Haim Meshulam

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O‐di‐isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O‐cyclohexyl methylphosphonofluoridate) and soman (O‐pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O‐isopropyl‐O‐(p‐nitrophenyl) methyl phosphonate (IMP‐pNP), DFP, and chlorpyrifos‐oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase‐inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was ∼ 10‐fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild‐type, and ninefold higher activity towards soman. L69V exhibited 100‐fold faster hydrolysis of DFP than the wild‐type. The active‐site mutant H115W exhibited 270–380‐fold enhancement toward hydrolysis of the P–S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino‐acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP‐pNP, indicating that PON1 is less active toward the more toxic optical isomers.

M1 Agonists for the Treatment of Alzheimer's Disease.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1‐m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151(S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of β/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau‐1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimers disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single‐blind placebo‐controlled, parallel‐group study in patients with probable AD. AFl02B was signiticantly effective at 40 and 60 mg, tid in the ADAS, ADAS‐cognitive and ADAS‐word recognition scales.

The anti-inflammatory and cholinesterase inhibitor bifunctional compound IBU-PO protects from β-amyloid neurotoxicity by acting on Wnt signaling components

Changes in signal transduction are implicated in neuronal responses to the Alzheimers amyloid-beta-peptide (Abeta), which include neurotransmitter systems and pathways involved in the maintenance of the nervous system. We report here that a new bifunctional compound IBU-PO, which combines a non-steroidal anti-inflammatory drug (NSAID) (Ibuprofen) and a cholinesterase (ChE) inhibitor (Octyl-Pyridostigmine), is neuroprotective against Abeta-neurotoxicity, and its activity is associated to Wnt signaling components in rat hippocampal and mouse cortical neurons. IBU-PO (0.01-1 microM) inhibits glycogen-synthase-kinase-3beta (GSK-3beta) and stabilizes cytoplasmic beta-catenin reverting the silencing of the Wnt pathway caused by Abeta-toxicity and GSK-3beta overexpression. In addition, IBU-PO enhances, dose-dependently, the non-amyloidogenic amyloid precursor protein (APP) cleavage by increasing secreted APP and decreasing endogenous Abeta1-40 in rat hippocampal neurons.

Selective Signaling via Unique Ml Muscarinic Agonistsa

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AP102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the ml AChR subtype. Notably, the selectivity of the new ml agonists is reflected also by activation of select second messenger systems via distinct G‐proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand‐selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimers disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Bifunctional compounds eliciting both anti-inflammatory and cholinergic activity as potential drugs for neuroinflammatory impairments

We tested two novel bifunctional compounds: ibuprofen-N-octyl-pyridostigmine bromide (IBU-PO) and ibuprofen-N-decyl-pyridostigmine bromide (IBU-PD). They both contain a non-steroidal anti-inflammatory drug (NSAID), ibuprofen (IBU) and pyridostigmine (PO), a cholinesterase inhibitor that acts as a cholinergic up-regulator (CURE). The two moieties are conjugated by a hydrocarbon spacer consisting of 8 (octyl) and 10 (decyl) carbons, respectively. The compounds were tested for their efficiency in reducing the neurological symptoms observed in experimental autoimmune encephalomyelitis induced in mice by myelin oligodendrocyte glycoprotein (MOG). IBU-PO and IBU-PD significantly ameliorated the clinical score (a 40-50% reduction in disease severity) over a period of 30 days, following daily administration of 1 and 0.1mg/kg, i.p., respectively. Clinical improvement was accompanied by reduced responsiveness of MOG-specific T-cells. In addition, IBU-PO and IBU-PD down-regulated the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in cultured astrocytes. To determine which moiety was responsible for these effects, we tested each of the two components, IBU and PO. Our findings indicate that combining NSAID with cholinergic intervention contributes an added therapeutic value for each distinct entity and that these bifunctional compounds act both on the peripheral immunological system and on the central nervous system (CNS) inflammatory pathways.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: An unifying hypothesis

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimers disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimers disease.

Rigid analogs of acetylcholine can be m1-selective agonists: implications for a rational treatment strategy in Alzheimer's disease

Abstract Rigid analogs of acetylcholine offer an opportunity for selective actions at muscarinic receptor subtypes, since restricted conformational mobility alters the capacity of ligands to adapt to subtle differences in receptor structure. AF102B , a highly rigid analog of acetylcholine, is a centrally active M1 agonist and is evaluated in light of some currently available therapeutic strategies in Alzheimers disease.

Selective Signaling Via Novel Muscarinic Agonists: Implications for Alzheimer’s Disease Treatments and Clinical Update

Five human muscarinic acetylcholine receptors (mAChR) (ml–m5), have been cloned and expressed in suitable cell systems (reviewed by Hulme et al., 1990). * mAChRs have two binding domains, a ligand-binding extracellular (and including membrane-spanning) domain and a G-protein binding intracellular domain. This second domain, by interaction with various G-proteins, controls and modulates second messenger systems (Hulme et al., 1990).

Characterization of asymmetric fluorogenic phosphonates as probes for developing organophosphorus hydrolases with broader stereoselectivity

Organophosphorus hydrolases (OPH) such as mammalian plama paraoxonase (PON1) detoxify asymmetric toxic organophosphorus (OP) nerve agents by preferentially hydrolyzing the less toxic P(+) optical isomer. In order to develop new OPHs with broader stereoselectivity we have prepared a series of asymmetric fluorogenic organophosphonates (Flu-OPs). Such Flu-OPs may serve as molecular probes for screening large libraries of OP hydrolases during directed evolution. Flu-OPs were prepared as methylphosphonates (MPs) diesters containing either ethyl (E), isopropyl (I), cyclohexyl (C) or pinacolyl (P) groups that are structural congeners of the nerve agents VX, sarin, cyclosarin and soman, respectively. The second ester bond was formed with fluorescent moieties that are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy 9,9-dimethyl-9H-acridin-2-one (DDAO). To further characterize the Flu-OPs as surrogates of their respective nerve agents, we have studied the reactivation of Flu-OP-inhibited AChE using 2-PAM and toxogonin (TOX). AChE was 90-95% inhibited by all Flu-OPs (0.36-0.9(M) and then was reactivated by either 2-PAM or TOX. TOX caused a more rapid reactivation than 2-PAM with the following rank order; EMP>IMP>CMP. TOX was also shown to be a better reactivator than 2-PAM for AChE inhibited by the nerve agents VX and cyclosarin. PMP-AChE could not be reactivated by either TOX or 2-PAM, similarly to aging of PMP-AChE formed by inhibition with soman. Racemic CMP-MeCyC was used for screening two new PON1 variants from a neutral library of PON1. These multiple mutation variants include replacement of active site amino acid residues. Neither mutation in these new variants appeared in PON1 variants previously discovered by directed evolution using symmetric Flu-OP. Detoxification rate of cylcosarin by these new PON1 variants was rather slow indicating the need to further screen PON1 clones using optically active Flu-OPs. Therefore, we have separated enzymatically the P(-) enantiomer of CMP-MeCyC and determined its 98% purity using chiral HPLC.

Ml Muscarinic Agonists: From Treatment Toward Delaying Progression of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized, inter alia, by synaptic loss, neurofibrillary tangles, amyloid plaques containing the β-amyloid peptide (Aβ), and degeneration of cholinergic neurons that ascend from the basal forebrain to cortical and hippocampal areas (reviewed by Court and Perry, 1991). A presynaptic cholinergic hypofunction, as one of the major neuronal events in AD, is reflected, inter alia, in reduced levels of acetylcholine (ACh), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT). As a result of neuronal degeneration, the density of presynaptic M2 muscarinic receptors (mAChR) is significantly decreased in AD, yet post-synaptic Ml mAChR are relatively unchanged in AD (review by Court and Perry, 1991) (vide infra for further discussion). Degeneration of cholinergic neurons in AD is presumably a principal cause of the dementia. The “cholinergic hypothesis’.in AD implies that a cholinergic replacement therapy might be beneficial in alleviating some of the cognitive dysfunctions in this disorder (Court and Perry, 1991). Highly selective ml agonists, producing cellular excitation, should be beneficial in AD, regardless of the extent of degeneration of presynaptic cholinergic projections to the frontal cortex or hippocampus. This represents the most relevant approach of cholinergic treatment due to the role of Ml mAChRs in memory and learning processing (Fisher and Barak, 1994, Fisher, 1997). The present overview is an attempt to address some of these findings and to propose an unifying hypotheses regarding ml selective agonists aimed at treatment and therapy of AD.