A Complex Process of the Development of New Acetylcholinesterase Reactivators - from Prediction to in Vivo Evaluation

The mechanism of intoxication with organophosphorus compounds, including highly toxic nerve agents, is based on the irreversible inhibition of acetylcholinesterase that is followed by an accumulation of acetylcholine at peripheral and central cholinergic synapses, which in turn leads to the clinical manifestation of various signs and symptoms summarized as acute cholinergic crisis. Nerve agent poisoning is commonly treated using a combination of a cholinolytic drug to counteract the accumulation of acetylcholine at muscarinic receptors and acetylcholinesterase reactivators (pralidoxime or obidoxime) to reactivate nerve agentinhibited acetylcholinesterase. There is a strong interest in developing new, more potent acetylcholinesterase reactivators. The development of acetylcholinesterase reactivators consists of several steps: description of nerve agent intoxication mechanism on molecular basis (molecular design), prediction of biologically active structure of acetylcholinesterase reactivators (artificial neural networks), their synthesis, in vitro and in vivo evaluation of their potency to counteract acute toxicity of nerve agents. INTRODUCTION Organophosphorous (OP) compounds are ubiquitous as pesticides, in veterinary medicine and in industry. The highly toxic OP compounds, called nerve agents, were developed, produced and weaponized as chemical warfare agents and, in addition, they have been misused by terrorists in two Japanese cities Matsumoto (1994) and Tokyo (1995) (Bajgar, 2004). OP compounds irreversibly inhibit the enzyme acetylcholinesterase (AChE, EC 3.1.1.7), which is responsible for terminating the neurotransmitter action of acetylcholine (ACh) at various cholinergic nerve endings. Irreversible AChE inhibition results in the accumulation of ACh at cholinergic receptor sites, producing continuous stimulation of cholinergic fibres throughout the central and peripheral nervous systems (Marrs, 1993). Currently , a combination of an antimuscarinic agent (preferably atropine), and an AChE reactivator (called an oxime according to its chemical structure) is recommended for the treatment of OP poisoning (Bajgar, 2004). Atropine blocks the effects of accumulated AChinduced overstimulation of peripheral muscarinic receptor sites, whereas reactivators repair the biochemical lesion by dephosphorylating the enzymatic molecule, AChE, and restoring its 2 www.jmedcbr.org JMedCBRDef, Vol 3, 2005 Journal of Medical Chemical, Biological and Radiological Defense activity (Kassa, 2002). Unfortunately, currently available oximes (pralidoxime, obidoxime and HI-6), used in clinical toxicology to counteract the acute effects of OP pesticides and introduced into some armies to counteract the acute effects of nerve agents, have been shown to be rather ineffective against certain nerve agents, especially soman, cyclosarin and tabun (Cabal et al., 2004; Kassa, 2002). As a result, the development of new, more potent AChE reactivators with sufficient efficacy to reactivate phosphonylated or phosphorylated AChE and thereby decrease the acute toxicity of OP compounds regardless of their chemical structure is still a very important task for toxicological research institutes throughout the world. Ideally, the process of the development of oximes should be as effective and quick as possible. Our Department of Toxicology has tried to optimize this process and the final structure of this process is described in this work. THE PROCESS OF THE DEVELOPMENT OF NEW AChE REACTIVATORS The entire developmental process consists of five phases • prediction of new AChE reactivator structures using artificial neural networks (ANN) • description of the reactivation process using molecular design • synthesis of new AChE reactivators • in vitro testing, and • in vivo testing. A short description of individual developmental steps is shown in Figure 1 and described below. Figure 1. Description of the developmental process JMedCBRDef, Vol 3, 2005 www.jmedcbr.org 3 Journal of Medical Chemical, Biological and Radiological Defense Prediction using artificial neural networks The first step of the developmental process is to use artificial neural networks (ANN) to predict AChE behavior. Using known biological activities of different substances, there is the possibility to “learn” how the ANN works without detailed knowledge of the exact interaction between a compound and an organism. Chemometric methods are able to estimate biological activity of chemical compounds. These are combined using calculations and are used to estimate the biological activities of potential antidotes without actually synthesizing them. In our development process, ANN are used for the prediction of the appropriate structure of new AChE reactivators. Biological activity and structure of currently used AChE reactivators are used as an input data set. Then, the model of relationships between chemical structure and biological activity is calculated. Afterwards, we are able to predict new, more potent reactivators of AChE inhibited by nerve agents based on these models (Dohnal and Kuca, 2004). Editors note: Another article describing the use of ANN and QSAR to AChE reactivators is "Structural bioinformatics and QSAR analysis applied to the acetylcholinesterase and bispyridinium aldoximes" by P.P. Mager and A. Weber in Drug Des Discov. 2003;18(4):127-50. Molecular design Molecular modeling is used for a study of AChE conformational changes caused by substances such as OP compounds (nerve agents, pesticides). This study is performed using molecular dynamics that calculate intramolecular energies of modified residues. Reconformational changes in AChE structure caused by reactivators are then examined. The influence of these substances towards the enzyme is evaluated on the basis of known structures and by docking method and subsequent molecular design simulations. The acquired description of interactions and their quantification obtained from interaction energies of model systems serve as proposition for new, more potent AChE reactivators (Wiesner et al., 2005). Synthesis of new AChE reactivators All the promising AChE reactivators predicted by ANN and molecular design methods are synthesized using special methods developed especially for the synthesis of currently used AChE reactivators. Over the last three years, we have synthesized more than twenty AChE reactivators (Kuca et al., 2003a; 2003b; 2004a; 2004b). All of these synthesized substances are mono or bis quaternary pyridinium rings, connected mostly with three or four membered linkage chains. In all their structures, the oxime group is functioning as a nucleophile able to split the bond between enzyme and inhibitor. The oxime group is usually located in the position two or four on the pyridinium ring. In vitro testing The reactivation potency of each synthesized AChE reactivator is first evaluated using in vitro experiments, with the help of potentiometric method of the measurements of AChE activity (Kuca and Kassa, 2003). The homogenates from rat, pig and human brains are used as a source of AChE, although commercially available pure enzymes can also be used. The protein in AChE homogenate (0.5 mL) is mixed with isopropanol solution of nerve agent to achieve 95% AChE inhibition and incubated at 25 °C for 30 min. After incubation, 2.5 mL of 3 M NaCl and distilled water are added to a final volume of 23 mL. Then, 2 mL of 0.02 M acetylcholine iodide is added and the enzyme activity is assayed. The activities of intact (ao) and nerve agent-inhibited (ai) AChE are estimated. After the nerve agent-inhibited AChE is incubated for 10 min with a solution of an oxime reactivator, the activity of the reactivated AChE (ar) is obtained. The activity values a0, ai and ar, are calculated from the slopes of initial 4 www.jmedcbr.org JMedCBRDef, Vol 3, 2005 Journal of Medical Chemical, Biological and Radiological Defense parts of titration curves. Each value is the arithmetic mean of two independent measurements. The kinetics of the reactivation process may be represented by the scheme: EI + R EIR E + P KR kR where EI is the nerve agent-inhibited enzyme, R is the reactivator, E is the reactivated enzyme, EIR is the intermediate complex, and P is the product, usually a phosphonylated, unstable oxime. KR and kR are the dissociation constants and the rate constants for decomposition of the intermediate complex, respectively. For all the oximes whose reactivation abilities are screened, the percentage of reactivation (% R) is calculated from equation