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Acetylcholinesterase (AChE) ensures the functioning of cholinergic synapses by limiting the duration of ACh action. Partial inhibition of this enzyme is used in medical practice for the treatment of Alzheimer's and Parkinson's disease, traumatic brain injury and myasthenia gravis. However, virtually all anti-AChE agents possess various side effects mostly as a result of lack of selectivity among various organs and tissues (Caldwell, 2009). The drawbacks could be overcome by using inhibitors capable of inactivating AChE selectively. A new set of promising compounds, the alkylammonium derivatives of 6-methyluracil (ADEMS), have been recently synthesized and identified as inhibitors of AChE in vitro with inhibitory constants between 7 × 108 and 3 × 109 M−1·min−1 (Anikienko et al., 2008). During exercise of dogs and rats on the treadmill, these compounds displayed surprising effects. Animals treated with ADEMS had no breathing problems and easily survived even when their limb muscles were paralysed. In particular, one ADEMS compound 1,3-bis[5(diethyl-o-nitrobenzylammonium)pentyl]-6-methyluracildibromide (C-547) (Figure 1) was very effective in this respect. The concentrations required for paralysis of the limb muscles (ED50) and those for respiratory failure (LD50) differed significantly and the LD50/ED50 ratio for C-547 was 300 (Kovyazina et al., 2004; Zobov et al., 2005).
In an assay of single quantum miniature endplate currents (MEPC), anti-cholinesterase action is detectable as a prolongation of the MEPC decay phase. This postsynaptic potentiation is a consequence of the repetitive binding and activation of postsynaptic receptors by non-hydrolysed ACh and can be therefore used as a measure of the anti-cholinesterase activity (Giniatullin et al., 1993; 2001; Kovyazina et al., 2003). C-547 increased the amplitude and prolonged the time course of MEPCs at the rat locomotor muscles [specifically the extensor digitorum longus (EDL) and soleus muscles] even at nanomolar concentrations, whereas a 100-fold greater concentration was required to affect the MEPCs in the respiratory diaphragm and intercostal muscles (Petrov et al., 2009).
The reasons for this differing sensitivity between the diaphragm and limb muscles are unknown. We therefore determined to what extent the anti-cholinesterase efficacy of ADEMS depends on the presence and position of 5(diethyl-o-nitrobenzylammonium)pentyl groups. For this question, three 6-methyluracil derivatives were compared in terms of their effect on MEPC. We also determined whether the difference in the sensitivity of the diaphragm and EDL to ADEMS compounds could be explained by the differing expression levels of butyrylcholine esterase (BuChE) in these muscles, as it was shown in vitro that some ADEMS inhibit BuChE at concentrations much higher than those inhibiting AChE (Anikienko et al., 2008). The differences in sensitivity of the AChE itself to ADEMS between the diaphragm and EDL were assessed. Finally, we estimated the sensitivity to C-547 of purified AChE from the brain and EDL, after BuChE was inhibited.
Our results showed that the high intermuscular differences in the presence of ADEMS is partly caused by a higher level of ADEMS-resistant BuChE activity in the diaphragm and also by differing sensitivities of muscle and brain AChEs to ADEMS. Surprisingly, the brain AChE was almost three orders of magnitude less sensitive to C-547 than the AChE from leg muscles.
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The compound C-547, a member of the ADEMS set, increased the amplitude and decay time of MEPC in a manner characteristic of AChE inhibition. However, it was effective in the diaphragm and in external intercostal muscles at concentrations that are greater by one or two orders of magnitude than on the EDL and soleus locomotor muscles (Petrov et al., 2006; 2009). In this work, two other ADEMS compounds, C-627 and C-857, were compared with C-547. Like C-547, these analogues also exhibited a greater potency on the EDL, in particular, the compound C-857, which has only one alkylammonium radical in position 1 of the uracil ring. This position is evidently more important than position 3 for observed sensitivity differences and points to fine variations in the cholinesterase binding sites between diaphragm and EDL muscles. Such variations were not detected with the other AChE inhibitors neostigmine, paraoxon and armin. Hence, the difference between respiratory and locomotor muscles is characteristic of ADEMS as AChE molecular sensors rather than the result of a differing accessibility of diaphragm and leg muscles to anti-cholinesterase agents, in general. Here we showed that there are two possible explanations for our findings a different ratio of ADEMS-sensitive AChE to ADEMS-insensitive BuChE and/or different sensitivities of the AChE subtypes.
The family of mammalian cholinesterases includes two closely related enzymes, capable of hydrolysing ACh and, consequently, of controlling its concentration in the synaptic cleft. These are AChE (EC 22.214.171.124) and BuChE (126.96.36.199). Irrespective of the fact that 65% of the amino acid sequences of these enzymes are homologous and the catalytic centres are virtually identical, they differ substantially in the efficiency of substrate hydrolysis and kinetics of their interaction with a number of inhibitors (Chatonnet and Lockridge, 1989). It is known that BuChE hydrolyses ACh at high concentrations and is not inhibited by an excess of substrate, while AChE functions better at a relatively low substrate concentration and is inhibited by an excess of substrate (Zdrazilova et al., 2006). Hence, it is generally accepted that the rapid hydrolysis of ACh released from nerve motor endings is mostly performed by AChE while the synaptic function of BuChE is not quite clear. BuChE could substitute for the AChE when the latter is insufficient (Minic et al., 2003; Girard et al., 2007). There is a decrease in AChE activity and simultaneously an increase in BuChE activity during Alzheimer's and Parkinson's diseases and during cerebral trauma (Giacobini, 2004). Importantly, addition of horse serum BuChE or recombinant human BuChE into the systemic circulation was able to protect animals from lethal doses of AChE inhibitors (Lenz et al., 2007; Masson and Lockridge, 2010).
As C-547 is a selective inhibitor of AChE (Anikienko et al., 2008) the activity of BuChE in the diaphragm could compensate for the loss of AChE activity. This possibility was supported by the fact that the level of mRNA of BuChE and the activity of the enzyme is several times greater in the diaphragm than in the EDL. Undoubtedly, C-547-resistant BuChE in the diaphragm can significantly counteract the prolongation of MEPCs. However, the elimination of BuChE before the application of C-547 increased the sensitivity of the diaphragm to C-547 but did not fully remove the differences between the EDL and diaphragm. In other words, without iso-OMPA, a proportion of the diaphragm AChE is already inhibited by 10 nM C-547. Because ACh is still hydrolysed by BuChE, the MEPCs are not prolonged. After the elimination of BuChE with iso-OMPA, 10 nM C-547 inhibits the diaphragm AChE sufficiently to increase the amplitude and time course of the MEPCs. Because this was not observed with the lower, 5 nM concentration of C-547 known to be fully effective on EDL (Figure 2E and F), BuChE activity can be viewed as important but not the only reason for the lower diaphragm sensitivity to ADEMS. Therefore, there must be another mechanism underlying the lower sensitivity of the diaphragm to the ADEMS and our assays of AChE in EDL and diaphragm homogenates showed that diaphragm AChE was actually more than 20 times less sensitive to C-547 than that from the EDL.
What could be the basis of this difference in AChE sensitivity? In vertebrates, AChE is coded by a single gene (Hasin et al., 2004; Krejci et al., 2006; Massoulie et al., 2008), but there is a heterogeneity of the molecular forms of AChE as a result of alternative splicing, the attachment of various non-catalytic subunits and post-translational modifications. In mammals, several alternative splicing variants of the AChE gene are known, as well as a single read-through transcript (Legay, 2000), Variants of the alternative splicing of the 5th or 6th exon usually designated ‘E’ and ‘S’ generate catalytic subunits, which contain the same catalytic domain associated with distinct C-terminal peptides. So, the first variant ‘E’ (the so-called ‘erythrocytic’ variant) codes the 32 amino acids at the C-terminus that is responsible for the attachment of the glycophosphatidylinositol anchor. The ‘E’ variant was found mainly at the surface of hematopoietic cells but the physiological function of the enzyme in these cells remains unclear. The ‘S’ variant (AChE-S) is expressed in the CNS as well as in skeletal muscles. This variant (the so-called ‘synaptic’ or ‘tailed’ variant) codes 40 amino acids in the C-terminus, which form a specific domain with the cysteine residue located three amino acids from the end of the protein. This cysteine residue enables disulfide bonding with other AChE subunits, giving rise to amphipathic homodimers and homotetramers that can interact with anchor subunits that are specific for this variant.
In addition, several reports describe alternative promoter usage and splicing at the 5′-end of the gene (Meshorer et al., 2004). However, these sequences located before the translation initiation ATG codon are absent in the AChE protein, and probably play a regulatory role.
In addition, in some species, including mice, rats and humans, a read-through transcript (AChE-R) of the 4th exon has been described. The C-terminus of AChE-R differs from those of other AChE variants and cannot tetramerize via disulfide bonding with other subunits, and so only produces a soluble monomeric molecule. The other 95% of the coding sequences of AChE are invariant, as is the catalytic domain of all AChE isoforms.
The AChE-R variant was found in hematopoietic cells of mice and rats but only expressed as 1% of the total AChE in electrically excitable cells under normal conditions. The expression of this variant rapidly increased (during 30 min) in the CNS during stress (Perrier et al., 2002) and in muscles during myasthenia gravis (Brenner et al., 2003). After exposure to anti-AChE agents, the AChE-R level slightly increases but remains minor (around 1%) (Perrier et al., 2005). Direct injection of human BuChE into the circulation provides protection against organophosphate poisoning (Evron et al., 2007), but the possibility of the involvement of soluble monomers of AChE-R in the rapid hydrolysis of ACh in the synaptic cleft remains an open question. For example, in mutant animals without AChE-S in the synapses, no changes in the amplitude and duration of synaptic potentials after AChE inhibitors were observed. This suggests that in the absence of AChE-S in the synaptic cleft, inhibitors of AChE did not cause a further increase in ACh concentration and, hence, the contribution of AChE-R to the hydrolysis of ACh, in these mutants at least, is not significant. Our PCR analysis of the AChE-R mRNA expression of this minor AChE variant showed no significant differences between all of the investigated organs. Because the differences in the sensitivity of AChE from the various organs to C-547 were observed in both electrophysiological and biochemical experiments, we can exclude the putative enhancement of AChE-R synthesis in response to the inhibition of AChE-S as a hypothesis to explain the differences in sensitivity to C-547. Thus, our analysis revealed no correlation between the efficiency of AChE inhibition in various organs by C-547 and the level of mRNA splicing (AChE-S vs. AChE-R).
Homotetramers of AChE-S can covalently bind with one of the anchor subunits – Col Q (collagen Q) or PRiMA (proline-rich membrane anchor) (Massoulie, 2002). ColQ forms a triple helical structure in a proline-rich domain in their C-terminus. As each ColQ can attach one AChE-S tetramer, hetero-oligomeric complexes may contain four, eight or twelve AChE subunits, which are designated the A4, A8 and A12 asymmetric forms, respectively (Bon et al. 2003). In muscles, the dominant one is A12, which consists of three tetramers anchored at the basal lamina via the collagen-like tail Col Q. The latter is a minority tetramer bound to the cell membrane by 20 kDA – PRiMA and its content varies in different types of muscles. On the other hand, G4 is highly abundant in the brain where it forms 80–90% of the total cholinesterase activity (Rotundo, 1988; Inestrosa et al., 1994). PRiMA induces the formation of the complex with AChE-S homotetramers (G4) and attaches them to the cell membrane via a transmembrane domain (Perrier et al., 2002). The PRiMA anchor is mainly synthesized by nervous tissue (Perrier et al., 2002; 2003) but Col Q by muscles (Feng et al., 1999). In muscle cells, the expression of PRiMA mRNA, as well as the level of PRiMA AChE was suppressed by myogenesis and innervation (Xie et al., 2008). Therefore, in neuromuscular junctions, the majority of the AChE is anchored to the basal lamina by Col Q with a relatively low level of PRiMA AChE that is most likely secreted by the nerve (Jevsek et al., 2004; Leung et al., 2009). In brain synapses, the enzyme is mainly attached to the plasma membrane through PRiMA (Grassi et al., 1982); moreover, this anchor is required for the intracellular processing of AChE in neurons (Dobbertin et al., 2009). However, the specific inhibitors for the catalytic subunits formed by Col Q and PRiMA-anchored molecular forms of AChE are not known. In this respect, it is interesting that treadmill exercise, which increases the amount of PRiMA AChE (G4), but not Col Q AChE (A12) in fast EDL, can dramatically decrease the sensitivity of the EDL to C-547. This suggests that AChE anchored by PRiMA is less sensitive to C-547 than the AChE anchored by Col Q. Therefore, it is not surprising that brain PRiMA-anchored AChE is three orders of magnitude less sensitive to C-547 (as we have demonstrated here in biochemical and electrophysiological experiments) than it would be if it were anchored by Col Q. Most likely the G4 form is less sensitive towards the inhibitory action of this compound. However, the purification and direct measurement of the sensitivity of isolated G4 to C-547 is a difficult task because of the relatively low level of activity of this subtype in the total muscular AChE. Experiments in this regard are underway.
What then is known about the differences between PRiMA and ColQ AChEs? ColQ and PRiMA appear to form similar but not the same quaternary complexes with AChE-S subunits, which differ in the number of prolines in the domains that form this complex (8 in ColQ, 14 in PRiMA). In addition, the number and positions of the cysteines are very different in ColQ (2 adjacent cysteines, located 4 and 5 residues upstream of the first proline) and in PRiMA (4 cysteines located 2, 4, 8 and 15 residues upstream of the first proline, plus a cysteine located 13 residues downstream of the last proline) (Massoulie, 2002). Therefore, the organization of the complex of AChE with one of the anchor subunits (PRiMA and ColQ) may contain some structural differences, but it is not yet known how these may influence the interaction with AChE inhibitors (in particular, with C-547).
Differences between the catalytic subunits of PRiMA and ColQ AChEs were described at the level of the post-translational modification of AChE. So, human, mouse and rat AChE carries three N-glycosylation sites, all of which were utilized in the recombinant version of the enzyme. Using lectin-agarose conjugates, it has been demonstrated that AChE anchored by Col Q binds to DBA and VVA-B lectins (specific for N-acetylgalactosamine), while AChE anchored by PRiMA does not (Scott et al., 1988). This means that the terminal residues in the N-glycans of PRiMA and ColQ AChE isoforms are different. These are N-acetylgalactosamine in ColQ AChE and another N-glycan in AChE anchored by PRiMA. Although differences in glycosylation have been shown to affect the efficiency of some AChE inhibitors (edrophonium, rivastigmine) or 2G8 antibody (Liao et al., 1991), the involvement of glycosylation in the differing sensitivities of PRiMA and ColQ AChEs to ADEMS requires verification.
To our knowledge, this report is the first evidence of the possibility of organ- and tissue-specific AChE inhibition. It also raises the issue of the molecular differences between the PRiMA and ColQ AChEs and, consequently, of the possibility of the targeted synthesis of new anti-cholinesterase agents, specific to the brain, say, for the treatment of Alzheimer's disease. At the same time, muscle-specific ADEMS could be tested for the ability to treat syndromes caused by either a decreased density of cholinoceptors or amount of transmitter released, such as myasthenia gravis, a number of congenital myasthenic syndromes (Engel et al., 2010) or Sjogren's syndrome with malfunctioning salivation (Dawson et al., 2005). Also patients with brain injuries (Giacobini, 2004) or in a post-stroke condition, whose AChE levels are reduced but BuChE levels elevated (Ben Assayag et al., 2010) may be affected by the various ADEMS in different ways. It would also be important to test whether the carriers of common debilitating variants of BuChE, such as the Kalow variants in the BuChE gene, having a 30% lower catalytic activity that might be a potential contributing factor to Alzheimer's disease) (Podoly et al., 2009) could be less susceptible to the effects of ADEMS.
Future experimental studies should be focused: (i) on the possible modulation of ADEMS binding to Col Q- vs. PRiMA-AChEs; (ii) on the differences in AChE N-glycosylation; and (iii) on uncovering other reasons for the differing sensitivities of AChEs to these compounds.