of BuChE as a natural antidote for OPc, but also the role of this enzyme in the lipid and lipoprotein metabolism. The research will be performed in vitro and in vivo on experimental animals.
The results of the proposed scientific research could be of great practical value for the use in clinical toxicology, and, consequently, of considerable commercial importance for the pharmaceutical industry.

022-1340036-2083 Frontotemporal Dementias

MINISTRY OF SCIENCE, EDUCATION AND SPORTS OF THE REPUBLIC OF CROATIA (2007-2014)

Principal Investigator: Rajka Liščić

SUMMARY
Background: A group of frontotemporal dementias (FTD) is a major cause of early non-Alzheimer dementia (AD) and is increasingly being recognized as an important cause of dementia. FTD may be mistaken clinically for AD, particularly in early stage of the disease. However, FTD is behaviourally and pathologically distinct from AD and encompasses three clinical syndromes with heterogeneous neuropathology according to Neary et al. 1998. A motor neuronopathy complicating FTD has been recognized as features of amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND). The disease develops gradually, where changes in personality and behaviour precede the development of symptoms and signs of anterior horn cell involvement. Patients with typical ALS may develop FTD, and vice versa ALS occurs in patients with FTD. There is, however, less awareness of the involvement of cognition and possible dementia in ALS, especially in early stage of the disease.

Hypothesis and aim: As FTD and AD have different prognoses and treatment; there is a need for accurate clinical diagnosis. The hypothesis is that patients, who present with bulbar palsy and ALS, rather than corticospinal features, may be most susceptible to the development of FTD.

Expected results:

  1. To establish criteria for FTD diagnosis.
  2. To determine if ALS patients have cognitive impairment. Follow-up study should tell us if deterioration of cognitive impairment exists and if so weather is correlated with deterioration of motoric functions or not.
  3. To determine the nature of the differing pathological entities behind these heterogeneous forms of lobar atrophy and to find a molecular biological basis that might explain them. With molecular markers becoming available, it will be possible to specify the exact nosological relationship between FTD-ALS and FTD and between FTD-ALS and the classical ALS. We’d like to establish whether or not they do indeed represent phenotypic variants of a common genotype.

Importance: We aim to distinguish between different FTD phenotypes and genotypes

022-0222148-2142 Toxic Effects of Mycotoxins on Humans and Animals

MINISTRY OF SCIENCE, EDUCATION AND SPORTS OF THE REPUBLIC OF CROATIA (2007-2014)

Principal Investigator: Maja Peraica

SUMMARY
Mycotoxins are the metabolites of moulds with toxic, carcinogenic, genotoxic, mutagenic, and teratogenic properties. In our earlier studies, we found low concentrations of fumonisin B1 (FB1), fumonisin B2, ochratoxin A (OTA), and zearalenon in food samples collected in various regions of Croatia. OTA is considered to be involved in the development of endemic nephropathy (EN). This kidney disease of unknown etiology and high frequency of tumors of the upper urothelial tract appears in the western part of Brodska Posavina. It is speculated that, in addition to OTA, another nephrotoxic mycotoxin such as FB1 should be involved in the development of EN.
The mechanism of the toxicity of OTA and FB1 is not fully understood. It was proved on cultured cells that OTA and FB1 cause oxidative damage of lipids and DNA. However, it is not know whether low concentrations of these mycotoxins usually found in food can also cause oxidative damage of lipids, proteins and DNA in experimental animals. OTA and FB1 are frequently found together in cereals, and therefore it would be important to see whether their effect is synergistic or not. OTA is an organic anion that accumulates in the kidney cells and partially excretes in urine. OTA is partially reabsorbed due to organic anion transporters (OAT) on the epithelial cells along the nephron, which may contribute to its toxicity in specific parts of the kidney. Recent investigations have shown that the localization of specific OATs in rats is sex-related. Toxic effects of OTA are more severe in male rats, which could be due to the higher expression of OAT in males.
The proposed project will use recently developed methods, some of which will be used for the first time in the research of the mechanisms of action of mycotoxins.
The aim of this project is to clarify the role of oxidative stress caused by mycotoxins in damage of macromolecules (DNA, lipids, and proteins), as well as to improve the understanding of the mechanism of toxicity of OTA by investigating the changes in kidney OAT system. The other aim is to find out whether exposure to OTA and FB1 is involved in the development of EN.
We expect that our results will: a) show whether oxidative stress is involved in the mechanism of OTA and FB1 toxicity, and whether this effect is synergistic, b) show whether OTA affects the expression of OATs in the rat kidney and whether this effect is sex-related, and c) improve the knowledge about the mechanism of EN development.