Synthesis and In Vitro Evaluation of 2-PAM Analogs as Potential Resurrecting Agents for. Aged Human Acetylcholinesterase

Transcription

1 Synthesis and In Vitro Evaluation of 2-AM Analogs as otential Resurrecting Agents for Aged uman Acetylcholinesterase S. ilanthi Yasapala Department of Chemistry University of Iowa 1

2 Introduction Acetylcholinesterase (AChE) is among the most catalytically powerful enzymes known to man. AChE has an amazing catalytic power with a turnover number over 10 4 /s. 1 The main role of AChE is the termination of neurotransmission by rapid hydrolysis of the neurotransmitter, acetylcholine, ACh (Figure 1). 2,3 This essential role of AChE makes it a target of numerous reversible and irreversible inhibitors, including most approved anti-alzheimer drugs. 4 5 and neurotoxins such as carbamates and organophosphates (s). 6 rganophosphates include both pesticides (e.g., parathion, chlorpyrifos) and chemical warfare agents (CWAs, e.g., sarin, soman) that lead to irreversible inhibition of AChE. 6,7 AChE has a catalytic triad of Ser200, is440 and Glu327, that is buried in the bottom of a deep narrow gorge (Figure 2). 2,6 In addition, it has a catalytic anionic subsite, in which the choline moiety of ACh is stabilized via cation-π and electrostatic interactions. 6 ACh is stabilized further by interaction of its carbonyl oxygen within an oxyanion hole, while the methyl moiety of its acetyl group in an acyl pocket. 2 ear the rim of the gorge lies a peripheral anionic site (AS) that interacts allosterically with the active site to exert a regulatory role on ACh hydrolysis. 6 Knowledge of these key interactions is in higher importance when developing specific and reactive alkylating agents (reversible inhibitors) for the treatment of poisoning. AChE s catalytic mechanism involves acylation and deacylation steps. 3 The overall catalytic process is initiated by nucleophilic attack on the carbonyl carbon of ACh by Ser200. 1,3 2

3 owever, the catalytic Ser200 which is crucial in AChE hydrolysis is also reactive towards the inhibitors in a similar way. 3 In the case of inhibitors (Figure 3), phosphylated enzyme resulting from inhibition is more stable than the acetylated enzyme. As a result, the AChE- complex does not undergo rapid spontaneous hydrolysis as that of the acylated enzyme. Thus, the failure of inhibited AChE to hydrolyze ACh results in over-stimulation of cholinergic receptors, and eventually causes death by respiratory failure. 8,9 umerous efforts have been taken to minimize or ban the use of pesticides. 10 owever, their extensive use results in up to 200,000 human fatalities annually worldwide. 11 Furthermore, great stocks of highly toxic based chemicals such as sarin and soman are still being used as chemical warfare agents (CWAs) and are considered a serious threat to national security 10 ; i.e. use of CWAs in Syria in The current human poisoning treatments include the use of a nucleophilic oxime such as 2-pyridinium aldoxime methyl chloride (2-AM, figure 4) to dephosphylate the AChE- adduct (termed reactivation). 5,7 owever, after phosphylation, some AChE- adducts undergo a rapid de-alkylation process called aging 6 (Figure 5); i.e. the half-life of aging for the nerve agent soman is about 2-4 minutes. 9 The aged-ache adduct is resistant to both spontaneous and oxime induced reactivation. 6,9 There is no known oxime or other antidote that is able to displace the phosphyl moiety from the active site of aged AChE adduct. 10,13,14 owever, if the aged adduct is re-alkylated, the known oximes such as 2-AM or monoisonitrosoacetone (MIA) could then be used to reactivate the -inhibited AChE. 10 3

4 bjectives: The major objective of this study is to synthesize methyl analogs of 2-AM to alkylate the aged AChE- adduct. The importance of this method is that methyl transfer from the methyl analog would convert the phosphyl mono-anion into a neutral moiety while producing 2-AM in situ. 2-AM can then reactivate the non-aged form of the enzyme resulting from the alkylation process. In this approach, a single agent would not only transfer methyl to the AChE- adduct but also provide the oxime reactivator. Experimental rocedure This research has the following objectives. 1. Synthesis of relevant alkylating agents 2. Evaluating alkyl transfer abilities in a model reaction 3. Biological evaluation to find the reactivating abilities of alkylating agents for aged-ache adducts. 1. Synthesis rocedure The -methyl oxime ethers were synthesized according to the literature 15 by reacting commercially available pyridine-2-carboxaldehyde with methoxyamine followed by alkylation with methyl triflate (MeTf) at room temperature (Figure 6). To enhance the methyl transfer ability, four more compounds were synthesized by substituting electron-withdrawing groups such as F and CF3 at different positions on the aromatic ring. All the compounds were purified, characterized by proton nuclear magnetic resonance ( 1 -MR), carbon nuclear magnetic resonance ( 13 C-MR) and high resolution electron spray ionization mass spectrometry (R- EIMS) and stored at 4 C. 4

5 2. Model Reaction The effectiveness of above synthesized compounds as methyl transfer agents was first evaluated by using sodium methyl methanephosphonate as an analogue of the aged AChE - adduct. Model reactions were conducted in an MR tube by mixing equimolar concentrations of phosphonate salt (27 mm in d6-dms) with the oxime ethers synthesized above. The reaction was monitored by 1 -MR for a few hours or days depending on the methyl transferring rate. 2. Biological Evaluation 2.1. Determination of half maximal inhibitory concentration (IC50): The abilities of the alkylating reagents to bind to AChE were evaluated by measuring IC50 values against hache by a well-known colorimetric method developed by Ellman et al. 16 erein, acetylthiocholine (ATCh) is used as the enzyme substrate. The rate of the thiocholine production from acetylthiocholine hydrolysis is measured by UV-visible spectroscopy. This was accomplished by continuous reaction of thiocholine with 5, 5 -dithiobis(2-nitrobenzoic acid) (DTB) and measuring the production of yellow color at 412 nm. General method: All stock solutions were prepared and stored at 4 or -4 C. Ten different concentrations of each alkylating agent were prepared and used to determine the IC50 value. Assay was conducted on a Molecular Devices Spectra Max micro-plate reader using polystyrene 96-well plates as follows. Each experimental well was loaded with phosphate buffer containing DTB, human AChE (hache) and the alkylating agent in each concentration. The control well was loaded in the same way but with only the solvent which was used for stock preparation of alkylating agent. Finally, ATCh was added to all wells and initial rate of the reaction at less than 10% turnover of 5

6 the initial substrate concentration was measured. Initial rate was corrected for background (non enzymetic) hydrolysis of ATCh and performed in duplicates. IC50 values were determined by nonlinear fit of data. Activity Recovery Assay The activity recovery assay includes inhibition of AChE with a sarin analog ( analog), aging of AChE- complex, and evaluation of the ability of the alkylating agents for the reactivation of aged-ache adduct. Briefly, hache was incubated with the sarin analog shown in Figure 7 for 30 minutes at room temperature. treated enzyme was passed through a Sephadex G-50 Quick Spin Column to remove residual inhibitor. The enzyme activity was checked by Ellman assay to make sure enzyme is fully inhibited. Then, treated enzyme was kept at 27 0 C for 48 hours to obtain aged- AChE adduct. -inhibited AChE was then treated with alkylating agent at ten times IC50. Treated enzyme was incubated with 2-AM for 30 minutes. The enzyme activity was referred to control activity and percentage reactivation was calculated at specific time periods (1h, 4h, 24h).The residual activity was measured in parallel at each time point to check spontaneous hydrolysis. Result and Discussion Unsubstituted methyl oxime ether and Compounds containing CF3 group on the 5 position of pyridine ring showed low methyl transfer in the model reaction while compounds having F atoms on the aromatic ring, did not show methyl transfer. Compounds bearing F atom on the aromatic ring showed nucleophilic aromatic substitution instead of methyl transfer and MR data from model study confirmed the formation of methyl sarin according to the proposed mechanism shown in Figure 8. 6

7 The IC50 values obtained in the study are given in Table 1.The IC50 values suggested that inhibition of hache by oxime ethers required micro molar concentrations that represent the ability of those alkylating agents to bind to the enzyme. Most of the alkylating agents have more or nearly same potency as 2-AM (IC µm). owever, none showed considerable reactivity for the aged adduct. Alkylating agents which bear an F atom on the aromatic ring, showed time dependent inhibition in preliminary assays. Therefore, the IC50 values were evaluated over 7 hours to determine their time dependent inhibition. The reduction in apparent IC50 value with time in compound which bears an Flourine atom at 3 position on pyridinium ring indicated that after 2 hours, it is about three fold more potent than at initial time. owever, inhibitory potency of Compound which has Flourine atom at 5 position on pyridinium ring remained mostly the same for the entire 2 hours. All the data suggested that substitution on the aromatic ring or even changing the position of substitution could change the inhibitory potency for AChE. Conclusion All the methyl oxime analogs were more potent inhibitors than the well-known antidote 2- AM. owever, none of them was able to resurrect the aged adduct. In addition, IC50 values indicated that substitution of the simple pyridinium ring by an electron-withdrawing group make them more potent. owever, nucleophilic aromatic substitution makes some inhibitors irreversible AChE inactivator. owever, none of the compounds was able to resurrect the aged-ache adduct from our preliminary screen. But, we hypothesized that inhibitors that contain an F atom on the pyridinium ring may arylate the aged AChE adduct by nucleophilic aromatic substitution, thereby making the non-aged form of the enzyme. But, due to the bulky arylating agent, 2-AM may not be able to nucleophilically attack to the enzyme adduct easily. Therefore, we decided to use a 7

8 smaller oxime such as monoisonitrosoacetone (MIA) than 2-AM for the resurrection study. This issue is currently under investigation. 8

9 Figure 3 C Figure 1: Chemical structure of acetylcholine, ACh Figure 2: Schematic view of the active-site gorge of AChE Figure 3: Chemical structures of inhibitors of AChE; a pesticide b,c,d- nerve agents Figure 4: Chemical structure of 2-AM 9

10 R' Ser R Ser R R' AChE- adduct aged-ache- adduct Figure 5: Scematic representation of aging process R 1 2.Cl R, a, rt, 4 h MeTf R C 2 Cl 2, rt Tf 4-20 h R=, F, CF 3 Figure 6: General reaction scheme for preparation of methyl oxime analogous and chemical structure of each alkylating agent 3 C Figure 7: sarin analog used in the study F F F + F F Figure 8: roposed mechanism for methyl sarin formation during model reaction Table 1: IC50 values of methyl oxime ethers Compound IC50 at t=0h (µm) IC50 at t=2h (µm) 1 (unsubstituted) 70 ± ± 40 2 (F atom at 3 position on the 110 ± ± 4 pyridine) 3 (F atom at 5 position on the 70 ± 10 9 ± 2 pyridine 4 (CF3 group at 5 position on the pyridine 90 ± 2 160± 20 10

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